JP2023541816A - Methods and compositions for treating autoimmune diseases and cancer - Google Patents

Abstract

Molecular constructs that target tumor necrosis factor receptor 1 (TNFR1) and/or tumor necrosis factor receptor 2 (TNFR2) are provided. The constructs are for treating diseases, disorders, and conditions in which these receptors and/or TNF are involved in the pathogenesis or their inhibition or activation is intended to treat diseases, disorders, and conditions or It can improve the symptoms. Constructs provided herein include TNFR1 antagonist constructs that are engineered to inhibit TNFR1 function and eliminate TNFR1 agonist activity. Constructs provided herein include agonists and antagonists of TNFR1 and TNFR2. TNFR1 antagonist constructs are engineered to inhibit TNFR1 function and, in some embodiments, to avoid agonist activity. Also included are agonists and antagonists of TNFR2. Agonists of TNFR2 enhance regulatory T cell function to control acute or chronic inflammation. Antagonists of TNFR2 are intended to reduce regulatory T cell function and increase immunity to treat cancer and certain immunodeficiency diseases. Also provided are methods of treating various diseases in which TNF and its receptors play a role. [Selection diagram] Figure 1

Description

Related Application Filed on August 27, 2020, inventor H. Michael Shepard and Applicant Enosi Life Sciences Corp. Priority is claimed to U.S. Provisional Application No. 63/071,313 entitled ``METHODS AND COMPOSITIONS TO TREAT AUTOIMMUNE DISEASES AND CANCER,'' filed by J.D.

This application was filed on February 19, 2020 and published on August 27, 2020 as International PCT Publication No. WO2020/172218, by the inventor H. Michael Shepard and Applicant Enosi Life Sciences Corp. Related to International PCT Application No. PCT/US2020/018739 entitled "ANTIBODIES AND ENONOMERS". This application is a U.S. national phase application PCT/US2020/018739 filed on February 19, 2020, claiming the benefit of Provisional Application No. 62/808,635, filed on February 21, 2019. , also related to U.S. Application No. 17/432,720, filed August 20, 2021.

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 submitted herein, the contents of which are incorporated by reference in its entirety. The electronic file was created on August 26, 2021, is 1.69MB in size, and has the title 5301SEQPC1. It was txt.

FIELD This application relates to nucleic acid constructs and encoded products for use as anti-TNF therapies. The diseases to be treated are those in which TNF receptors and/or TNF or TNF/TNF receptor pathways are involved or play a role in their pathogenesis.

Anti-TNF therapy/TNF blockers (a type of biological disease-modifying antirheumatic drugs; DMARDs) are usually prescribed after failure of conventional DMARDs. These therapies include monoclonal antibodies (mAbs), such as the chimeric mAb infliximab (Remicade®); murine variable regions and human IgG1 constant regions, and the fully humanized mAb (IgG1) adalimumab (e.g., the trademarked Humira®), and golimumab (Simponi® antibody); PEGylated humanized Fab' fragment of mAb targeting TNF, certolizumab pegol (Cimzia® antibody) and TNFR2 fusion proteins, such as the TNFR2-Fc fusion protein etanercept (sold under the trademark Enbrel®), which contains an extracellular receptor region containing a binding site for human TNFR2 fused to the Fc of human IgG1. The drug, sold under the trademarks Remsima® and Inflectra®, is a derivative of infliximab, which is approved for use in the European Union for the treatment of various autoimmune and chronic inflammatory diseases and disorders. It is a biosimilar. These TNF inhibitors, which sequester TNF, are effective in treating, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA), and/or inflammatory bowel disease (IBD; Crohn's disease and ulcerative arthritis). used in the treatment of a variety of diseases or conditions, including colitis (such as colitis).

However, such treatments have serious side effects, such as sepsis and serious infections such as listeriosis, reactivation of tuberculosis, reactivation of hepatitis B/C, Reactivation of herpes zoster, as well as invasive fungal and other opportunistic infections, such as M. with an increased risk of reactivation of T. tuberculosis infection. These treatments have been shown to induce macrophage apoptosis in rheumatoid synovium. Infliximab is associated with increased apoptosis in inflammatory cell infiltrates in the intestines of patients with Crohn's disease. Other antirheumatic drugs such as methotrexate and glucocorticoids can also induce apoptosis of immune cells (see, e.g., Vigna-Perez et al. (2005) Clin. Exp. Immunol. 141(2):372-380). thing). These therapeutic agents can cause severe congestive heart failure, drug-induced lupus, and demyelinating central nervous system (CNS) disease, as well as worsening of lymphoma and non-melanoma skin cancers (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. Source ( ncbi.nlm.nih.gov/books/NBK507863/). Other adverse side effects include liver damage, demyelinating diseases/CNS disorders, lupus, psoriasis, sarcoidosis, and increased susceptibility to the development of additional autoimmune diseases as well as cancer, including lymphoma and solid malignancies. (e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113(43):12304-12309; Zalevsky et al. (2007) J. Immunol. 179:1872-1883; Zoran et al. (2019) Sci. Rep. 9:17231). Therefore, the use of these therapeutics is limited, especially for chronic diseases/conditions that require long-term administration, such as arthritis and inflammatory bowel disease (IBD). Approximately 30% of RA patients are non-responsive or do not see sustained treatment benefit with the use of anti-TNF therapy (e.g. McCann et al. (2014) Arthritis & Rheumatology 66(10 ):2728-2738). Nonresponse also occurs in non-RA patients receiving anti-TNF therapy. Depending on the anti-TNF agent, 13-33% of treated patients respond to treatment. up to 46% stop responding, leading to discontinuation or dose escalation (see, e.g., Richter et al. (2019) MABS 11(4):653-665). Thus, improved treatment Treatments that are effective and safe are needed.

SUMMARY Molecular constructs that target tumor necrosis factor receptor 1 (TNFR1) and/or tumor necrosis factor receptor 2 (TNFR2), and nucleic acids encoding them, are provided. This construct treats diseases, disorders, and conditions in which these receptors and/or TNF are involved in the pathogenesis or their inhibition or activation may ameliorate the disease, disorder, and/or condition or its symptoms. It is for treatment. Constructs provided herein include agonists and antagonists of TNFR1 and TNFR2. TNFR1 antagonist constructs are designed to inhibit TNFR1 function and avoid TNFR1 agonist activity. Also included are agonists and antagonists of TNFR2. Agonists of TNFR2 enhance regulatory T cell function to control acute or chronic inflammation. Antagonists of TNFR2 reduce regulatory T cell function to enhance immunity and are used in the treatment of cancer and certain immunodeficiency diseases.

There are two TNF receptors on cells, TNFR1 and TNFR2. These pathways balance each other in normal physiology. TNF/TNFR1 promotes inflammation, whereas TNF/TNFR2 is anti-inflammatory. TNFR2 is generally activated later than TNFR1, so it does not immediately affect beneficial TNF-induced inflammation, but activates later to suppress overactivation of inflammatory pathways. Simultaneous inhibition of both pathways abolishes the anti-inflammatory effect of TNFR2. Existing TNF blockers are limited in their effectiveness because the Treg generator (TNFR2), which is anti-inflammatory, is turned off/off.

The constructs provided herein inhibit TNFR1 signaling or activity without compromising the ability of treated subjects to fight opportunistic infections, among other properties that distinguish them from conventional therapeutics that target TNF/TNFR. do. Some constructs provided herein specifically target and inhibit TNFR1 but do not antagonize TNFR2, thereby preventing transient activation of TNFR1 through receptor clustering. One type is a modified single chain antibody. The constructs provided herein silence the TNF inflammatory pathway mediated by TNFR1, but preserve, and in some embodiments enhance, the TNFR2 healing pathway. These constructs may be administered to treat indications that TNF blockers have become ineffective. Among the constructs provided herein are constructs that specifically inhibit tumor necrosis factor receptor type 1. Use of the methods and constructs for treating diseases, disorders, and conditions in which TNF or its receptor plays a role in the pathogenesis or symptoms is provided.

Existing anti-TNF drugs are used to treat diseases such as rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, axial spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohn's disease, pediatric Crohn's disease, and ulcerative colitis. Blocks excessive inflammation that occurs in autoimmune diseases. The constructs herein may be used to treat the same disease but avoid deleterious or adverse side effects. The constructs provided herein are more effective at suppressing inflammatory cytokines in vivo than conventional therapeutics such as the TNFR2-Fc fusion protein etanercept (sold under the trademark Enbrel®). and maintain regulatory T cell function. The constructs may include activity modifiers or property modifiers that increase serum half-life and have shown activity in blocking TNFR1 signaling, such as in TNF assays comparing activity to adalimumab and/or etanercept.

As established in mouse models, we show that this construct preserves macrophage function better than adalimumab and does not cause opportunistic infections; these constructs also preserve Treg function to a greater extent than adalimumab or etanercept. and is effective in treating diseases, disorders, and conditions such as rheumatoid arthritis. In some embodiments, the Kd is ≦1 nM and the in vivo t1/2 is about 10-12 days. The construct may be administered by any suitable route for the particular indication. Routes include, but are not limited to, subcutaneous, intravenous, intratumoral, intrahepatic, topical, mucosal, intradermal, and any other suitable route.

Among the constructs provided herein are: Constructs are provided that are tumor necrosis factor receptor 1 (TNFR1) antagonist constructs of formula 1: (TNFR1 inhibitor) n-linker p-(activity modifier) q, where n and q are each integers; , each independently 1, 2, or 3; p is 0, 1, 2, or 3; a TNFR1 inhibitor is a molecule that binds to TNFR1 and inhibits (antagonizes) the activity of TNFR1. an activity modifier is a moiety that modulates or alters the activity or pharmacological properties of the construct compared to the construct in the absence of the activity modifier; a linker increases the plasticity of the construct; and /or alleviate or reduce steric effects or interactions of the construct with the receptor, and/or increase the solubility of the construct in aqueous media]. A linker may include multiple components. Linkers include chemical linkers, polypeptide linkers, and combinations thereof. Constructs may be attached via chemical and/or physical bonds. This construct may be a fusion protein.

TNFR1 inhibitors may include domain antibodies (dAbs) or single chain antibodies. Constructs include those in which the TNFR1 inhibitor is a domain antibody (dAb) and the activity modifier is neither an unmodified single Fc region nor a human serum albumin antibody. For example, the activity modifier (or property modifier) is a modified Fc region or human serum albumin. In the construct, the TNFR1 inhibitor can be one that inhibits TNFR1 signaling and/or the activity modifier increases the serum half-life of the construct. For example, the construct may include an activity modifier that is modified to reduce or eliminate albumin, or ADCC (antibody-dependent cytotoxicity) activity, and/or CDC (complement-dependent cytotoxicity) activity to be reduced or eliminated. Examples include constructs that are Fc. A TNFR1 inhibitor can be an inhibitor that inhibits TNFR1 activity but does not antagonize tumor necrosis factor receptor 2 (TNFR2) activity. A TNFR1 inhibitor can be an inhibitor that inhibits TNFR1 signaling.

Multispecific constructs are also provided. For example, a multispecific construct comprising a TNFR1 inhibitor and a Treg expander is provided, wherein the bispecific construct interacts with two different target receptors or antigens or epitopes on the receptors. Ru. Among the multispecific constructs are constructs that are bispecific for TFNR1 and Treg expanders. The Treg expander may be a TNFR2 agonist.

The construct may include linkers to provide plasticity, increase solubility, and/or alleviate and/or reduce steric hindrance and/or van der Waals interactions. Constructs optionally generally include activity modifiers to alter or modulate the activity or properties of the construct. Constructs having the following formula 2 are provided: (TNFR1 inhibitor)n-(activity modifier)r1-(linker (L))p-(activity modifier)r2-(TNFR2 agonist)q, or (TNFR1 inhibitor) n-(activity modifier)) r1-(linker (L)) p-(activity modifier) r2-(Treg expander) q [where n=1, 2, or 3 and p= 1, 2, or 3, q=0, 1, or 2, and each of r1 and r2 independently is 0, 1, or 2]; this component The specified order or any other order may be as long as they interact to antagonize TNFR1, agonize TNFR2, or have Treg expander activity. For example, constructs in which the TNFR1 inhibitor moiety inhibits binding of TNFα binding to TNFR1 and/or inhibits signal transduction are included among those provided herein.

Constructs of formula 3a or 3b are also provided: (TNFR2 agonist or Treg expander) n-linker p-(activity modifier) q, formula 3a, or (activity modifier) q-linker p-(TNFR2 agonist or Treg expander) Expander) n, Formula 3b, where n and q are each an integer, each independently 1, 2, or 3; p is 0, 1, 2, or 3; the activity modifier is , a moiety that alters the pharmacological properties or activity of the construct; the TNFR2 agonist interacts with TNRFR2 and results in TNFR2 activity]. Treg expanders are molecules that include TNFR2 agonists and result in the expansion of Treg cells. The linker increases plasticity and/or alleviates or reduces steric effects or interaction of the construct with the receptor; and/or alters the solubility of the construct. In some embodiments, the activity modifier is an Fc region or a modified Fc region or a short FcRnBP. The linker is a linker that includes a hinge region or includes G and S residues. An example of a linker is a linker that increases the serum half-life of the construct. For example, the linker may have a sequence set forth in any of SEQ ID NOs: 812-834, 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 the serum half-life of the construct. The Fc region can be an Fc dimer. The Fc region may be modified to have reduced ADCC and/or CDC activity, such as an Fc modified to have reduced or no ADCC activity.

Constructs provided herein include those in which the TNFR1 inhibitor is defined in the Sequence Listing, listed below, or any of those known in the art. The Treg expander is any known in the art, is a TNFR2 agonist, or is listed in the Sequence Listing, or is a Treg expander known in the art; the linker is any Treg expander known in the Sequence Listing or listed below. The activity modifier is any shown in the Sequence Listing, known in the art, and/or shown below.

A construct is provided that is a TNFR1 antagonist construct, the 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 Prevents transient activation of TNFR1 via receptor clustering. In such constructs, the antibody or antigen-binding portion thereof includes modifications that improve the pharmacological properties and/or structure of the construct. In any of the constructs provided herein, the construct agonizes TNFR2 signaling, thereby increasing expression of regulatory T cells (Tregs), thereby causing TNFR1 antagonism and concomitant (or (concomitant with) a component that provides for increased expression of Tregs. In the constructs provided herein, a TNFR1 inhibitor inhibits TNFR1 by inhibiting TNFR1 signaling, e.g., when the antibody or antigen binding portion of the construct inhibits binding of TNFα binding to TNFR1. It can be a single chain antibody. Among the constructs are constructs in which the TNFR1 inhibitor is an antibody or antigen binding moiety that does not inhibit TNFα binding to TNFR1, but inhibits TNFR1 signaling. The property or activity that can be modulated/altered may be serum half-life.

The construct may include an Fc modified to eliminate ADCC and/or CDC activity. The construct may include Fc dimers, such as one Fc monomer containing a hole and the other containing a knob, forming a heterodimer. For example, knob mutations are selected from S354C, T366Y, T366W, and T394W (according to EU numbering), and hole mutations are selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering). , whereby the Fc monomers form a heterodimer. In some embodiments where the construct includes Fc, the Fc is derived from trastuzumab. This construct can be dimerized by fusion of the N-terminus and C-terminus of trastuzumab.

In some embodiments where the construct includes a linker, the linker is or includes a hinge region from an Fc region. For example, the hinge region is derived from trastuzumab and is linked to the Fc region. Constructs include constructs that include a linker connected to an 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 can be linked to the Fc region directly or through the hinge region. The Fc region or modified Fc region includes, for example, the sequence of amino acids shown in any of SEQ ID NOs: 10, 12, 14, 16, 27, 30, 1469, and 1470.

Constructs that bind neonatal Fc receptors (FcRn) are also provided. For example, TNFR1 constructs are provided that include a short FcRn-binding peptide (FcRnBP), where the short FcRn-binding peptide (FcRnBP) provides interaction with the FcRn of the construct and comprises 6-25 or 10-20 amino acid residues. Contains groups. For example, FcRnBP contains 12-20 residues or 15 or 16 residues. Examples of these are TNFR1 antagonist constructs in which the FcRn binding peptide (FcRnBP) comprises or consists of any of the peptides of SEQ ID NOs: 48-51. This construct includes a TNFR1 construct containing Fc heterodimers, one Fc monomer containing a hole and the other knob, so that the resulting Fc dimer is a heterodimer.

A TNFR1 antagonist construct is provided, the construct comprising a TNFR1 inhibitor; an Fc dimer; and a Treg expander, the Fc dimer comprising two complementary Fc monomers, and the TNFR1 inhibitor comprising an Fc dimer; The Treg expander is linked to one of the monomers and the Treg expander is linked to the other Fc monomer. In such constructs, the Treg expander can be a TNFR2 agonist. It may further comprise a second Treg expander linked to the same Fc monomer as the TNFR1 inhibitor, the first and second Treg expanders being the same or different. The second Treg expander may be a TNFR2 agonist. In some embodiments, the Treg expanders are the same. A TNFR1 inhibitor can be one that inhibits or blocks TNFR1 signaling. In some embodiments, a TNFR1 inhibitor binds TNFR1 and blocks or inhibits TNFα binding and TNFR1 signaling. In some embodiments, the TNFR1 inhibitor binds to TNFR1, does not prevent or interfere with TNFα binding, and blocks or inhibits TNFR1 signaling. In some embodiments of these constructs, the Treg expander is a TNFR2 agonist. A TNRF2 agonist can be one that stimulates or induces TNFR2 signaling. Examples of Treg expanders are TNFR2 agonists that are scFvs, VHH single domain antibodies, or Fabs of TNFR2 agonist monoclonal antibodies. In these constructs, the Treg expander can be a small molecule, a TNFR2 agonist, or a nucleic acid aptamer, or a peptide aptamer.

Also provided are any of these constructs that are or are also TNFR2 agonists. The TNFR2 agonist is a construct of formula 3a or 3b, where formula 3a is (Treg expander) n-linker p-(activity modifier) q and formula 3b is (activity modifier) q-linker p-(Treg expander )n. In these formulas, n and q are each integers, each 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 pharmacological properties of the construct compared to the construct in the absence of the activity modifier; a linker increases the plasticity of the construct and/or Alleviating or reducing steric effects of the construct or its interaction with its receptor and/or increasing the solubility of the construct in aqueous media. In both of these constructs, the Treg expander in the construct is a TNFR2 agonist. For example, a TNFR2 agonist stimulates or induces TNFR2 signaling. In other examples, the Treg expander is a TNFR2 agonist, which is an scFv, a VHH single domain antibody, or a Fab of a TNFR2 agonist monoclonal antibody. Treg expanders can be TNFR2 agonists that are small molecules, or nucleic acids, or peptide aptamers. In constructs containing all or a portion of trastuzumab, such as the Fc portion and/or the Fc and hinge region or modified forms thereof, the construct may be dimerized by N-terminal fusion with the C-terminus of trastuzumab.

A construct comprising a TNFR1 inhibitor moiety linked to one or more Treg expanders via a central PEG linker, or a construct comprising at least two TNFR1 inhibitors that are the same or different, or a construct comprising two Treg expanders that are the same or different. is provided. Constructs that include a PEG moiety, such as a central PEG linker, may include branched PEG moieties that link the TNFR1 inhibitor and one or more Treg expanders. Examples include those having structures selected from formulas 4A-4D:
Formula 4A:

n is 1 to 5;
R1 is H or CH3, or CH2CH3 or other C1-C5 alkyl;

is a TNFR1 inhibitor (TNFR1 antagonist);

is a Treg expander; or formula 4B:

is a TNFR1 inhibitor (TNFR1 antagonist),

is a Treg expander;
n is 1-5; or formula 4C:

is a TNFR1 inhibitor (TNFR1 antagonist) or a Treg expander;
n is 1-5; or formula 4D:

During the ceremony, each

are the same or different, each independently selected from a TNFR1 inhibitor (TNFR1 antagonist), and a TNFR2 agonist;
Activity modifiers are optional and may be linked to any suitable locus in the molecule; n is 1-5.

In the TNFR1 antagonist constructs and other constructs provided herein, the Treg expander can be a TNFR2 agonist. These constructs can be used, for example, when the activity modifier is an Fc region, or an Fc region that includes a hinge region or other linker; may include activity modifiers, such as Fc modified to reduce or eliminate . Examples thereof are constructs in which the Fc or modified Fc is an IgG Fc or an IgG1 or IgG4 Fc, and/or constructs that bind to neonatal Fc receptors (FcRn). Examples of these constructs include the constructs comprising short FcRn binding peptides (FcRnBPs), the short FcRn binding peptides (FcRnBPs) providing the interaction of the construct with FcRn, and containing between 6 and 25, such as between 10 and 20 amino acid residues. groups; including.

a) a TNFR1 inhibitor moiety that is TNFR1 selective; b) optionally one or more linkers; and c) optionally a half-life extending moiety, comprising at least one of b) and c); and Also provided are TNFR1 antagonists of any of the formulas in this application. In such constructs, the TNFR1 selective antagonist selectively binds to and inhibits TNFR1 signaling rather than TNFR2 signaling. As described for the constructs above, the TNFR1 inhibitor, linker, and other components can be as described above. These include constructs containing antigen-binding fragments in which selective antagonist TNFR1 inhibitors selectively bind to and inhibit TNFR1 signaling rather than TNFR2 signaling. For example, antigen-binding fragments that selectively bind to and inhibit TNFR1 rather than TNFR2 signaling can include domain antibodies (dAbs), scFv, or Fab fragments. 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, a human anti-TNFR1 antagonist monoclonal antibody may be directed to H398 comprising SEQ ID NO: 678, or ATROSAB, or an antigen-binding portion thereof, or SEQ ID NO: 31, or 32, or 673 or 678, or an antigen-binding portion thereof that binds to TNFR1. A sequence having at least 95% sequence identity to. Examples of TNFR1 inhibitors include domain antibodies (dAbs) or antigen-binding portions thereof, or sequences of amino acids set forth in any of SEQ ID NOs: 52-672, or at least a domain antibody (dAb) thereto that retains TNFR1 inhibitor activity. scFv comprising a sequence with 95% sequence identity; and/or at least 90% or 95% sequence identity thereto, and/or retains TNFR1 inhibitor activity; contains variants of these polypeptides with the same identity; and/or retains at least 90% or a Nanobody comprising a sequence with 95% sequence identity; and/or having the sequence set forth in SEQ ID NO: 683 or 684; A TNFR1 inhibitor comprising sequences with sequence identity. Some TNFR1 inhibitors, for example, confer selective inhibition of TNFR1;
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, 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/Q88 N/T89Q (Based on the sequence of soluble TNF shown in SEQ ID NO: 2)
a dominant-negative tumor necrosis factor (DN-TNF) or a TNF mutein (e.g., a DN-TNF or TNF mutein is a soluble TNF molecule) containing one or more amino acid substitutions selected from . For example, a TNFR1 inhibitor comprises a sequence of residues set forth in any one of SEQ ID NOs: 701-703, or a sequence of residues set forth in any one of SEQ ID NOs: 701-703 that retains TNFR1 inhibitor activity. A TNF mutein that includes a sequence that has at least about 90% or 95% sequence identity to the sequence or a fragment thereof.

Any of the foregoing constructs provided herein may include a linker, which may include all or a portion of the trastuzumab hinge sequence, SCDKTH corresponding to residues 222-227 of SEQ ID NO: 26, or trastuzumab up to the complete sequence of the hinge region of the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 sequences thereof. Contains or has a sequence having at least 98% or 99% sequence identity thereto of contiguous residues, or residues ESKYGPPCPPCP residues 212-223 of SEQ ID NO: 29, or a linker. For example, the construct may include a linker that includes the sequence SCDKTH corresponding to residues 222-227 of SEQ ID NO:26. The construct may include a GS linker, a linker containing glycine and serine (GS) residues, instead of or in addition to another linker. Exemplary GS linkers for any of the constructs provided herein include (GlySer)n, where n=1-10; (GlySer2); (Gly4Ser)n, where n=1 ~10); (Gly3Ser) n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG ; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; Also included are GS linkers and linkers that include all or a portion of the hinge sequence of trastuzumab corresponding to residues EPKSCDKTHTCPPCP (residues 219-233 of SEQ ID NO: 26), e.g., the linker includes a GS linker, or , contains only the sequence SCDKTH corresponding to residues 217-222 of SEQ ID NO: 31 from the hinge sequence. Such linkers include, for example, GS linkers and linkers that include all or a portion of the nivolumab hinge sequence corresponding to residues 212-223 of SEQ ID NO:29.

Constructs herein may include activity modifiers. Activity modifiers described anywhere herein include those described above and below, as well as others known to those of skill in the art; activity modifiers alter the activity or properties of the construct. The modified modifying activity can be a half-life extending moiety, which is an IgG Fc, a polyethylene glycol (PEG) molecule, or human serum albumin (HSA). Examples of IgG Fc are IgG1 or IgG4 Fc. The IgG1 Fc can be the Fc of trastuzumab shown in SEQ ID NO: 27, or a sequence of amino acids having at least 95% sequence identity thereto; the IgG4 Fc can be the Fc of nivolumab shown in SEQ ID NO: 30, or at least 95% sequence identical thereto. % sequence identity. For example, IgG1 Fc is human IgG1 Fc shown in SEQ ID NO: 10, and IgG4 Fc is human IgG4 Fc shown in SEQ ID NO: 16.

Constructs described herein include constructs that are or include TNFR1 inhibitors. These include constructs in which the TNFR1 inhibitor is monovalent. These include linkers such as where the linker comprises (Gly4Ser)3, and/or linkers comprising (Gly4Ser)3 and SCDKTH (residues 217-222 of SEQ ID NO: 31); and/or (Gly4Ser)3 and SEQ ID NO: 26; and/or (Gly4Ser)3 and nivolumab, corresponding to residues 212-223 of SEQ ID NO:29. Examples of constructs provided herein that inhibit TNFR1 include constructs that inhibit TNFR1 and include the sequence of residues set forth in any of SEQ ID NOs: 704-764; A construct having a sequence that has at least 95% or at least about 95% sequence identity to the sequence of residues shown in .

Provided herein are TNFR1 antagonist constructs. These include constructs in which the TNFR1 construct contains a short FcRn-binding peptide (FcRnBP); -25, e.g., the FcRnBP comprises 12-20 residues, or 15 or 16 residues, e.g., the FcRn-binding peptide (FcRnBP) is any of the peptides of SEQ ID NOs: 48-51 or at least about 95% thereof or an FcRn-binding peptide (FcRnBP) consisting of a peptide having sequence identity of SEQ ID NOs: 48 to 51.

Other exemplary TNFR1 inhibition constructs provided herein include constructs that include: a) domain antibodies that inhibit TNFR1; b) increase plasticity; reduce steric effects; a linker that increases solubility; and c) a half-life extending moiety. Constructs in which the half-life extending moiety is not a human serum albumin antibody or unmodified Fc are included. These constructs include a) domain antibodies (dAbs) of any of SEQ ID NOs: 52-672, or scFvs of any of SEQ ID NOs: 673-678, or Fabs of any of SEQ ID NOs: 679-682; 683 or 684 Nanobody, or a TNF mutein of any of SEQ ID NOs: 685-703; b) a GS linker, wherein (GlySer)n (wherein n=1-10); (GlySer2); (Gly4Ser)n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1-5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; TNFR1 antagonist constructs comprising a half-life extending moiety that is a G Fc. It will be done. In these constructs, or any provided herein containing one or more of these components, the GS linker can be (GGGGS)3; the IgG Fc can be the Fc of trastuzumab, or It may be Fc of nivolumab.

Other constructs provided herein that are TNFR1 antagonist constructs include: a) a domain antibody (dAb) of any of SEQ ID NOs: 52-672, or an scFv or sequence of any of SEQ ID NOs: 673-678; Fabs of any of numbers 679-682, or Nanobodies of SEQ ID NOs: 683 or 684, or TNF muteins of any of SEQ ID NOs: 685-703; b) all or part of the hinge sequence of trastuzumab, and of the hinge sequence of nivolumab; and c) a half-life extending moiety that is an IgG Fc. In such constructs, the linker may include all or part of the trastuzumab hinge sequence, where the IgG Fc is the trastuzumab Fc. In other embodiments, the linker may include all or part of the nivolumab hinge sequence and the IgG Fc is the nivolumab Fc.

Any construct provided herein that is a TNFR1 antagonist construct, comprising:
a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) A first linker that is a GS linker, wherein (GlySer)n (wherein n=1 to 10); (GlySer2); (Gly4Ser)n (wherein n=1 to 10); (Gly3Ser); ) n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG a first linker selected from; GGSSGGSGGSGSSG; GSSSGSGSGGSSSGSGSG;
c) a second linker selected from all or a portion of the hinge sequence of trastuzumab and all or a portion of the hinge sequence of nivolumab; and d) a construct comprising a half-life extending moiety that is an IgG Fc is provided. Ru.

In some embodiments, these constructs can include a first linker that is a GS linker that is (GGGGS)3; a second linker that has the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31). Contains; IgG Fc is the Fc of trastuzumab. In other embodiments, the first linker is a GS linker and is (GGGGS)3; the second linker comprises all or a portion of the nivolumab hinge sequence; the IgG Fc is a nivolumab Fc .

a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG;GGSSGGSSSGGGSSSGGSSG; 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 skilled in the art, such as (GGGGS)3. The PEG molecule may have a molecular weight of at least 25 kDa, generally at least 30 kDa or more, such as at least 40 kDa or 50 kDa, or 60 kDa, or 80 kDa, or more.

a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG;GGSSGGSSSGGGSSSGGSSG; and GSSSGS and c) a half-life extending moiety that is human serum albumin. An example of a linker is any described herein, such as when the GS linker is (GGGGS)3.

The primary amino acid sequence of any of the constructs provided herein (those above and below) can be optimized or modified to eliminate immunogenic sequences or epitopes. For example, in constructs containing IgG Fc, the IgG Fc can be modified to: a) introduce a knob-into-hole; b) to increase or enhance neonatal Fc receptor (FcRn) recycling. and c) modifications to reduce or eliminate immune effector function. In any such construct and containing IgG Fc, knob mutations may be selected among S354C, T366Y, T366W, and T394W (according to EU numbering); hole mutations may include Y349C, T366S, L368A, F405A , Y407T, Y407A, and Y407V (according to EU numbering). These TNFR1 Antagonist Constructors have T250Q, T250R, T250R, M252W, M252Y, M252Y, S254T, T256D, T256D, T256D, T256D, T256Q, V308I, V308F, E. 380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M 428L, M428L/N434S, V259I/V308F, It may be a construct selected from one or more of V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering). one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP) TNFR1 antagonist constructs that can be modified to reduce or eliminate immune effector functions, such as selected immune effector functions. For example, in these TNFR1 antagonist constructs, modifications to reduce or eliminate immune effector function are
Among 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 (according to EU numbering); and among IgG4: L235E, F234A/ L235A, S228P /L235E, and S228P/F234A/L235A (according to EU numbering)
selected from one or more of the following.

The TNFR1 antagonist or multispecific construct may include a central PEG linker portion; the construct may include a modified Fc region such as those described above, where the Fc region is a modified IgG Fc, and the modified IgG Fc is Modifications below:
a) Modification to introduce knob into hole,
Knob mutations were selected among S354C, T366Y, T366W, and T394W (according to EU numbering);
a modification in which the hole mutations are selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering),
modification selected from one or more of;
c) a modification to increase or enhance one or more immune effector functions,
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to increase or enhance immune effector function are
Among IgG1, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P 396L; F243L/R292P/Y300L; L234Y/G236W/S298A (in the first heavy chain) and S239D/A330L/I332E (in the second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in the first heavy chain) and D270E/K326D/A330M/K334E (in second heavy chain); A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A /I332E;G236A/ S239D/I332E; G236A/S239D/A330L/I332E; introduction of biantennary glycan at residue N297; introduction of defucosylated 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 (according to EU numbering),
one or more of the modifications selected from one or more of the following.

In some embodiments, in any of the constructs that include an Fc region, the construct may include an IgG1 Fc that includes one or more modifications to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. For example, one or more modifications that increase binding to FcγRIIb include one of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K (according to EU numbering). selected from one or more.

Also provided are constructs that are Treg expander constructs. Such constructs include: a) Treg expanders; b) increasing the plasticity of the construct and/or mitigating or reducing steric effects of the construct or its interaction with receptors; and c) a moiety that modulates or alters the activity or pharmacological properties of the construct as compared to the construct in the absence of the activity modifier. included. Treg expanders can be TNFR2 agonists. These constructs may further include a TNFR1 inhibitor. In some embodiments, the TNFR2 agonist is a TNFR2 selective agonist.

a) a TNFR2 agonist; b) a linker that increases the plasticity of the construct and/or alleviates or reduces the steric effects of the construct or its interaction with the receptor and/or increases the solubility of the construct in an aqueous medium. and c) an activity modifier, the moiety modulating or altering the activity or pharmacological properties of the construct as compared to the construct in the absence of the activity modifier. Constructs described herein are provided that are agonist constructs. In the TNFR2 agonist construct, the TNFR2 agonist may be a TNFR2 selective agonist. The construct may include an activity modifier, such as an activity modifier that is a half-life extending moiety. The construct can be a TNFR2 agonist construct that selectively activates or antagonizes TNFR2 without activating or antagonizing TNFR1. Included are TNFR2 agonist constructs in which the TNFR2 agonist binds to one or more epitopes within TNFR2. These include human TNFR2. Such epitopes include, for example, epitopes selected from one or more of the epitopes comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 839-865, 1202, and 1204.

A TNFR2 agonist construct is provided, the TNFR2 agonist comprising an antigen-binding fragment of an agonist human or humanized anti-TNFR2 antibody, or an antigen-binding portion thereof, or a single chain form thereof. An example of such an antibody is an agonist anti-TNFR2 antibody selected from MR2-1 (also referred to as ab8161; US Patent No. 9,821,010) or MAB2261 (US Patent No. 9,821,010). be done. A 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. Selective agonists can include TNFR2 agonist TNF muteins. Exemplary TNFR2 selective agonist muteins include, but are not limited to, 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, A 145R/S147T, E146D/S147D, D143V/ A soluble TNF variant comprising one or more TNFR2 selective mutations selected from F144L/A145S, S95C/G148C, and D143V/A145S, and any combination of the foregoing (all according to SEQ ID NO: 2) Can be mentioned. For example, TNFR2 agonists are TNF muteins, including mutein D143N/A145R.

In the TNFR2 agonist construct, the linker includes any described herein or known to those skilled in the art for use as a linker. Exemplary linkers include all or a portion of the trastuzumab hinge sequence corresponding to residues 219-233 of SEQ ID NO: 26, or all of the nivolumab hinge sequence corresponding to residues 212-223 of SEQ ID NO: 29. or a portion thereof, or a sequence having at least 95% sequence identity thereto. Other exemplary linkers include or consist of the sequence SCDKTH, which corresponds to residues 217-222 of SEQ ID NO:31. The linker can be a glycine-serine (GS) linker, such as, but not limited to, (GlySer)n (wherein n=1-10); (GlySer2); (Gly4Ser)n (wherein n=1). ~10); (Gly3Ser) n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG and GSSSGS. The linker can be, for example, a GS linker corresponding to residues 219-233 of SEQ ID NO: 26 and a linker comprising all or part of the hinge sequence of trastuzumab, or a GS linker and a sequence corresponding to residues 217-222 of SEQ ID NO: 31. It may also include a combination of linkers such as SCDKTH, or a GS linker and all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29.

All constructs provided herein may include activity modifiers that alter or modulate the properties or activities of the construct. For example, a half-life extending moiety is an activity modifier or a property modifier. Examples of the foregoing and below are IgG Fc, polyethylene glycol (PEG) molecules, and parts or derivatives of human serum albumin (HSA), or variants thereof. For example, in some cases the IgG Fc is an IgG1 or IgG4 Fc. An example of an IgG1 Fc is the Fc of trastuzumab shown in SEQ ID NO: 27; the Fc of an IgG4 Fc is the human version of nivolumab Fc shown in SEQ ID NO: 30; IgG1 Fc is human IgG4 Fc shown in SEQ ID NO: 16.

In some embodiments of TNFR2 agonist constructs, the TNFR2 agonist is monovalent. In others, it is multivalent, such as divalent or trivalent. TNFR2 constructs can include linkers as described herein. For example, the linker can be attached to a Gly-Ser, such as (Gly4Ser)3, or (Gly4Ser)3 and SCDKTH (residues 217-222 of SEQ ID NO: 31), or (Gly4Ser)3 and residues 219-233 of SEQ ID NO: 26. Corresponding hinge sequence of trastuzumab, or the hinge sequence of nivolumab corresponding to (Gly4Ser)3 and residues 212-223 of SEQ ID NO: 29, or a mutation of any of the foregoing having at least 95% sequence identity thereto. May include the body. These constructs may also include activity modifiers, such as modifiers that are half-life extending moieties such as PEG or HSA mentioned above. As above and below, the PEG moiety has a size of at least 20 kDa, typically at least 30 kDa or more.

a) a TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG;GGSSGGSSSGGGSSSGGSSG; and GSSSGS and c) a half-life extending moiety that is an IgG Fc. As described above, in an exemplary embodiment, the GS linker may be (GGGGS)3; the IgG Fc is the Fc of trastuzumab or the Fc of nivolumab.

Other TNFR2 agonist constructs include
a) a TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) a linker selected from 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.
Examples of linkers and activity modifiers are the trastuzumab hinge sequence; the IgG Fc is all or part of the trastuzumab Fc, or the nivolumab hinge sequence; the IgG Fc is the nivolumab Fc.

In other embodiments, the TNFR2 construct is
a) a TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; G; GGSSGGSSGGGSSGGSSG; and GSSSGS a first linker that is a selected GS linker;
c) a second linker selected from all or part of the hinge sequence of trastuzumab and all or part of the hinge sequence of nivolumab; and d) an activity modifier that is a half-life extending moiety that is an IgG Fc. include. Examples of such constructs include constructs 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); There is a construct that is Fc of trastuzumab. In other embodiments, the first linker is (GGGGS)3 and the second linker comprises all or part of the nivolumab hinge sequence; the IgG Fc is nivolumab Fc.

In some embodiments, the construct is
a) a TNFR2 agonist comprising an antigen binding fragment of an agonist human anti-TNFR2 antibody selected from MR2-1 or MAB2261;
b)
i) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG; GGSSGGSSGGGSSGGSSG; and GSSSGS a selected GS linker; and/or ii) a linker comprising all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and c) an IgG1 or IgG4 Fc, a PEG molecule, and human serum albumin. an activity modifier that is a half-life extending moiety selected from (HSA),
IgG1 Fc is human IgG1 Fc shown in SEQ ID NO: 10, or trastuzumab Fc shown in SEQ ID NO: 27;
A TNFR2 agonist construct comprising an activity modulator, wherein the PEG molecule has a molecular weight of at least 30 kDa or at least about 30 kDa.

In some embodiments, the construct is
a) 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, A14 5Q/E146D/S147D, A145T/E146D/ S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G1 48C, and D143V/A145S, (SEQ ID NO. a TNFR2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from the following:
b)
i) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG; GGSSGGSSGGGSSGGSSG; and GSSSGS a selected GS linker; and/or ii) a linker comprising all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and c) an IgG1 or IgG4 Fc, a PEG molecule, and human serum albumin. an activity modifier that is a half-life extending moiety selected from (HSA),
IgG1 Fc is human IgG1 Fc shown in SEQ ID NO: 10, or trastuzumab Fc shown in SEQ ID NO: 27;
A TNFR2 agonist construct comprising an activity modulator, wherein the PEG molecule has a molecular weight of at least 30 kDa or at least about 30 kDa.

In some embodiments, the construct is
a) TNFR2 TNF mutein containing mutations D143N/A145R;
b) a (GGGGS)3 linker; and c) a half-life extending moiety that is the Fc of trastuzumab or the Fc of nivolumab.

In some embodiments, the construct is
a) TNFR2 selective TNF mutein containing mutations D143N/A145R;
b) a (GGGGS)3 linker and a second linker comprising the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and c) a TNFR2 agonist comprising an activity modifier that is the Fc half-life extending portion of trastuzumab. It is a construct.

In some embodiments, the construct is
a) TNFR2 selective TNF mutein containing mutations D143N/A145R;
b) a (GGGGS) 3 linker and a second linker comprising all or part of the hinge sequence of nivolumab; and c) a TNFR2 agonist construct comprising an activity modifier that is the Fc half-life extending portion of nivolumab.

In some embodiments, the construct is
a) TNFR2 selective TNF mutein containing mutations D143N/A145R;
b) a linker comprising all or part of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO: 26; and c) a TNFR2 agonist construct comprising a half-life extension portion that is the Fc of trastuzumab.

In some embodiments, the construct is
a) TNFR2 selective TNF mutein containing 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) a TNFR2 agonist comprising an activity modifier that is the Fc half-life extending portion of nivolumab. It is a construct.

TNFR1 antagonist constructs, TNFR2 agonist constructs, and both, where the IgG Fc is monomeric or dimeric are provided. Constructs provided herein may include a dAb (or Vhh). The construct may include a Vhh single chain or double chain containing the dAb. These constructs may include HSA linked directly to the dAb or via a linker. The HSA and dAb can be linked in any order, such as to the N-terminus of the HSA and the C-terminus of the dAb linked directly or via a linker, as described above. An example of such a construct is a construct containing:
a) Residues 20-732 of dAb Dom1h-131-206 of SEQ ID NO: 59, or the construct of SEQ ID NO: 1475 or the remainder of SEQ ID NO: 1475, linked to HSA via a linker as shown in SEQ ID NO: 1475. a construct having at least 95%, 96%, 97%, 98%, 99% sequence identity to groups 20-732 and having TNFR1 antagonist activity; or b) a construct 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 having the sequence shown in any of SEQ ID NOs: 57-59 and variants thereof having 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 has at least 95%, 96%, 97%, 98%, 99% sequence identity thereto and has TNFR1 antagonist activity. or e) any combination of a) to d); or f) a humanized sequence of any of a) to f), or a sufficient portion of the construct for administration to humans is humanized. If so, a sufficient portion of the sequence is sufficient to eliminate or reduce an immune response to the construct when administered to a human.

A construct provided herein that is a TNFR1 construct may further include a TNFR2 agonist, or the construct may be a TNFR2 agonist construct. In constructs containing TNFR2 agonists, the TNFR2 agonist can be modified to eliminate amino acid or epitope sequences that are immunogenic in the subject being treated, such as for administration to a human subject. In constructs that include a TNFR2 agonist, the construct can be a TNFR2 selective agonist. These constructs may include modified IgG Fc. For example, IgG Fc has the following modifications:
a) Modification to introduce knob into hole;
b) modifications to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent It may include one or more modifications to reduce or eliminate immune effector functions selected from one or more of cell-mediated phagocytosis (ADCP). Examples of such modifications are:
a) Modifications to introduce knob into hole are:
one or more knob mutations selected from S354C, T366Y, T366W, and T394W (according to EU numbering); and selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering); Fc is selected from one or more hole mutations that form dimers;
b) Modifications to increase or enhance FcRn recycling include:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering)
selected from one or more of;
c) Modifications to reduce or eliminate immune effector functions are
Among 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 (according to EU numbering); and among IgG4, L235E, F234A/ L235A, S228P /L235E, and S228P/F234A/L235A (according to EU numbering)
selected from one or more of the following.

Constructs provided herein include TNFR2 agonist constructs that include modified IgG Fc, where the IgG Fc is modified as follows:
a) one or more modifications to introduce a knob-into-hole,
Knob mutations were selected among S354C, T366Y, T366W, and T394W (according to EU numbering);
Whole mutations are selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering), modifications;
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering); and c) a modification for increasing or enhancing immune effector function,
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to increase or enhance this immune effector function are
Among IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/ P396L; F243L/R292P/Y300L ; L234Y/G236W/S298A (in first heavy chain) and S239D/A330L/I332E (in second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in 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; G236A; /I332E;G236A /S239D/I332E; G236A/S239D/A330L/I332E; introduction of biantennary glycan at residue N297; introduction of defucosylated 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;S 267E/H268F/S324T; G236A/I332E/ S267E/H268F/S324T; E345R; and E345R/E430G/S440Y (according to EU numbering),
one or more of the modifications selected from one or more of the following.

Constructs provided herein that are TNFR2 agonist constructs may include modifications that increase binding to FcγRIIb, where the Fc is modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. may include a modified IgG1 Fc, such as. Examples of such modifications are modifications selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K (according to EU numbering). be.

Constructs that are or include TNFR2 agonist constructs that selectively activate or agonize TNFR2 without activating or antagonizing TNFR1 are provided. These constructs include constructs that include an activity modifier that is a) a TNFR2 agonist; b) one or more linkers; and c) a half-life prolonging moiety;
The TNFR2 agonist construct is a fusion protein comprising a single chain TNFR2 selective TNF mutein trimer fused to a multimerization domain and has the following 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, each of which may be the same or different; TNFmut is a TNFR2-selective TNF mutein; L1, L2 and L3 are linkers which may be the same or different. . The TNF muteins are 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/S14 7D, A145Q/E146D/S147D, A145T/ E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S 95C/G148C, and D143V/A145S (sequence 2), such as TNFR2 selective mutein D143N/A145R. In these constructs, the multimerization domain is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; 805), or the trimerization domain of human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807), or at least 95%, 96%, 97%, 98%, 99% sequence identity thereto may be selected from mutants having the same sex. For example, the multimerization domain is an IgG1 Fc or an IgG4 Fc, and an IgG1 Fc or an IgG4 Fc is also a half-life extending moiety. These constructs include linkers, including any linkers described herein and any linkers known to those of skill in the art. Examples of these constructs are those in which the L1, L2 and/or L3 linkers are (GGGGS)n, where n=1-5, and all or part of the stalk region of TNF (SEQ ID NO: 812) or to Constructs independently selected among variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity. These constructs include those in which the linker between the TNFR2 agonist and the half-life extending moiety is: a GS linker, where (GlySer)n (where n=1-10); (GlySer2 ); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) GS linker selected from n (wherein n=1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSGSGG; GGSSGG; GGSSGGSGGSSSSG; ; or all or part of the trastuzumab hinge sequence and the nivolumab hinge A linker selected from all or part of the sequence; or a combination thereof. The half-life extending moiety may be selected from: an IgG1 Fc, which is the Fc of human IgG1 as shown in SEQ ID NO: 10, or an Fc of trastuzumab, as shown in SEQ ID NO: 27; a Fc of human IgG4, as shown in SEQ ID NO: 16. , or an IgG4 Fc that is the Fc of nivolumab shown in SEQ ID NO: 30; a PEG molecule of at least 30 kDa or at least about 30 kDa in size; human serum albumin (HSA), and at least 95%, 96%, 97% thereto; Variants of polypeptide moieties with 98%, 99% sequence identity.

Constructs that are or include TNFR2 agonist constructs are provided. These constructs include the following expressions:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (formula III) [wherein
a) MD is a multimerization domain; TNFmut is a TNFR2-selective TNF mutein; L1, L2, and L3 are linkers that may be the same or different, where:
i) MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or human TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
ii) L1, L2 and L3 are each (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812), or a mixture thereof;
iii) the TNF mutein comprises the TNFR2 selective mutation D143N/A145R];
b)
IgG1 Fc which is human IgG1 Fc shown in SEQ ID NO: 10 or trastuzumab Fc shown in SEQ ID NO: 27;
IgG4 Fc that is human IgG4 Fc shown in SEQ ID NO: 16 or nivolumab Fc shown in SEQ ID NO: 30;
a PEG molecule of at least 30 kDa or at least about 30 kDa in size; and human serum albumin (HSA);
a half-life extending moiety selected from; and c) a linker between the TNFR2 selective agonist and the half-life extending moiety,
(GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5); ( SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; ;GGSSGGSSSGGGSSGGGSSG; and GSSSGS. or a linker selected from 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.

Constructs include TNFR2 agonist constructs comprising the following 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; L1, L2 and L3 are linkers, which may be the same or different;
i) the MD is selected from IgG1 Fc or IgG4 Fc;
ii) L2 and L3 in formula II and L1 and L2 in formula III are each independently (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812); or a combination thereof;
iii) Each of L1 of formula II and L3 of formula III is
(GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5); ( SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; ;GGSSGGSSSGGGSSGGGSSG; and GSSSGS. or a linker selected from 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;
iv) TNF muteins include TNFR2 selective mutations D143N/A145R]. In these constructs, the MD is
IgG1 Fc that is the Fc of human IgG1 shown in SEQ ID NO: 10, or the Fc of trastuzumab shown in SEQ ID NO: 27; or Fc of human IgG4 shown in SEQ ID NO: 16, or Fc of nivolumab shown in SEQ ID NO: 30. IgG4 Fc; or combinations or variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto.

An exemplary construct is one that includes a MD that is an IgG1 Fc of trastuzumab, and the linker between the MD and the adjacent TNF mutein is of the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO:26. The MD, in whole or in part, or 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 a construct that includes a MD that is an IgG1 Fc of trastuzumab, and the linker between the MD and the adjacent TNF mutein corresponds to (Gly4Ser)3 and residues 219-233 of SEQ ID NO: 26. Contains the trastuzumab hinge sequence. In some embodiments, the MD is an 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); The MD is the IgG4 Fc of nivolumab, and the linker between the MD and the adjacent TNF mutein is all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29, or the MD is the IgG4 Fc of nivolumab. The linker between the MD and the adjacent TNF mutein, including a linker that is Fc, includes (Gly4Ser)3 and all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29.

Constructs herein, including agonist constructs, can be modified to eliminate immunogenic sequences, such as immunogenic to humans. Provided herein are TNFR2 agonist constructs in which the TNFR2 agonist is modified to eliminate immunogenic sequences or epitopes that are immunogenic in a subject, such as a human subject.

In the constructs provided herein that are TNFR2 agonist constructs and include modified IgG Fc, the IgG Fc has the following modifications:
a) Modification to introduce knob into hole,
the knob mutation is selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering);
a modification in which the hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering)
c) a modification to reduce or eliminate immune effector function;
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to reduce or eliminate immune effector function are
Among 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 (according to EU numbering); and among IgG4, L235E, F234A/ L235A, S228P /L235E, and S228P/F234A/L235A (according to EU numbering),
may include one or more of the modifications selected from one or more of the following.

Provided are any of the foregoing constructs that are TNFR2 agonist constructs comprising a modified IgG Fc, wherein the IgG Fc has the following modifications:
a) Modification to introduce knob into hole,
the knob mutation is selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering);
a modification in which the hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering)
c) a modification for increasing or enhancing immune effector function;
The immune effector function is selected from one or more of CDC, ADCC and ADCP; the modification to increase or enhance the immune effector function is in the IgG1, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A (in first heavy chain) and S239D/A330L/ I332E (in second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in first heavy chain) and D270E/K326D/A330M/K334E (in second heavy chain); A327Q/ P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A 330L/I332E; two at residue N297 Introduction of branched glycans; introduction of defucosylated glycans at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C2 20D /D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; /E430G/S440Y (according to EU numbering)
one or more of the modifications selected from one or more of the following.

There is provided any of the foregoing TNFR2 agonist constructs according to any of the claims, comprising an IgG1 Fc modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. Examples of such are S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K (according to EU numbering). selected from one or more.

The constructs provided herein can be multispecific in that they interact with more than one target. An example of such a multispecific construct is a multispecific TNFR1 inhibitor/TNFR2 agonist construct, which is of any of the following formulas:
(TNFR1 inhibitor) n-linker (L) p- (TNFR2 agonist) q (Formula I), or (TNFR1 inhibitor) n-linker (L) p- (TNFR2 agonist) q, or (TNFR1 inhibitor) n - (TNFR2 agonist) q-linker (L)p, or (TNFR2 agonist) q-(TNFR1 inhibitor) n-linker (L)p, or any of the above containing an appropriate activity modifier [wherein n = 1 or 2, p = 1, 2, or 3, and q = 1 or 2; the TNFR1 inhibitor interacts with TNFR1 and inhibits its activity; A linker is a moiety that modulates or alters the activity or pharmacological properties of the construct compared to the construct in the presence of a linker; [Change effect]. These constructs include constructs that are multispecific TNFR1 inhibitor/TNFR2 agonist constructs, where the TNFR1 inhibitor selectively inhibits TNFR1 signaling without inhibiting or antagonizing TNFR2 signaling. TNFR1 inhibitors do not interfere with the activation or agonization of TNFR2; TNFR2 agonists selectively activate or agonize TNFR2 signaling without activating or agonizing TNFR1 signaling. and TNFR2 agonists do not interfere with either inhibition or antagonism of TNFR1. Examples of such constructs are constructs a) to c) below:
a) the TNFR1 inhibitor is
i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB, or a polypeptide having a sequence with at least 95% sequence identity thereto; or ii) a domain of any of SEQ ID NOs: 52-672. An antibody (dAb), or a scFv of any of SEQ ID NOs: 673-678, or a Fab of any of SEQ ID NOs: 679-682, or a Nanobody of SEQ ID NOs: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 701-703. , or a polypeptide having a sequence that has at least 95% sequence identity with any of the aforementioned polypeptides and is a TNFR1 inhibitor; or iii) confers selective inhibition of TNFR1;
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, 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/Q88 N/T89 (Based on the sequence of soluble TNF shown in SEQ ID NO: 2)
a) a dominant-negative tumor necrosis factor (DN-TNF) or a TNF mutein comprising a soluble TNF molecule having one or more amino acid substitutions selected from b) a linker;
i) GS linker, (GlySer)n (in the formula, n=1 to 10); (GlySer2); (Gly4Ser)n (in the formula, n=1 to 10); (Gly3Ser)n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; SSSGSGSG;GGSSGGSSSGGGSSSGGSSG and GSSSGS; and/or ii) all or part of the trastuzumab hinge sequence corresponding to residues 219-233 of SEQ ID NO: 26, or corresponding to residues 212-223 of SEQ ID NO: 29. all or part of the hinge sequence of nivolumab; and iii) an IgG1 or IgG4 Fc,
the IgG1 Fc is selected from a human IgG1 IgG1 Fc as set forth in SEQ ID NO: 10, or a trastuzumab IgG1 Fc as set forth in SEQ ID NO: 27;
the IgG4 Fc is selected from a human IgG4 IgG4 Fc set forth in SEQ ID NO: 16, or a nivolumab IgG4 Fc set forth in SEQ ID NO: 30;
Optionally, one for Fc to introduce knob-into-hole and/or to increase or enhance neonatal Fc receptor (FcRn) recycling and/or to reduce or eliminate immune effector function. IgG1 or IgG4 Fc containing the above modifications
selected from;
c) the TNFR2 agonist is
i) an antigen binding fragment that binds to one or more epitopes within human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202, and 1204; or ii) selected from MR2-1 or MAB2261. or iii) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D14 3N/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, E 146D/S147D, D143V/F144L/A145S, or iv) a TNFR2 selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from S95C/G148C, and D143V/A145S (see SEQ ID NO: 2); or iv) mutation D143N. /A145R, wherein the TNF mutein is (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812). or v) a single-chain TNFR2-selective TNF mutein trimer linked by the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III)
a TNFR2 selective agonist comprising: wherein MD is a multimerization domain; TNFmut is a TNFR2 selective TNF mutein; L1, L2, and L3 are linkers that may be the same or different; ,
The MD is 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. merization domain (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
L1, L2 and L3 are each (GGGGS)n (wherein n=1 to 5), or are all or part of the stalk region of TNF (SEQ ID NO: 812), or a mixture thereof;
TNF mutein contains TNFR2 selective mutation D143N/A145R]
selected from.

Other such constructs include constructs that are multispecific TNFR1 antagonist/TNFR2 agonist constructs;
a) The TNFR1 inhibitor is a domain antibody (dAb) of any of SEQ ID NOs: 52-672, or an scFv of any of SEQ ID NOs: 673-678, or a Fab of any of SEQ ID NOs: 679-682, or SEQ ID NO: 683 or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) the linker comprises (GGGGS)3, a polypeptide comprising the sequence SCDKTH (residues 222-227 of SEQ ID NO: 26), and the Fc of trastuzumab;
c) TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A1 45R/S147T, Q88N/T89S/ A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E14 6S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A 145S, S95C/G148C, and D143V/A145S (See SEQ ID NO: 2).

Other such multispecific constructs include
a) The TNFR1 inhibitor is a domain antibody (dAb) of any of SEQ ID NOs: 52-672, or an scFv of any of SEQ ID NOs: 673-678, or a Fab of any of SEQ ID NOs: 679-682, or SEQ ID NO: 683 or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) the linker comprises (GGGGS)3, all or part of the hinge sequence of nivolumab, and the Fc of nivolumab;
c) TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A1 45R/S147T, Q88N/T89S/ A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E14 6S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A 145S, S95C/G148C, and D143V/A145S (See SEQ ID NO: 2), which is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from
There is a construct.

Other such multispecific constructs include
a) The TNFR1 inhibitor is a domain antibody (dAb) of any of SEQ ID NOs: 52-672, or an scFv of any of SEQ ID NOs: 673-678, or a Fab of any of SEQ ID NOs: 679-682, or SEQ ID NO: 683 or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) the linker comprises (GGGGS)3 and the Fc of trastuzumab;
c) TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A1 45R/S147T, Q88N/T89S/ A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E14 6S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A 145S, S95C/G148C, and D143V/A145S (See SEQ ID NO: 2), which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from:
There is a construct.

Other such multispecific constructs include
a) The TNFR1 inhibitor is a domain antibody (dAb) of any of SEQ ID NOs: 52 to 672, or an scFv of any of SEQ ID NOs: 673 to 678, or a Fab of any of SEQ ID NOs: 679 to 682, or SEQ ID NO: 683 or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) the linker comprises (GGGGS)3 and the Fc of nivolumab;
c) The TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A1 45R/S147T, Q88N/T89S/ A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E14 6S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A 145S, S95C/G148C, and D143V/A145S , and TNFR2-selective TNF muteins that are soluble TNF variants comprising one or more TNFR2-selective mutations selected from any combination of the foregoing mutations (see SEQ ID NO: 2).
There is a construct.

These multispecific constructs may include modified Fc, where the IgG Fc has the following modifications:
a) Modification to introduce knob into hole;
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) a modification to reduce or eliminate immune effector function.

Examples of Fcs containing knob-into-hole modifications are:
the knob mutation is selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering);
The hole mutations are selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering).

Other examples are multispecific constructs that include Fcs, e.g., Fcs that include modifications to increase or enhance FcRn recycling include T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q , V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/ Y436H, N434F/Y436H, T250Q/M428L , T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering). Fc is an immune effector selected from one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). May include modifications to functionality. The Fc may include modifications to reduce or eliminate immune effector function in IgG1 and/or IgG4:
Among 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 (according to EU numbering); and/or among IgG4, L235E, F234 A/L235A , S228P/L235E, and S228P/F234A/L235A (according to EU numbering)

IgG Fc has the following modifications:
a) Modification to introduce knob into hole,
The knob mutation is selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering);
The hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering); and c) a modification for increasing or enhancing immune effector function,
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to increase or enhance immune effector function are
Among IgG1, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/ P396L; F243L/R292P/Y300L ; L234Y/G236W/S298A (in first heavy chain) and S239D/A330L/I332E (in second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in 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; G236A; /I332E;G236A /S239D/I332E; G236A/S239D/A330L/I332E; introduction of biantennary glycan at residue N297; introduction of 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 (according to EU numbering),
may include one or more of the modifications selected from one or more of the following.

Other such multispecific constructs are the following constructs: Constructs comprising an IgG1 Fc modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. Examples of such are that modifications that increase binding to FcγRIIb include: A construct selected from one or more of the following.

Also provided are constructs that are multispecific TNFR1 antagonists/TNFR2 agonists (TNFR1 antagonists are monovalent; TNFR2 agonists are monovalent). Also provided are multispecific constructs that are multispecific TNFR1 antagonist/TNFR2 agonist constructs (wherein the TNFR1 antagonist is monovalent; the TNFR2 agonist is bivalent).

In some embodiments, the multispecific construct is a multispecific TNFR1 antagonist/TNFR2 agonist construct;
a) TNFR1 antagonist is
i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB; or ii) a domain antibody (dAb) of any of SEQ ID NOs: 52-672, or an scFv of any of SEQ ID NOs: 673-678; or a Fab of any of SEQ ID NOs: 679-682, or a TNF mutein of any of SEQ ID NOs: 683 or 684, or SEQ ID NOs: 701-703, or having at least 95% or at least about 95% sequence identity thereto. or iii) confers selective inhibition of TNFR1;
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, 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/Q88 N/T89 (See the sequence of soluble TNF shown in SEQ ID NO: 2)
selected from dominant-negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule having one or more amino acid substitutions selected from;
b) the linker is a branched PEG molecule having a size of at least 30 kDa or at least about 30 kDa;
c) TNFR2 agonist is
i) an antigen binding fragment that binds to one or more epitopes within human TNFR2 selected from among the epitopes shown in SEQ ID NOs: 839-865, 1202 and 1204; or ii) selected from MR2-1 or MAB2261 or iii) an antigen-binding fragment of an agonistic human anti-TNFR2 antibody; or iii) a TNFR2-selective TNF mutein,
K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q8 8N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E14 6D/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 D 143V/A145S (see sequence number 2) or iv) a single-chain TNFR2-selective TNF mutein trimer comprising one or more TNFR2-selective mutations selected from A single chain TNFR2 comprising D143N/A145R, in which the TNF mutein is linked by (GGGGS)n, where n=1-5, or comprising all or part of the stalk region of TNF (SEQ ID NO: 812). or v) a TNFR2 selective agonist having the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III)
[where MD is a multimerization domain; TNFmut is a TNFR2-selective TNF mutein; L1, L2, and L3 may be the same or different linkers, where MD is a multimerization domain of EHD2 (sequence 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 (SEQ ID No. 805); Residues 110-139 of number 806, SEQ ID NO: 807);
L1, L2 and L3 are each (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812), or a mixture thereof;
TNF muteins include TNFR2 selective mutations D143N/A145R]
selected from TNFR2 selective agonists including.

Also provided are multispecific constructs in which the TNFR1 antagonist and TNFR2 agonist are each monovalent. Also provided are constructs in which the TNFR1 antagonist is monovalent and the TNFR2 agonist is bivalent.

The constructs provided herein can be used for and for use in treating a variety of diseases, disorders, and conditions. Provided are chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or conditions characterized by TNF overexpression or deregulated TNFR1 signaling in the pathogenesis thereof. or a multispecific construct that is a multispecific TNFR1 antagonist/TNFR2 agonist for use in the treatment of disorders. Treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders or diseases, conditions or disorders characterized by overexpression of TNF or deregulated TNFR1 signaling in their pathogenesis Provided is the use of multispecific TNFR1 antagonist/TNFR2 agonist constructs for.

Compositions comprising any of the constructs provided herein in a pharmaceutically acceptable carrier or vehicle are also provided. These compositions include, but are not limited to, the modulation of TNF overexpression or TNFR1 signaling in chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, and the pathogenesis thereof. It may be used for or in a method for the treatment of diseases, disorders, and conditions, such as diseases, conditions, or disorders characterized by relief. Examples of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders characterized by overexpression of TNF or deregulated TNFR1 signaling in pathogenesis diseases, disorders, and conditions that are associated with These include 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 (IBD), uveitis, fibrotic diseases, endometriosis, lupus, multiple sclerosis (MS), congestive heart failure, cardiovascular disease, myocardial infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), SARS-CoV-2, influenza, acute and chronic neurological Degenerative 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, lung diseases and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viruses or bacterial infections, pneumonia, genetic diseases with TNF/TNFR1 as the causative pathological mediator, periodic fever syndrome, or cancer. Specifically, the constructs provided herein, such as, but not limited to, 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 include a TNFR2 antagonist and optionally a linker and optionally an activity modifier. Such a construct has, for example, formula 5:
(TNFR2 antagonist) n-linker p-(activity modifier) q, or linker p-(activity modifier) q-(TNFR2 antagonist) n [wherein
n and q are each integers, each independently 1, 2, or 3;
p is 0, 1, 2, or 3;
TNFR2 antagonists are molecules that interact with TNFR2 to inhibit (antagonize) its active TNFR2, thereby inhibiting Treg proliferation and/or inducing Treg death, and inhibiting the proliferation and/or inducing Treg death of TNFR2-expressing tumor cells. may induce death of TNFR2-expressing tumor cells;
An activity modifier is a moiety that modulates or alters the activity or pharmacological properties of the construct as compared to the construct in the absence of the activity modifier;
The linker increases the plasticity of the construct, and/or alleviates or reduces steric effects or interactions of the construct with the receptor, and/or increases the solubility of the construct in aqueous media].

In these constructs, each of the activity modifiers and linkers are as defined and described for the constructs above and below. They can be used in methods of treatment and use, as well as pharmaceutical compositions.

TNFR2 antagonists are useful for a variety of diseases, disorders, and conditions, for example, to reduce and/or inhibit the proliferation of myeloid-derived suppressor cells (MDSCs) and/or on the surface of MDSCs present in the tumor microenvironment. to induce apoptosis in MDSCs by binding to expressed TNFR2; and/or to induce the expansion of T effector cells, including cytotoxic CD8+ T cells, through inhibition of Treg amplification and activity. can be used. The TNFR2 antagonists in the constructs are directed to residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4), or to larger epitopes comprising residues 130-149, 137-144 or 142-149, or within these epitopes. At least five contiguous or non-contiguous residues of does not bind; or 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/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and/or residues 75-128 of SEQ ID NO: 4, Includes antibodies, antigen-binding fragments thereof, or single chain antibodies that do not bind to epitopes containing at least five consecutive or discontinuous residues within 86-103, 111-128, or 150-190. For example, an antibody, fragment thereof, or single chain form thereof is directed against an epitope comprising one or more residues of the KCRPG sequence (SEQ ID NO: 840), while the same is directed against a peptide comprising the KCSPG sequence of human TNFR2 (SEQ ID NO: 839). Binds with an affinity that is at least 10 times higher than that of the antibody or antigen-binding fragment. In some embodiments of the TNFR2 antagonist construct, the TNFR2 antagonist is an antibody or a fragment or single chain form of an antibody selected from:
TNFRAB1 (see SEQ ID NO: 1212 and 1213 for the heavy and light chain sequences of TNFRAB1, respectively), TNFRAB2 and TNFR2A3 (see, e.g., U.S. Patent Publication No. 2019/0144556 for a description of these antibodies) );
CDR-H3 sequence of TNFRAB1 (QRVDGYSSYWYFDV; corresponds to residues 99-112 of SEQ ID NO: 1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223), or at least about 85% thereof. Same sequence An antibody, a fragment thereof, or a single chain form thereof, comprising a CDR-H3 sequence with a specificity. For example, TNFRAB1 specifically binds residues 130-149, including residues KCRPG, of TNFR2 with a 40-fold higher affinity than residues 48-67, including residues KCSPG of TNFR2. In some embodiments, the TNFR2 antagonist is
Epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851)
one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), positions 91-98 (PECLSCGS; SEQ ID NO: 1248), and/or positions 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR2; and an epitope selected from the first epitope in which TNFR2A3 comprises residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and comprises a KCRPG motif, and/or residues 159-171 of human TNFR2 ( VVCKPCAPGTFSN; SEQ ID NO: 1464).

In some embodiments, the TNFR2 antagonist in the construct is a CDR-H1 amino acid having a sequence set forth in any of SEQ ID NOs: 1214, 1215, and 1231-1233; A CDR-H2 having the sequence shown, a CDR-H3 sequence shown in any of SEQ ID NOs: 1217, 1223, and 1225-1229, and/or a CDR-H3 sequence of TNFRAB1 corresponding to residues 99-112 of SEQ ID NO: 1212. H3; CDR-L1 sequence of TNFRAB1 corresponding to the CDR-L1 sequence shown in any of SEQ ID NO: 1218 and 1234 to 1236 and/or residues 24 to 33 of SEQ ID NO: 1213; SEQ ID NO: 1219, 1220, 1237 and/or any of SEQ ID NO: 1221, 1222, and 1241-1244; or the CDR-L3 sequence of TNFRAB1, corresponding to residues 88-96 of SEQ ID NO: 1213; and/or the CDR-H1 and CDR-L3 sequences of the human antibody heavy chain variable domain consensus sequence of SEQ ID NO: 1245. CDR-H2 sequence (replaced with the corresponding CDR sequence of a phenotype-neutral TNFR2-specific antibody) and/or CDR-L1, CDR-L2 and CDR-L3 sequences of the human antibody light chain variable domain sequence of SEQ ID NO: 1246. (substituted with the corresponding CDR sequences of a phenotypically neutral TNFR2-specific antibody) to produce a humanized, antagonistic TNFR2 antibody. For example, the construct includes a TNFR2 antagonist that specifically binds to an epitope within TNFR2 set forth in any one of SEQ ID NOs: 1247-1464. In some embodiments, the TNFR2 antagonist is
(a) one or more epitopes within human TNFR2 comprising one or more of the residues KCRPG corresponding to residues 142-146 of SEQ ID NO: 4, or comprising residues 130-149, 137-144 or 142-149; epitopes that do not bind to larger epitopes, or at least five contiguous or discontinuous residues within these epitopes, including residues KCSPG corresponding to residues 56-60 of SEQ ID NO: 4; and/or (b) one or more TNFR2 epitopes comprising a 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 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 residues 75-128, 86 of SEQ ID NO: 4 specifically binds to an epitope selected from epitopes containing at least 5 contiguous or discontinuous residues within ~103, 111-128, or 150-190.

In some embodiments, the TNFR2 antagonist construct comprises a TNFR2 antagonist that is a small molecule. For example, a TNFR2 antagonist is thalidomide or its analogs, such as lenalidomide and pomalidomide.

In some embodiments, the TNFR2 antagonist construct comprises a TNFR2 antagonist that decreases FoxP3 expression and inhibits the suppressive activity of Tregs. Examples of such antagonists are histone deacetylase inhibitors that reduce FoxP3 expression and inhibit the suppressive activity of Tregs. Examples of such inhibitors are panobinostat or cyclophosphamide or triptolide.

TNFR2 constructs can be used in treatment methods and uses to treat infectious diseases, as well as to treat cancers that express TNFR2. Examples of such cancers are: T-cell lymphomas 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, neurological cancer. The cancer is selected from glioma, head and neck cancer, liver cancer, and lung cancer.

The claims set forth below and in the priority application, and their subject matter, are incorporated by reference into this Abstract.

Figure 1 shows a plasmid map of the pCBL-1 expression plasmid containing the CMV promoter, which is the inserted fragment of TE19080L.

FIG. 2 shows an exemplary bispecific construct with a linker (part of the hinge region) and an activity modifier joining two ligands, such as a TNFR1 inhibitor (TNFR1 antagonist) and a TNFR2 agonist.

FIGS. 3A-D illustrate exemplary PEG-centered multispecific constructs for displaying/providing one or more targets or two or more moieties that interact with one target at multiple sites. show. Figure 3A shows an exemplary bivalent construct. One of the circles is, for example, a polypeptide agonist, antagonist, or binding protein, such as an antibody or antigen-binding fragment thereof, or an aptamer (nucleic acid or peptide). The other circle represents the polysaccharide or receptor ligand or other moiety that interacts with the target of interest. The bivalent nature provides clustering of targets for receptor activation. In embodiments provided herein, targets include TNFR1 and TNFR2; as described throughout this disclosure, moieties include TNFR1 inhibitors, such as moieties that inhibit TNFR1 signaling; and other moieties that are TNFR2 agonists or Treg expanders. Figure 3B shows a monovalent single ligand such as CD3+ for preventing cytokine release syndrome coupled to an agonist, antagonist, or binding protein via a PEG moiety, which is dual for receptor clustering. It is worth it. Again, exemplary targets include TNFR1 and/or TNFR2. Figure 3C shows a heterobifunctional PEG for cross-linking 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 checkpoint control receptors for either stimulation or inhibition of an immune response, or to cross-link two different receptors to achieve suppression of receptor activity. (i.e., to deliver CD3 versus CD450, or two different ligands, such as stimulatory and co-stimulatory ligands, to two different receptors on the same cell). Figure 3D shows homobifunctional PEGs for clustering identical receptors on the same or different cells depending on chain length, or for capturing circulating disease targets such as soluble receptors or ligands such as TNF. shows. Additionally, in all of these embodiments, additional PEG side chains optionally linked to a serum half-life extending moiety such as HSA or another reactive or functional group such as an FcRn polypeptide can be included in these constructs. can. The PEG moiety can be modified or replaced with a moiety with similar properties to present a binding moiety.

FIG. 4 shows additional exemplary configurations and structures of PEG-centered constructs for displaying or providing binding or reactive moieties such as TNFR1 inhibitors and/or TNFR2 agonists described herein.

FIG. 5 shows additional exemplary configurations and structures of PEG-centered constructs for displaying or providing binding or reactive moieties such as TNFR1 inhibitors and/or TNFR2 agonists. X and Y can be a ligand and a reactive moiety.

Detailed explanation summary A. Definition B. Structure and method overview C. Tumor Necrosis Factor (TNF) and Chronic Inflammatory and Autoimmune Diseases and Disorders 1. Tumor necrosis factor (TNF)
2. Tumor necrosis factor receptor (TNFR)
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)
ii. Endometriosis iii. Cardiovascular disease iv. Acute respiratory distress syndrome (ARDS)
v. Severe Acute Respiratory Syndrome (SARS) and COVID-19
D. Treatment of rheumatoid arthritis and other chronic inflammatory and autoimmune diseases and disorders 1. Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs)
2. Anti-TNF therapy/TNF blockerE. Treatment methods targeting TNFR1/TNFR2 1. TNFR1 selective antagonist a. TNFR1 antagonistic antibody b. Monovalent TNFR1 antagonizing antibody/antibody fragment 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 named GSK1995057 and GSK2862277 iii. Nanobodies (Nbs)
iv. Anti-TNFR1 Nanobody-Anti-Albumin Nanobody Fusion Construct c. Dominant-negative inhibitor of TNF (DN-TNF)/TNF mutein 2. TNFR2 selective agonist a. TNFR2 agonist antibody b. TNFR2-selective TNF muteins and fusions thereof 3. Anti-TNFR2 Antagonizing Antibodies and Small Molecule InhibitorsF. Selective targeting of TNFR1 and/or TNFR2 axis 1. Selective blockade of TNFR1 by TNFR1 antagonists 2. Selective activation of TNFR2 by TNFR2 agonist 3. TNFR1 antagonist constructs, TNFR2 agonist constructs; multispecific, e.g. bispecific, TNFR1 antagonist and TNFR2 agonist constructs 4. Components of TNFR1 Antagonist Constructs, TNFR2 Agonist Constructs, and Multispecific, e.g., Bispecific, TNFR1 Antagonist/TNFR2 Agonist Constructs a. TNFR1 inhibitor moiety (TNFR1 antagonist)
b. TNFR2 agonist and TNFR2 antagonist constructs c. Linker i. Peptide linkers a) Flexible linkers b) Rigid linkers ii. Chemical linker d. Activity modifier i. Modifications of the Fc portion a) Knob-in-hole b) Modifications that enhance neonatal Fc receptor (FcRn) recycling c) Enhancement or reduction/absence of Fc immune effector function ii. Other modifications of the Fc portion iii. Human serum albumin e. Multispecific TNFR1 Antagonist/TNFR2 Agonist Constructs Multispecific Constructs, PEG-centered Multispecific Constructs, e.g., Bispecific, TNFR1 Antagonist/TNFR2 Agonist Constructs f. Additional activity modifiers - fusion proteins containing part or all of the polypeptide that increases serum half-life 5. Prediction and elimination of immunogenicity in protein therapeutics a. B cell and T cell epitopes b. In Silico Epitope Prediction Method 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 Method i. In vitro B cell epitope prediction method ii. In vitro T cell epitope prediction method MHC/HLA binding assay iii. In vitro T cell assay d. In vivo epitope prediction method e. Removal of predicted B and T cell epitopes (immunodepletion)
G. Pan-growth factor trap polypeptide 1. receptor tyrosine kinase (RTK)
a. Human Epidermal Growth Factor Receptor (HER) Family b. Diseases related to the human epidermal growth factor receptor (HER) family and its ligands 2. Pan-growth factor inhibition a. RB242 ligand trap b. RB200 and RB242 for the treatment of autoimmune diseases
c. RB242 ligand trap 3. Optimized multispecific, e.g. bispecific, growth factor trap constructs a. Extracellular domain (ECD) polypeptide b. Modifications to the extracellular domain c. Multimerization domain d. Modifications to the Fc domain i. Introduction of knob-in-hole ii. Neonatal Modifications that enhance Fc receptor (FcRn) recycling iii. Effector Function 4 Compositions, Therapeutic Uses and Treatment Methods a. Pharmaceutical composition b. Therapeutic Uses and Methods of Treatment 5. Combination therapyH. Evaluation of activity and efficacy of TNFR1 antagonist and TNFR1 antagonist/TNFR2 agonist constructs 1. Disease activity score (DAS28)
2. SOMAscan® proteomics analysis and other proteomics tools to quantify analytes 3. Transcriptome analysis to predict response to treatment and 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 TNFR1 antagonist/TNFR2 agonist constructs 9. Antibody Dependent Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) Assays 10. Disease model a. Collagen-induced arthritis (CIA)
b. Rheumatoid arthritis synovial mononuclear cell culture c. Tg197 mouse model of arthritis d. ΔARE Mouse Model of Arthritis/IBD e. Humanized TNF/TNFR2 mouse I. Methods of Producing Nucleic Acids Encoding TNFR1 Antagonist and TNFR1 Antagonist/TNFR2 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 encoded polypeptides 3. Vectors and cells 4. Expression a. Prokaryotic cells b. Yeast cells c. Insects and insect cells d. Mammalian expression cells e. Plant 5. Purification 6. Additional modifications a. PEGylation b. Albuminization c. Purification tag 7. Nucleic acid molecules and gene therapy J. Compositions, formulations and dosages 1. Preparation 2. Administration of TFNR1 antagonist constructs, TNFR2 agonist constructs, multispecific, eg, bispecific, 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 immunosuppressive diseases, disorders, and conditions 4. Combination therapy L. Example

A. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials referenced throughout this disclosure, unless otherwise noted, are incorporated herein by reference. It is incorporated in its entirety. If there is more than one definition for a term in this document, the definition in this section takes precedence. When referring to a URL or other such identifier or address, such identifiers may change and certain information on the Internet may come and go, but equivalent information may not be found by searching the Internet. It is understood that this is possible. Reference thereto evidences the availability and dissemination of such information to the public.

As used herein, a construct is a product that includes one or more components, generally at least two components. This component can be a polypeptide, small molecule, aptamer, nucleic acid, and/or other such components described herein or known to those skilled in the art. Various constructs are described and illustrated herein. Its components and its diversity will be apparent from the description herein. Those skilled in the art, in view of the description, may envision other constructs within the scope of the disclosure and claims herein. The term construct is used because the product may include various types of components.

As used herein, a construct that is a TNFR1 construct or a TNFR2 antagonist construct is a construct that includes a TNFR1 inhibitor moiety, a moiety that inhibits or reduces TNFR1 activity, such as signal transduction.

As used herein, a construct that is a TNFR2 construct or a TNFR2 agonist construct is a construct that includes a TNFR2 agonist moiety, which activates an activity of TNFR2, e.g., an activity that results in signal transduction or expansion of Treg cells. Or the guiding part.

As used herein, a construct that is a TNFR2 antagonist construct is a construct that includes a TNFR2 antagonist.

As used herein, a construct that is a multispecific construct is a construct that includes two or more antagonists or agonists or portions of both, e.g., a construct that includes a TNFR1 inhibitor and a TNFR2 agonist, or two TNRF1 antagonists. for example, when each interacts with a different epitope on TNFR1, or when each has different TNFR1 antagonist activity, or when two TNFR2 agonists (when each interacts with a different TNFR2 epitope, or when each such as those with different TNFR2 agonist activities.

As used herein, "tumor necrosis factor", "tumor necrosis factor alpha", "TNF", "TNF-alpha", "TNF-α" and "TNFα" are used interchangeably, and TNF refers to a pleiotropic proinflammatory cytokine that is a member of the superfamily and is associated with inflammatory and immunomodulatory activities, including regulation of tumorigenesis/cancer, host defense against pathogenic infections, apoptosis, autoimmunity, and septic shock . If other members of the TNF superfamily are intended, they will be identified by name. TNF is involved in the coordination of innate and adaptive immune responses and especially in the organogenesis of lymphoid organs. TNF is produced as a homotrimeric membrane-bound protein containing 233 amino acids that can be cleaved by the protease TACE (also known as TNF alpha converting enzyme, ADAM17) to release soluble TNF (solTNF) containing 157 amino acids; Membrane-bound and soluble forms of TNF are biologically active. The homotrimer of TNF binds to and transmits signals through two high-affinity specific receptors, TNFR1 and TNFR2; membrane-bound TNF primarily activates TNFR2, and soluble TNF primarily activates TNFR2. Activates TNFR1. Uncontrolled or dysregulated production of TNF is associated with, for example, septic shock, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and inflammatory bowel disease (IBD), as well as neurological disorders. Some chronic inflammatory and autoimmune diseases and conditions, including but not limited to degenerative and demyelinating diseases and conditions, such as, but not limited to, Alzheimer's disease, Parkinson's disease , associated with stroke and multiple sclerosis.

As used herein, a "TNF mutein" or "TNF-α mutein" or "modified TNF polypeptide" refers to a TNF from a particular species that differs in amino acid sequence from the corresponding wild-type TNF (TNFα). Refers to a polypeptide having an amino acid sequence that differs in one or more amino acids. Generally, such modified TNF polypeptides retain the ability to activate or inhibit TNFR1 and/or TNFR2. Certain mutations in TNF may render the resulting TNF mutein selective for binding to TNFR1 or TNFR2, resulting in a TNF mutein with antagonizing or agonistic properties. For example, as described herein, there are TNFR1 selective antagonizing TNF muteins, and TNFR2 selective agonist TNF muteins.

As used herein, a "dominant-negative inhibitor of TNF" or "DN-TNF" is a TNF that has one or more mutations that abrogate binding to and signaling through TNFR1 and/or TNFR2. It is a mutein. DN-TNF generates soluble TNF (sTNF or solTNF) by rapidly exchanging subunits with native TNF homotrimers to form inactive mixed TNF heterotrimers with disrupted receptor-binding surfaces. ), thereby preventing its interaction with TNF receptors. DN-TNF does not affect transmembrane TNF (tmTNF), maintaining the protective role of TNF signaling through TNFR2. An example of DN-TNF is SEQ ID NO: 1 (with reference to the sequence of solTNF shown in SEQ ID NO: 2, corresponding to residues L57Y, S86Q, Y87H, I97T, Y115Q, and A145R), which impairs binding to TNFR. , a TNF mutant containing one or more of the following substitutions: L133Y, S162Q, Y163H, I173T, Y191Q and A221R.

As used herein, "modification" refers to an alteration of the amino acid sequence in a polypeptide or the nucleotide sequence in a nucleic acid molecule, including amino acid or nucleotide deletions, insertions, rearrangements, substitutions, and, respectively, including combinations thereof. Methods of modifying polypeptides or nucleic acids are routine to those skilled in the art, such as using recombinant DNA methodologies.

As used herein, "deletion," when referring to a nucleic acid or polypeptide sequence, refers to one or more nucleotides compared to a target polynucleotide or polypeptide, or a sequence such as a native or wild-type sequence. or refers to the deletion of amino acids.

"Insertion," as used herein, when referring to a nucleic acid or amino acid sequence, refers to 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 the wild-type sequence contains one or more additional nucleotides within the linear length of the sequence.

"Addition," as used herein, when referring to a nucleic acid or amino acid sequence, refers to the addition of one or more nucleotides or amino acids to either terminus as compared to another sequence.

As used herein, "substitution" or "replacement" refers to a native, target, wild-type or Refers to the substitution of one or more nucleotides or amino acids in another nucleic acid or polypeptide sequence with another nucleotide or amino acid. Thus, one or more substitutions within a molecule do not change the number of amino acid residues or nucleotides in the molecule. Amino acid substitutions relative to a particular polypeptide may be expressed in terms of the number of amino acid residues along the length of the polypeptide sequence. For example, a modified polypeptide having a modification in which the amino acid at position 100 of the amino acid sequence is substituted/replaced from tyrosine (Tyr; Y) to glutamic acid (Glu; E) can be expressed as Y100E, Tyr100Glu, or 100E. Y100 can be used to indicate that the modified 100th amino acid is tyrosine. For purposes of this specification, modifications are also in the heavy chain (HC) or light chain (LC) of the antibody, so that modifications are also referred to by reference to HC- or LC- to indicate the chain of the polypeptide. can be shown.

As used herein, "at the corresponding position" or the statement that a nucleotide or amino acid position "corresponds" to a nucleotide or amino acid position in a disclosed sequence as shown in a sequence listing refers to , refers to nucleotide or amino acid positions that are identified in alignment with a reference sequence to maximize identity using standard alignment algorithms such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues using, for example, conserved identical amino acid residues as a guide. Generally, to identify corresponding positions, sequences of amino acids are aligned to obtain the highest-order match (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, H.G., 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 ) SIAM J. Applied Math 48:1073).

As used herein, sequence alignment 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. A set of aligned sequences refers to two or more sequences aligned at corresponding positions and may include aligned sequences from RNA, such as ESTs and other cDNAs, aligned with genomic DNA sequences. Related or variant polypeptides or nucleic acid molecules may be aligned by any method known to those skilled in the art. Such methods typically maximize matching and include methods using manual alignment, and methods using the numerous alignment programs available (e.g., BLASTP) and other methods known to those skilled in the art. . By aligning polypeptide or nucleic acid sequences, one skilled in the art can identify similar moieties or positions using identical conserved amino acid residues as a guide. Additionally, one skilled in the art can also use conserved amino acid or nucleotide residues as a guide to find corresponding amino acid or nucleotide residues between and within human and non-human sequences. Corresponding positions may also be based on structural alignments, eg, by using computer-simulated alignments of protein structures. In other examples, corresponding regions may be identified. One of ordinary skill in the art can also use conserved amino acid residues as a guide to find corresponding amino acid residues between and within human and non-human sequences.

As used herein, the statement that proteins are "compared under like conditions" means that any one or more conditions that may affect the activity or properties of the protein or drug are Unchanged or substantially unchanged means that different proteins are treated the same or substantially the same way. For example, when comparing the activity of an antibody to another antibody, any one or more conditions such as the amount or concentration of the polypeptide; excipients, carriers, or other components other than the active agent in the formulation (e.g. temperature; pH; storage time; storage container; storage characteristics (such as agitation); and/or other conditions associated with exposure or use. are the same or substantially the same between and between.

As used herein, "adverse effect" or "side effect" or "adverse event" or "adverse side effect" refers to harmful, deleterious and/or undesirable effects associated with the administration of a therapeutic agent. It refers to an action that does not exist. For example, side effects associated with the administration of anti-TNF antibodies such as adalimumab (eg, sold under the trademark Humira®) are known to those skilled in the art, and some are described herein. Such harmful side effects include, for example, serious infections such as tuberculosis and other infections caused by viruses, fungi, and bacteria, including upper respiratory tract infections, and rashes, headaches, and nausea. Skin and skin toxicity include. Accordingly, "adverse effect" or "side effect," refers to a harmful, deleterious, and/or undesirable effect of administration of a therapeutic agent. Side effects or adverse effects are graded by toxicity, and various toxicity scales exist and definitions for each grade are provided. Examples of such scales are the National Cancer Institute Common Toxicity Criteria version 2.0 toxicity scale and the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Assigning a grade of severity is within the skill of an experienced physician or other health care professional. Symptom severity may be quantified using the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system. CTCAE is a descriptive term used for reporting adverse events (AEs). A grade (severity) scale is provided for each AE term. CTCAE displays grades 1-5 and clinically describes the severity of each adverse event based on the following general guidelines: Grade 1 (mild AE). Grade 2 (moderate AE); Grade 3 (severe AE); Grade 4 (life-threatening or disabling AE); and Grade 5 (AE-related death/fatal).

As used herein, "properties" of polypeptides, such as antibodies, include binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermotolerance, and Refers to any property exhibited by a polypeptide, including, but not limited to, resistance to pH conditions. Changes in properties can alter the "activity" of a polypeptide. For example, changes in the binding specificity of an antibody polypeptide can alter various binding activities, such as the ability to bind antigen and/or the polypeptide's affinity or avidity, or in vivo activity.

As used herein, "activity" or "functional activity" of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. Such activity can be determined empirically. Exemplary activities include, but are not limited to, the ability to interact with biomolecules through antigen binding, DNA binding, ligand binding, or dimerization; and enzymatic activities, such as kinase activity or proteolytic activity. can be mentioned. For antibodies (including antibody fragments), activity includes, but is not limited to, the ability to specifically bind a particular antigen, the affinity of antigen binding (e.g., high or low affinity), avidity of binding (e.g., high or low affinity), on-rate, off-rate, effector function, e.g., antigen neutralization or clearance, virus neutralization, and in vivo activity, e.g., pathogen infection or invasion. or the ability to promote clearance, or to penetrate particular tissues or fluids or cells within the body. Activity can be determined by ELISA, flow cytometry, surface plasmon resonance, or equivalent assays that measure on-rate or off-rate, immunohistochemistry and immunofluorescence histology and microscopy, cell-based It may be evaluated in vitro or in vivo using recognized assays such as assays, flow cytometry and binding assays (eg, panning assays). For example, for antibody polypeptides, activity can be determined by measuring binding affinity, avidity, and/or binding coefficient (e.g., for on-rate/off-rate), and other activities in vitro or in vivo to determine various effects. , measuring, for example, immune effects such as antigen clearance; penetration or localization of antibodies into tissues; protection from diseases such as infections; serum or other body fluid antibody titers; or other assays well known in the art. It can be evaluated by The results of such assays indicating that the polypeptide exhibits activity may be correlated with the activity of the polypeptide in vivo, which in vivo activity may be referred to as therapeutic activity or biological activity. The activity of the modified polypeptide is 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60% of the activity compared to the unmodified polypeptide. %, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, It can be any level of percentage of the activity of the unmodified polypeptide, including, but not limited to, 500% or more activity. Assays for determining the functionality or activity of modified (or variant) antibodies are well known in the art.

As used herein, "bind," "bound," and their grammatical variations involve a molecule engaging in any attractive interaction with another molecule, bringing the two molecules together with each other. It refers to bringing about stable meetings in close proximity. Binding interactions include, but are not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and include, but are not limited to, binding proteins, nucleic acids, carbohydrates, lipids, and drugs. It involves interactions between molecules, including small molecules such as compounds that contain them. Exemplary bindings are antibody-antigen interactions and receptor-ligand interactions. When an antibody "binds" to a particular antigen, "binding" refers to the specific recognition of the antigen by the antibody through cognate antibody-antigen interactions at the antibody combining site. Binding can also include the association of multiple chains of a polypeptide, such as antibody chains, that interact through disulfide bonds.

As used herein, "binding activity" refers to characteristics of a molecule, eg, a polypeptide, that relate to whether and how it binds one or more binding partners. Avidity can include the ability to bind to a binding partner, the affinity (e.g., high affinity) to bind to a binding partner, the avidity to bind to a binding partner, the strength of binding to a binding partner, and/or the ability to bind to a binding partner. specificity of binding.

As used herein, "affinity" or "binding affinity" refers to the strength of interaction between two or more molecules, such as binding partners, and typically between two binding partners. Represents the strength of noncovalent interactions. The affinity of an antibody or antigen-binding fragment thereof for an antigenic epitope is a measure of the strength of all non-covalent interactions between a single antibody combining site and the epitope. Low affinity antibody-antigen interactions are weak and the molecules tend to dissociate quickly, whereas high affinity antibody-antigen binding is strong and the molecules remain bound for long periods of time. Binding affinities are determined by the rate of association (ka or kon) and/or dissociation (kd or koff), half-maximal effective concentration (EC50) values, and/or thermodynamic data (e.g., Gibbs free energy (ΔG), enthalpy (ΔH), entropy (-TΔS), and/or calculation of association (Ka) or dissociation (Kd) constants. ng/mL), where 50% of maximal binding is observed for a fixed amount of antigen. Typically, EC50 values are determined from sigmoidal dose-response curves, with the EC50 being 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 are calculated using standard kinetic methodologies of antibody reactions, e.g. It can be determined by immunoassay such as ELISA followed by curve fitting analysis.

As used herein, "affinity constant" refers to the association constant (Ka) used to measure the affinity of an antibody for an antigen. The higher the affinity constant, the higher the antibody's affinity for the antigen. Affinity constants are expressed in inverse molar units (i.e., M-1) and are determined using standard kinetic methodologies of antibody reaction (e.g., immunoassay, surface plasmon resonance, or other kinetic methods known in the art). (interaction assay), it can be calculated from the rate constant of the association-dissociation reaction. The binding affinity of an antibody may be expressed as a dissociation constant, or Kd. The dissociation constant is the reciprocal of the association constant, ie Kd=1/Ka. Therefore, the affinity constant may also be expressed as Kd. Affinity constants can be determined by standard kinetic methodologies of antibody reactions, e.g. immunoassay, surface plasmon resonance (SPR) (e.g. Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) )Analyst.123:1599), isothermal titration calorimetry (ITC) or other dynamic interaction assays known in the art (e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989)); see also US Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (e.g., BIAcore 2000, BIAcore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Tr. ans. 27:335).

Methods of calculating affinity are well known, such as methods of determining EC50 values or methods of determining association/dissociation constants. For example, in terms of EC50, high binding affinity means that the antibody is approximately 10ng/mL, 9ng/mL, 8ng/mL, 7ng/mL, 6ng/mL, 5ng/mL, 3ng/mL, 2ng/mL, 1ng/mL. means specifically binding to a target protein with an EC50 of less than or equal to EC50. High binding affinity may also be characterized by an equilibrium association constant (Kd) of 10 M or less, such as 10 M, 10 M, 10 M, 10 M, 10 M, or 10 M or less. . In terms of equilibrium association constant (Ka), high binding affinity is generally greater than or equal to about 10M, greater than or equal to about 10M, greater than or equal to about 10M, or about 10M, 10M, 10M, or 10M. It is associated with a Ka value of -1 or higher. Affinity can be estimated empirically, or affinity can be calculated, e.g. by comparing the affinities of two or more antibodies for a particular antigen, e.g. by calculating the affinity contrast of the antibodies tested. By doing so, you can compare and decide. For example, such affinity can be determined by using conventional techniques such as ELISA; equilibrium dialysis; surface plasmon resonance; by radioimmunoassay using radiolabeled target antigen; or other methods known to those skilled in the art. can be easily determined by Affinity data can be found, for example, in Scatchard et al. (1949) Ann N. Y. Acad. Sci. , 51:660 or by curve fitting analysis using a four-parameter logistic nonlinear regression model using the following equation: y=((AD)/(1+((x/ C)^B)))+D, where A is the minimum asymptote, B is the slope coefficient, C is the inflection point (EC50), and D is the maximum asymptote.

As used herein, "antibody avidity" refers to the multiple binding sites between a multivalent antibody and its cognate antigen, such as an antibody that contains multiple binding sites associated with an antigen with repetitive epitopes or epitope arrays. Refers to the strength of interaction. High avidity antibodies have a higher strength of such interactions compared to low avidity antibodies.

As used herein, "specificity for a target" such as TNFR1 refers to a preference for binding to the target, higher binding affinity, compared to non-targets. Selective binding generally refers to binding to a target with an affinity of at least about 107-108 M-1. It also means that if the affinity of a moiety or molecule for one target molecule is compared to its affinity for another and the difference is of a certain magnitude, such as about a 10-fold, then the moiety or molecule is It is said to have high specificity for the first target.

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 an antibody or antigen-binding fragment thereof. refers to the ability of an antibody to form one or more non-covalent bonds with its cognate antigen through non-covalent interactions between the antibody combining site and the antigen. Typically, an antibody that immunospecifically binds (or specifically binds) to, for example, TNFR1 is an antibody having a dissociation constant (Kd) of about or greater than or equal to 1×10 M−1 or 1×10 M−1 (or with a dissociation constant (Kd) of 1× The antibody binds to TNFR1 with an affinity constant (Kd) of 10-7M or 1×10-8M or less. Antibodies or antigen-binding fragments that immunospecifically bind to a particular antigen can be prepared using immunoassays such as, for example, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance (SPR), or other methods known to those skilled in the art. Can be identified by other techniques.

As used herein, "steric effect" refers to the effect of the size of an atom or group on a molecule. Steric effects include, but are not limited to, steric hindrance and van der Waals repulsion. Steric effects are effects that result from the fact that atoms occupy space. When atoms are brought close together, energy is wasted as the electrons near the atoms repel each other.

As used herein, "exhibits at least one activity" or "retains at least one activity" means that a mutant antibody or other refers to the activity exhibited by an antibody polypeptide, such as a therapeutic polypeptide. A modified or variant polypeptide that retains the activity of the target polypeptide may exhibit improved activity, decreased activity, or maintain the activity of the unmodified polypeptide. In some cases, the modified or variant polypeptide may retain increased activity compared to the target or unmodified polypeptide. In some cases, a modified or variant polypeptide may retain reduced activity compared to an unmodified or target polypeptide. The activity of a modified, or variant, polypeptide can be a percentage of any level of activity of the unmodified or target polypeptide, including, but not limited to, the activity of the unmodified or target polypeptide. In comparison, 1% of activity, 2% of activity, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, Includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more activity. In other embodiments, the change in activity is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold over the unmodified or target polypeptide. , 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x More than once big. Assays for retention of activity depend on the activity retained. Such assays can be performed in vitro or in vivo. Activity may be measured using assays known in the art and described below for activity, such as, but not limited to, ELISA and panning assays. The activity of a modified or variant polypeptide compared to an unmodified or target polypeptide can also be evaluated with respect to in vivo therapeutic or biological activity or outcome following administration of the polypeptide.

As used herein, "surface plasmon resonance" refers to an optical phenomenon that allows analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix. Commercial systems are available. For example, the BIAcore system (GE Healthcare Life Sciences) is a typical commercial system.

As used herein, an "antibody" means a variable heavy chain of an immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, to specifically bind to an antigen. immunoglobulins and immunoglobulins, whether naturally occurring or produced synthetically, such as partially or completely recombinantly, and/or any fragment thereof comprising at least a portion of a variable light chain region. Points to a fragment. Thus, an antibody includes any protein that has 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 includes two heavy chains (which can be designated as H and H') and two light chains (which can be designated as L and L'), where each heavy chain has a full-length It may be a globulin heavy chain or a sufficient portion thereof to form an antigen binding site (for example, heavy chains include, but are not limited to, VH chain, VH-CH1 chain, and VH-CH1- (including, but not limited to, CH2-CH3 chains), and each light chain may be a full-length light chain or a sufficient portion thereof to form an antigen binding site (e.g., a light chain may include , including, but not limited to, VL chains and VL-CL chains). Each heavy chain (H and H') is paired with one light chain (L and L', respectively). Typically, an antibody minimally comprises all or at least a portion of a variable heavy (VH) chain and/or a variable light (VL) chain. Antibodies may also contain other regions, such as, for example, all or part of a constant region and/or all or part of a hinge region (sufficient to provide plasticity).

For purposes of this specification, the term "antibody", unless otherwise specified, includes full-length antibodies and portions thereof, including, for example, antibody fragments, such as anti-TNFR1 antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide bonded Fv (dsFv), Fd fragments, Fd' fragments, single chain Fv ( scFv), single chain Fabs (scFab), hsFv (helix-stabilized Fv), single domain antibodies (dAbs, or sdAbs), minibodies, diabodies, anti-idiotypic (anti-Id) antibodies, nanobodies, and camelids. antibodies, free light chains, VHH antibodies (or Nanobodies), or antigen-binding fragments of any of the above. Antibody fragments also include, for example, tandem scFv, Fab-scFv (HC C-terminus or LC C-terminus), Fab-(scFv)2 (C-terminus), scFv-Fab-scFv, Fab-CH2-scFv, scFv fusions (C combinations of any of the above fragments, such as Fab fusions (HC C-terminus, or LC C-terminus), scFv-scFv-dAb, scFv-dAb-scFv, dAb-scFv-scFv, and tribodies. Can be mentioned. The term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific and heteroconjugate antibodies (e.g., bispecific, trispecific and tetraspecific 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., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subsubclasses (eg, IgG2a and IgG2b).

As used herein, "antibody form" refers to the specific structure of an antibody. Antibodies herein include full-length antibodies and portions thereof, such as, for example, Fab fragments or other antibody fragments. Fabs are therefore a specific form of antibody.

As used herein, reference to a "corresponding form" of an antibody means that when comparing the properties or activities of two antibodies, the properties are compared using the same form of the antibody. . For example, if an antibody is stated to have reduced activity compared to the activity of the corresponding form of a first antibody, it means that a particular form of that antibody, such as a Fab, has less activity compared to the Fab form of the first antibody. It means low activity.

As used herein, a full-length antibody refers to 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 antibodies with a hinge region, such as human antibodies produced by antibody-secreting B cells, and antibodies with the same domain produced synthetically.

As used herein, "multispecific construct" refers to a construct that includes an antibody or a portion of an antibody that exhibits affinity for multiple target antigens such that it can specifically interact with the target. Refers to the construct. The multispecific constructs herein may have a structure similar to a complete immunoglobulin molecule, including an Fc region, e.g. an IgG Fc region, and an antigen binding region, such as a portion that specifically binds TNFR1 or TNFR2. May include.

As used herein, "bispecific construct" refers to a multispecific construct that has binding specificities for two different antigens. Bispecific constructs include, for example, monoclonal antibodies or antigen-binding fragments thereof linked to a polypeptide region such as an Fc or modified Fc that alters the activity of the construct. For human therapy, the construct is derived from a human source, or is derived from a human source or humanized, and the construct has binding specificity for at least two different antigens. The bispecific constructs/molecules provided herein may have binding specificity for TNFR1 and TNFR2. For example, a bispecific construct includes a TNFR1 antagonist and a TNFR2 agonist. Bispecific antibodies or constructs include antibodies and antigen-binding fragments thereof, which contain two separate antigen-binding domains (e.g., two scFvs joined by a linker, or two dAbs, or two Fab). Antigen binding domains may bind the same antigen or different antigens.

As used herein, an "antibody fragment" or "antibody portion" is one that is shorter than the full length but sufficient to form an antigen-binding site (e.g., one or more complementarity determining regions (CDRs)). Refers to any portion of a full-length antibody that contains at least a portion of a sufficient antibody variable region and thus retains the binding specificity and/or activity of the full-length antibody; antibody fragments include those produced by enzymatic treatment of the full-length antibody. antibody derivatives produced synthetically, eg, recombinantly. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab)2, single chain Fv (scFv), Fv, dsFv, diabody, triabody, affibody, nanobody, aptamer, dAb. , Fd and Fd fragments (see, eg, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; pp. 3-25, Kipriyanov). Fragments may include multiple chains linked together such as by disulfide bridges and/or peptide linkers. Antibody fragments generally contain at least about 50 amino acids, such as about or at least 100 amino acids, 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 (VH) and one variable light (VL) domain linked by non-covalent interactions.

As used herein, dsFv (disulfide bonded Fv) refers to an Fv with engineered intermolecular disulfide bonds that stabilize the VH-VL pair.

As used herein, "scFv fragment" refers to an antibody fragment that includes a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker. The linker is of such length that the two variable domains are cross-linked without substantial interference. An exemplary linker is a (Gly-Ser)n residue, with some Glu or Lys residues dispersed throughout to increase solubility.

A "diabody" as used herein is a dimeric scFv. Diabodies usually have shorter peptide linkers than scFvs and preferentially dimerize.

As used herein, a "triabody" is a trimeric scFv. They contain three peptide chains, each one VH domain and one VL domain, joined by a short linker (e.g., a linker consisting of 1-2 amino acids) to form a VH domain within the same peptide chain. Contains a domain that allows intramolecular association of the domain and the VL domain. Triabodies normally trimerize.

As used herein, a "Fab fragment" is an antibody fragment resulting from digestion of full-length immunoglobulin with papain, or a fragment having the same structure produced synthetically, eg, by recombinant methods. Fab fragments include a light chain (comprising VL and CL) and another chain that includes the variable domain of the heavy chain (VH) and one constant region domain of the heavy chain (CH1).

As used herein, "F(ab')2 fragment" refers to antibody fragments resulting from digestion of immunoglobulins with pepsin at pH 4.0-4.5, or synthetically, e.g., by recombinant methods. Fragments with the same structure are generated. The F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain portion providing flexibility, e.g., in the hinge region containing cysteine residues, forming a disulfide bond connecting the two fragments. an additional 2-3 amino acids, all or in part, sufficient to

As used herein, a Fab' fragment is a fragment that includes half of an F(ab')2 fragment (ie, one heavy chain and one light chain).

As used herein, an Fd fragment is a fragment of an antibody that includes the variable domain (VH) and one constant region domain (CH1) of the antibody heavy chain.

As used herein, an Fd' fragment is a fragment of an antibody that contains the heavy chain portion of one of the F(ab')2 fragments.

As used herein, an Fv' fragment is a fragment that contains only the VH and VL domains of an antibody molecule.

As used herein, hsFv (helix-stabilized Fv) refers to an antibody fragment in which the constant domain normally present in Fab fragments is replaced with a heterodimeric coiled-coil domain (e.g., Arndt et al. 2001) J. Mol. Biol. 7:312:221-228).

As used herein, "domain antibody," "single domain antibody," "sdAb," or "dAb" are used interchangeably and refer to an antibody heavy chain (VH) or an antibody light chain (VL). refers to a small monomeric antibody fragment containing the variable domain of dAbs are the smallest antigen-binding fragments of antibodies; they are approximately 11-15 kDa (approximately 100-150 amino acids) in size, approximately one-tenth the size of a complete monoclonal antibody (mAb). There are three complementarity determining regions (CDRs) in each VH and each VL. Each dAb contains three of the six CDRs from the antibody's VH-VL pair, highly diversified loop regions that bind to the target antigen.

As used herein, camelid antibodies, also referred to as nanobodies or VHHs, lack light chains and are composed of two identical heavy chains. They occur naturally in camelids such as camels and alpacas.

As used herein, a polypeptide "domain" refers to a structurally and/or functionally distinct or definable polypeptide (a sequence of 3 or more, generally 5, 10, or more amino acids) ). Exemplary polypeptide domains form independently folded structures within a polypeptide that are composed of one or more structural motifs (e.g., a combination of alpha helices and/or beta strands connected by loop regions). and/or is recognized by a specific functional activity such as enzymatic activity, dimerization, or antigen binding. A polypeptide may have one or more, typically two or more, distinct domains. For example, a polypeptide can have one or more structural domains and one or more functional domains. Single polypeptide domains can be distinguished on the basis of structure and function. A domain can include a linear sequence of contiguous amino acids. Alternatively, a domain can include multiple non-contiguous amino acid portions that are non-contiguous along the linear sequence of amino acids of the polypeptide. Polypeptides typically contain multiple domains. For example, each heavy chain and each light chain of an antibody molecule includes multiple immunoglobulin (Ig) domains, each approximately 110 amino acids long. Those skilled in the art are familiar with polypeptide domains and can identify them by structural and/or functional homology with other such domains. Although definitions are provided for purposes of illustration herein, it is understood that it is well within the skill of those skilled in the art to recognize particular domains by name. If desired, domains may be identified using appropriate software.

As used herein, a "functional region" of a polypeptide includes at least one functional domain (e.g., through antigen binding, DNA binding, ligand binding, or dimerization, or through enzymatic activity, e.g., kinase activity). or proteolytic activity that confers a specific function, such as the ability to interact with biomolecules; exemplary functional regions of a polypeptide include VH, VL, CH, CL, etc. antibody domains, 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 includes at least one structural domain.

As used herein, an "Ig domain" is a domain recognized as such by those skilled in the art that is distinguished by a structure called the immunoglobulin (Ig) fold, which consists of two beta-pleated folds. It contains sheets, each containing antiparallel sequence beta strands of amino acids connected by loops. The two beta sheets within the Ig fold are sandwiched by hydrophobic interactions and conserved intrachain disulfide bonds. Individual immunoglobulin domains within an antibody chain can be further distinguished on the basis of function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), whereas a heavy chain contains one variable region domain (VH) and three or four constant region domains. Contains regional domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.

As used herein, "variable domain", with respect to antibodies, is a specific immunoglobulin (Ig) domain of an antibody heavy or light chain that contains amino acid sequences that differ between different antibodies. Each light chain and each heavy chain has one variable region domain (VL and VH, respectively). The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region includes 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" refer to each variable region within each variable region that together forms the antigen-binding site of an antibody. used interchangeably to refer to one of multiple parts of. Each variable region domain contains three CDRs named CDR1, CDR2, and CDR3. The three CDRs are discontinuous along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within the loops that connect the parallel strands of the beta sheets of the variable domain.

As used herein, "antigen-binding domain," "antigen-binding site," "antigen-binding fragment," "antigen-binding site," and "antibody-combining site" are used synonymously and refer to domains within an antibody. A domain that recognizes and physically interacts with a cognate antigen. A natural conventional full-length antibody molecule has two conventional antigen-binding sites, each comprising a portion of the heavy chain variable region and a portion of the light chain variable region. Traditional antigen binding sites include loops connecting antiparallel beta chains within variable region domains. The antigen binding site may include other portions of the variable region domain. Each conventional antigen binding site includes three hypervariable regions of the heavy chain and three hypervariable regions of the light chain. Hypervariable regions are also called complementarity determining regions (CDRs).

As used herein, "a portion thereof", with respect to an antibody heavy or light chain, or a variable heavy or light chain, includes the heavy and light chains and includes all six CDRs when assembled into an antibody. at least one or two, typically three, four, variable heavy chains (VH) and variable light chains (VL) sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody comprising: , refers to a contiguous portion thereof sufficient to include all five or six CDRs to form an antigen binding site. Generally, a sufficient antigen binding site requires heavy chain CDR3 (CDRH3). Usually, an additional light chain CDR3 (CDRL3) is required. As described herein, those skilled in the art will know and be able to identify CDRs based on Kabat or Chothia numbering (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 al. (1987) J. Mol. Biol. 196:901-917).

A "framework region" or "FR" as used herein is a domain within an antibody variable region domain that is located within the beta sheet. FR regions are relatively more conserved than hypervariable regions with respect to their amino acid sequences. Each variable region contains four framework regions that separate three hypervariable regions.

As used herein, a "constant region" domain is a domain of an antibody heavy or light chain that contains amino acid sequences that are more conserved among antibodies than the variable region domains. 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, including CH1, CH2, CH3, and CH4. Full-length IgA, IgD, and IgG isotypes include CH1, CH2, and CH3 domains and a hinge region, whereas IgE and IgM include CH1, CH2, CH3, and CH4 domains. The CH1 and CL domains extend the Fab arm of the antibody molecule and contribute to interaction with antigen and rotation of the antibody arm. Antibody constant regions can provide effector functions such as, but not limited to, clearance of antigens, pathogens, and toxins to which the antibody specifically binds, for example, through interaction with various cells, biomolecules, and tissues.

As used herein, "antibody hinge region" or "hinge region" refers to the polypeptide region in the heavy chain of gamma, delta, and alpha antibody isotypes that lies between the CH1 and CH2 domains. and binds the Fab region and Fc region. There is no homology with other antibody domains. This region is rich in proline residues and provides plasticity to IgG, IgD, and IgA antibodies, allowing the two "arms" of the Fab portion (each containing one antibody binding site) to be mobile and relative to each other. whereas they assume different angles as they bind to the antigen. This plasticity allows the Fab arms to move to align the antibody binding site to interact with epitopes on the cell surface or other antigens. Two interchain disulfide bonds within the hinge region stabilize the interaction between the two heavy chains. In some embodiments provided herein, synthetically produced antibody fragments include one or more hinges, e.g., to promote stability through interactions between two antibody chains. Contains areas. A hinge region is an example of a portion of a dimerization domain, and for purposes herein is part of a linker.

As used herein, "fragment crystallizable region" or "Fc" or "Fc region" or "Fc domain" includes the constant region of an antibody heavy chain and includes the first constant region immunoglobulin domain. Refers to polypeptides excluding . Fc consists of the last two constant region immunoglobulin domains of IgA, IgD, and IgG (CH2 and CH3, also called Cγ2 and Cγ3) or the last three constant region immunoglobulin domains of IgE and IgM (CH2, CH3 and CH4). Optionally, Fc domains may include all or part of a flexible hinge region at the N-terminus of these domains. For IgA and IgM, the Fc may include the J chain. For an exemplary Fc domain of IgG, the Fc comprises immunoglobulin domains CH2 and CH3, and optionally all or part of the hinge between CH1 and CH2 (also referred to as Cγ1 and Cγ2). The boundaries of the Fc region can vary, but typically include at least a portion of the hinge region. For purposes herein, Fc also refers to any allelic form, such as any variant or modified form of Fc that has altered binding to an Fc receptor (FcR) or that has altered Fc-mediated effector function. Including gene or species variants, or any mutant or modified forms. 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 are linked directly or indirectly to an Fc region or derivative thereof. Typically, an Fc chimera combines the Fc region of an immunoglobulin with another polypeptide. Derivatives of Fc polypeptides or modified Fc polypeptides are known to those skilled in the art.

As used herein, "Kabat numbering" refers to the index numbering of IgG1 Kabat antibodies (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); just as chymotrypsin numbering allows comparisons between proteases, it facilitates comparisons between antibodies. Those skilled in the art can identify regions of constant regions using Kabat numbering.

As used herein, "EU numbering" or "EU index" refers to the EU antibody numbering described in Edelman et al., (1969) Proc. Natl. Acad. Sci. USA 63:78-85. Refers to the scheme. "EU Index in Kabat" is the human IgG1 Kabat antibody described in Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Refers to the EU antibody numbering scheme. EU numbering, or EU numbering in Kabat, is frequently used by those skilled in the art to number the amino acid residues of the Fc region of antibody light and heavy chains. For example, one skilled in the art can identify regions of constant regions using EU numbering. For example, the CL domain of the Igκ light chain corresponds to residues R108-C214 according to Kabat and EU numbering (see, eg, 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 table below defines the numbering of IgG1 and IgG4 heavy chain constant domains and Ig kappa light chain constant domains by EU, Kabat, and sequential numbers. Table 1 shows the IgG1 heavy chain constant domains according to EU, Kabat and sequential numbering, where the sequential numbering is with respect to the sequence of amino acids shown in SEQ ID NO: 9, residues within the CH1, CH2 and CH3 domains. , as well as identifying the hinge region. Table 2 shows immunoglobulin (Ig) kappa light chain constant domains by EU, Kabat and sequential numbering, with the sequential numbering being with respect to the sequence of amino acids shown in SEQ ID NO:17. In Table 2, the top row (bold) shows the amino acid residue numbers by sequential numbering (see SEQ ID NO: 17); the second row (bold) shows the numbers of the top row. provides the one-letter code for the amino acid residue at the position indicated; the third line (italics) gives the corresponding Kabat number according to Kabat numbering; the fourth line gives the corresponding EU Indicates the index number. Table 3 shows the IgG4 heavy chain constant domains by EU, Kabat and sequential numbering, where the sequential numbering is with respect to the sequence of amino acids shown in SEQ ID NO: 15 and residues within the CH1, CH2 and CH3 domains. , as well as identifying the hinge region.

As used herein, the phrase "derived from" when referring to an antibody fragment derived from another antibody, such as a monoclonal antibody, refers to an antibody fragment that retains the binding specificity of the original antibody (e.g., a Fab , F(ab'), F(ab')2, single chain Fv (scFv), Fv, dsFv, dAb, diabody, Fd and Fd' fragments). Such fragments can be obtained by various methods known in the art, including, but not limited to, enzymatic cleavage, chemical cross-linking, recombinant means, or combinations thereof. Generally, derived antibody fragments share the same or substantially identical heavy chain variable regions (VH) and light chain variable regions (VL) of the parent antibody such that the antibody fragment and the parent antibody bind to the same epitope.

As used herein, "parent antibody" or "source antibody" refers to antibody fragments (e.g., Fab, F(ab'), F(ab)2, single chain Fv (scFv), Fv, dsFv, dAb, diabody, Fd and Fd' fragments) refer to antibodies from which they are derived.

The term "epitope" as used herein refers to any antigenic determinant on an antigen or protein to which the paratope of an antibody can bind. Epitopic determinants usually include chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics and specific charge characteristics.

As used herein, "humanized antibodies" and human therapeutics refer to antibodies and other antibodies that have been modified to contain "human" sequences of amino acids so that administration to humans does not elicit an immune response. refers to protein therapeutics. For example, humanized antibodies typically contain complementarity determining regions (CDRs or hypervariable loops) derived from immunoglobulins of non-human species, and the remainder of the antibody molecule derived primarily from human immunoglobulins. Methods for humanizing proteins, including antibodies, and producing them are well known and readily available to those skilled in the art. For example, DNA encoding a monoclonal antibody can be altered by recombinant DNA techniques so that the amino acid composition of the non-variable regions encodes an antibody based on a human antibody. Methods for identifying variable and non-variable regions of immunoglobulins are known, including computer programs designed to identify such regions. Thus, in general, a humanized antibody comprises 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 (FR) are of human immunoglobulin sequences. Humanized antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

As used herein, a "multimerization domain" is a sequence of amino acids that facilitates stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each of which is identical or a different multimerization domain, referring to a sequence containing a complementary multimerization domain that can form a stable multimer with the first domain. Generally, a polypeptide is linked directly or indirectly to a multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. Multimerization domains include, for example, immunoglobulin constant regions or domains, such as Fc domains or portions thereof derived from IgG, including IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD and IgM, and modifications thereof. It can be a form, etc.

As used herein, a "dimerization domain" is a multimerization domain that promotes interaction between two polypeptide sequences, such as, but not limited to, antibody chains. . Dimerization domains include, but are not limited to, all or a portion of a full-length antibody hinge region, or one or more dimerization domains that are sequences of amino acids known to promote interactions between polypeptides. Included are amino acid sequences containing cysteine residues that promote the formation of disulfide bonds between two polypeptide sequences, such as merization sequences (eg, leucine zipper, GCN4 zipper).

As used herein, "chimeric polypeptide" refers to a polypeptide that includes portions from at least two different polypeptides, or from two non-contiguous portions of a single polypeptide. Thus, a chimeric polypeptide generally comprises a sequence of amino acid residues derived from all or part of one polypeptide and an amino acid sequence derived from all or part of another, different polypeptide. The two moieties can be linked directly or indirectly, by a peptide bond, other covalent bond, or a substantial amount of the chimeric polypeptide under equilibrium and physiological conditions, e.g., isotonic pH 7 buffered saline. may be linked through other non-covalent interactions of sufficient strength to maintain the integrity of the moieties.

As used herein, a "fusion protein" is a polypeptide that has been engineered to contain sequences of amino acids that correspond to two different polypeptides that are in close proximity to each other along the length of a vector. For example, by expressing a fusion protein from a vector containing two adjacent nucleic acids encoding two polypeptides. Thus, fusion protein refers to a chimeric protein comprising two moieties derived from two or more proteins or peptides linked directly or indirectly via peptide bonds. The two molecules may be adjacent in the construct or separated by a linker or spacer polypeptide.

As used herein, "linker," "linker unit," or "link" is a chain of atoms that covalently joins an antibody or antigen-binding fragment thereof to another therapeutic moiety or another antibody or fragment thereof. refers to a peptide or chemical moiety that contains Linkers are included, for example, to increase plasticity, modify steric effects including steric hindrance, and increase solubility in aqueous media.

As used herein, "linker peptide" or "spacer peptide" refers to a short sequence of amino acids (or a nucleic acid encoding an amino acid sequence, etc.) that joins two polypeptide sequences. "Peptide linker" refers to a short sequence of amino acids that joins two polypeptide sequences. Examples of polypeptide linkers are linkers that join a peptide transduction domain to an antibody, or linkers that join two antibody chains in a synthetic antibody fragment, such as an scFv fragment. Linkers are well known, and any known linker may be used in the methods provided. An exemplary polypeptide linker includes a (Gly-Ser)n amino acid sequence with several 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 "epitope tag" is typically added to the N-terminus or C-terminus of a polypeptide, such as the antibodies and antibody fragments/constructs provided herein. Refers to the sequence of amino acids. Including a tag fused to a polypeptide may facilitate purification and/or detection of the polypeptide. Typically, a tag or tag polypeptide will have sufficient residues to provide an epitope recognized by an antibody, or to aid in detection or purification, but will not be limited to the number of residues of the polypeptide to which it is linked. Refers to polypeptides that are short enough not to interfere with activity. The tag polypeptide is typically sufficiently unique that antibodies that specifically bind to it do 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, usually about 8-50 amino acid residues, typically 9-30 amino acid residues. A tag may be linked to one or more chimeric polypeptides in a multimer, allowing detection of the multimer or its recovery from a sample or mixture. Such tags are well known and can be easily synthesized and designed. Exemplary tag polypeptides include tag polypeptides used for affinity purification, such as the FLAG tag; His tag; influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5 (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 (e.g. Evan et al. (1985) Molecular and Cellular Biology 5:3610-3616); and the herpes simplex virus glycoprotein D (gD) tag and antibodies thereof (see, eg, Paborsky et al. (1990) Protein Engineering 3:547-553). The antibodies used to detect epitope-tagged antibodies are typically referred to herein as secondary antibodies.

As used herein, a "label" or "detectable moiety" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof, such as an anti-TNFR1 antibody or antigen-binding fragment thereof provided herein). A detectable marker (e.g., a fluorescent molecule, chemiluminescent molecule, bioluminescent molecule, contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product). This detection method may include known in vivo and/or in vitro detection methods (e.g. visual inspection imaging, magnetic resonance (MR) spectroscopy, ultrasound signals, X-rays, gamma ray spectroscopy (e.g. positron emission tomography)). It can be any method known in the art, including PET) scanning, single photon emission tomography (SPECT), fluorescence spectroscopy, or absorption). Indirect detection refers to the measurement of a physical phenomenon, such as the emission or absorption of energy or particles, of an atom, molecule, or composition that binds directly or indirectly to a detectable moiety (e.g., a labeled secondary antibody or a primary refers to the detection of an antigen-binding fragment thereof that binds to an antibody (eg, an anti-TNFR antibody or antigen-binding fragment thereof provided herein).

As used herein, "nucleic acid" refers to at least two linked nucleotides or nucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. Refers to derivatives. The term "nucleic acid" also includes analogs of nucleic acids such as peptide nucleic acids (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids also include, for example, nucleotide analogues or "backbone" linkages other than phosphodiester linkages, such as phosphotriester linkages, phosphoramidate linkages, phosphorothioate linkages, thioester linkages, or peptide linkages (i.e., peptide nucleic acids). DNA and RNA derivatives containing. The term also includes, as equivalents, nucleotide analogs, derivatives, variants and analogs of either RNA or DNA made from single-stranded (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. In the case of RNA, the uracil base is uridine.

As used herein, an "isolated nucleic acid molecule" is a nucleic acid molecule that is separated from other nucleic acid molecules present in the nucleic acid molecule's natural source. An "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or culture medium, if produced by recombinant techniques, or free of chemical precursors, if chemically synthesized. Or it may be substantially free of other chemicals. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

"Operably linked," as used herein with reference to nucleic acid sequences, regions, elements, or domains, means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide may be operably linked to a nucleic acid encoding a polypeptide such that the nucleic acid can be transcribed and translated to express a functional fusion protein; results in secretion of polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to a nucleic acid encoding a second polypeptide, and the nucleic acid is transcribed as a single mRNA transcript. However, one of the two polypeptides can be expressed by translation of the mRNA transcript. For example, an amber stop codon can be introduced into a partial amber suppressor cell, and the resulting single mRNA transcript can be translated to yield a fusion protein comprising the first and second polypeptides, or It may be located between a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide such that it can be translated to produce only the first polypeptide. In another example, a promoter may be operably linked to a nucleic acid encoding a polypeptide, such that the promoter regulates or mediates transcription of the nucleic acid.

As used herein, "synthetic" refers to a nucleic acid molecule, or gene or polypeptide molecule, produced by recombinant and/or chemical synthesis methods, e.g., with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide. .

As used herein, naturally occurring α-amino acid residues refer to the 20 α-amino acid residues found in nature that are incorporated into proteins by specific recognition of a charged tRNA molecule by its cognate mRNA codon in humans. It is an amino acid residue.

As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

As used herein, "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 that contains an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids in polypeptides, such as provided antibodies, include the 20 natural amino acids (Table 4), unnatural amino acids, and amino acid analogs (e.g., those in which the α-carbon is amino acids). As used herein, amino acids appearing 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). Nucleotides present in various nucleic acid molecules and fragments are designated by the standard one-letter designation routinely used in the art.

As used herein, "amino acid residue" refers to an amino acid formed by chemical digestion (hydrolysis) of a polypeptide at its peptide bonds. The amino acid residues described herein are generally in the "L" isomeric form. Residues of the "D" isomeric type may be substituted for any L amino acid residue so long as the desired functional properties are 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. J. Biol. Chem., 243:3557-59 (1968), and in accordance with the standard polypeptide nomenclature described in 35 U.S.C. Shown below.

All sequences of amino acid residues represented by formulas herein have a left-to-right orientation in the conventional direction from amino-terminus to carboxyl-terminus. Additionally, the phrase "amino acid residue" is defined to include the amino acids listed in the Table of Correspondence (Table 4), modified amino acids, unnatural amino acids and unusual amino acids. Additionally, a dash at the beginning or end of an amino acid residue sequence may indicate 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, e.g. shows a peptide bond. Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (e.g., Watson et al., Molecular Biology of the Gene, 4th Edition). , 1987, The Benjamin/Cummings Pub. Co., p. 224).

Such substitutions may be made according to the exemplary substitutions set forth in Table 5 below.

Other substitutions are also permissible and may be determined empirically or according to other known conservative or non-conservative substitutions.

As used herein, "naturally occurring amino acids" refers to the 20 L-amino acids that are present in polypeptides.

As used herein, the term "unnatural amino acid" refers to an organic compound that has a structure similar to a natural amino acid, but that has been structurally modified to mimic the structure and reactivity of the natural amino acid. Thus, non-naturally occurring amino acids include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids, including, but not limited to, D stereoisomers of amino acids. Exemplary unnatural amino acids are known to those skilled in the art and include, but are not limited to, 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine/β-amino-propionic acid ( Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid/piperidic acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoiso Butyric 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-stranded or double-stranded linear or circular DNA molecule that includes segments of DNA joined and juxtaposed in a manner not found in nature. be. 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 part of a larger DNA molecule that has specific attributes. For example, a DNA segment encoding a particular polypeptide is part of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read in the 5' to 3' direction, encodes the amino acid sequence of the particular polypeptide.

As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from 5' to 3' end. Polynucleotides include RNA and DNA and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of polynucleotide molecules is given herein in terms of nucleotides (abbreviated as "nt") or base pairs (abbreviated as "bp"). The term nucleotide is used for single-stranded and double-stranded molecules where the context allows. When this term is applied to double-stranded molecules, it is used to represent the entire length and is understood to be equivalent to the term base pair. It will be appreciated by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that their ends may be staggered. Therefore, not all nucleotides within a double-stranded polynucleotide molecule can pair. Such unpaired ends generally do not exceed 20 nucleotides in length.

As used herein, production by recombinant means by using recombinant DNA methods refers to the use of well-known methods of molecular biology to express proteins encoded by cloned DNA.

As used herein, "expression" refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods include, but are not limited to, quantitation of polypeptides in cell lysates by ELISA, Coomassie Blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

As used herein, a "host cell" is a cell used to receive, maintain, replicate, and/or amplify a vector. Host cells can also be used to express polypeptides encoded by vectors. The nucleic acid within the vector is replicated when the host cell divides, thereby amplifying the nucleic acid.

As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed once the vector is transformed into a suitable host cell. Reference to vectors includes vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digestion and ligation. Reference to vectors also includes vectors containing nucleic acids encoding polypeptides such as modified anti-TNFR1 antibodies. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell to amplify the nucleic acid or to express/display the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but can be designed to integrate the gene or portion thereof into the genome's chromosomes. Vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles is well known to those skilled in the art. Vectors also include "viral vectors" or "viral vectors." A viral vector is a virus that is operably linked to an exogenous gene and designed to transfer the exogenous gene into cells (as a vehicle or shuttle).

As used herein, "expression vector" includes vectors capable of expressing DNA operably linked with regulatory sequences, such as promoter regions, capable of effecting the expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Accordingly, expression vector refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus, or other vector that results in the expression of the cloned DNA when introduced into a suitable host cell. Suitable expression vectors are well known to those skilled in the art and include vectors that are replicable in eukaryotic and/or prokaryotic cells, and vectors that remain episomal or that 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 acid or nucleotide bases in a comparison between a test and a reference polypeptide 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. Alignment may be local or global. Matches, mismatches, and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between residues of aligned sequences so that identical or similar letters are aligned. In general, there may be internal gaps and terminal gaps. If a gap penalty is used, sequence identity may be determined without end gap penalties (eg, terminal gaps are not penalized). Alternatively, sequence identity may be determined as the number of identical positions/length of the entire aligned sequence x 100, without considering gaps.

As used herein, a "global alignment" is an alignment that aligns two sequences from beginning to end, aligning each character of each sequence only once. Generate alignments regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on a "global alignment" means that in a complete sequence alignment of the two compared sequences, each 100 nucleotides in length, 50% of the residues are the same. means. It is understood that global alignments may be used in determining sequence identity even if the lengths of the aligned sequences are not the same. Differences at the ends of the sequences are taken into account when determining sequence identity, unless "no penalty for end gaps" is selected. Generally, global alignments are used for sequences that share significant similarity over most of their length. An exemplary algorithm for performing global alignment includes the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48:443). An example program for performing global alignment is publicly available and is available on the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/). Tool, and deepc2. psi. iastate. edu/aat/align/align. Examples include programs available in html.

As used herein, a "local alignment" is an alignment that aligns two sequences, but aligns only the portions of the sequences that share similarity or identity. Therefore, local alignment determines whether a subsegment of one sequence is present in another sequence. If there is no similarity, no alignment is returned. Local alignment algorithms include BLAST or the Smith-Waterman algorithm (Adv. Appl. Math. 2:482 (1981)). For example, 50% sequence identity based on a "local alignment" means that in a full sequence alignment of two compared sequences of any length, a region of similarity or identity of 100 nucleotides in length is In regions of similarity or identity it is meant to have 50% of the residues that are the same.

For purposes herein, sequence identity may be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. The default parameters of the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity), and Gribskov et al. Nucl. Weighted comparison matrix of Acids Res. 14:6745 (1986) (described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979)); (2) for each gap 3.0 penalty for , and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Any two nucleic acid molecules have nucleotide sequences or any two polypeptides are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" or other similar variations enumerating percent identity can be determined using known computer algorithms based on local or global alignment (e.g., wikipedia.org/wiki/Sequence_alignment_software). References are provided, links to a number of known and published alignment databases and programs). Generally, for purposes herein, sequence identity is determined using the Needleman-Wunsch Global Sequence Alignment tool available from NCBI/BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&Page_TYP E=BlastHome ); LAalign (William Pearson (Adv. Appl. Math. (1991) 12:337-357) implementing the Huang and Miller algorithm); and Xiaoqui Huang's program (deepc2.psi.iastate.edu/aat/align/a lign (available at .html) using a computer algorithm based on global alignment. Typically, the full-length sequences of each polypeptide or nucleotide that are being compared are aligned over the entire length of each sequence in a global alignment. Local alignment may also be used when the sequences being compared are of substantially the same length.

The term "identity" as used herein refers to a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one non-limiting example, "at least 90% identical" refers to a percent identity of 90% to 100% compared to a reference polypeptide or polynucleotide. Identity at a level of 90% or greater is assumed for illustrative purposes when comparing test and reference polypeptides or polynucleotides having a length of 100 amino acids or nucleotides. or less than 10% (ie, 10 out of 100) of the nucleotides differ from that of the reference polypeptide or polynucleotide. Similar comparisons may be made between test and reference polynucleotides. Such differences may be expressed as point mutations randomly distributed over the entire length of the amino acid sequence, or they may be expressed up to the maximum allowed, e.g. 10/100 amino acid differences (approximately 90% identical). characteristics), may be clustered at one or more positions of varying length. Differences may also be due to deletions or truncations of amino acid residues. Differences may also be due to deletions or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. Depending on the length of the sequences compared, at levels of homology or identity greater than about 85-90%, results can be independent of program and gap parameter set. Such high-level identity can often be easily assessed without relying on software.

As used herein, a "disulfide bond" (also referred to as an SS bond or disulfide bridge) is a covalent single 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 "through covalent or non-covalent interactions" It means to combine.

As used herein, the phrases "conjugated to an antibody" or "linked to an antibody" or grammatical variations thereof refer to a moiety, such as a diagnostic or therapeutic moiety, to an antibody or antigen-binding fragment thereof. when the moiety is attached to an antibody or antigen-binding fragment thereof by any known means for joining a peptide, such as by production of a fusion protein by recombinant means or post-translation by chemical means. means to be combined. Conjugation may use any of a variety of linking agents to effectuate the conjugation, including, but not limited to, peptide or compound linkers, or chemical crosslinkers.

As used herein, "antibody-dependent cell-mediated cytotoxicity," "antibody-dependent cytotoxicity," and "ADCC" refer interchangeably to Fc receptors (FcR) (e.g., natural killer Non-specifically cytotoxic cells expressing (NK) cells, neutrophils, and macrophages exhibit cell-mediated reactivity that recognizes antibodies bound to target cells and subsequently causes lysis of the target cells. NK cells, the primary cells that mediate ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. (1991) Annu. Rev. Immunol, 9:457-492. In vitro ADCC assays may be performed to assess the ADCC activity of molecules of interest (see, eg, US Pat. Nos. 5,500,362 and 5,821,337). Exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model as disclosed in Clynes et al. (1998) Proc. Natl. Acad. Sci. USA 95:652-656. obtain.

As used herein, complement dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When such antibodies bind to surface antigens on target cells, such as bacterial cells or virus-infected cells, the binding of protein C1q to these antibodies triggers the classical complement pathway, leading to the membrane attack complex ( MAC) is formed, followed by cell lysis.

As used herein, antibody-dependent cellular phagocytosis (ADCP) is a cellular process in which phagocytic effector cells, such as monocytes and macrophages, internalize target cells. Once phagocytosed, target cells reside in phagosomes, which fuse with lysosomes and degrade the target cells via oxygen-dependent or independent mechanisms.

"Therapeutic activity" as used herein refers to the in vivo activity of a therapeutic polypeptide. Generally, therapeutic activity is an activity associated with the treatment of a disease or condition. The therapeutic activity of the modified polypeptide may be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30% of the therapeutic activity compared to the unmodified polypeptide. %, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, The percentage of any level of therapeutic activity of the unmodified polypeptide can be 200%, 300%, 400%, 500% or more.

As used herein, the term "assessment" refers to obtaining the absolute value of the activity of a protein, such as an antibody or antigen-binding fragment thereof, present in a sample, and also the use of an index, ratio, percentage, or visual representation indicating the level of activity. It is intended to include quantitative and qualitative determinations in the sense of obtaining a target or other value. Evaluation may be performed directly or indirectly.

As used herein, "disease or disorder" is due to a cause or condition, including but not limited to infectious diseases, acquired conditions, and genetic conditions, and is characterized by identifiable symptoms. , refers to a pathological condition in an organism.

As used herein, "treating" a subject with a disease or condition means that the subject's symptoms are partially or completely alleviated or remain static after treatment. . Treatment therefore includes prevention, treatment and/or cure. Prevention refers to prevention of underlying disease and/or prevention of worsening of symptoms or progression of disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof, or composition provided herein.

As used herein, treatment means amelioration of symptoms or signs of a disease, disorder, or condition.

As used herein, "prevention, prevention, prevention" or "prophylaxis" refers to a method by which the risk of developing a disease or condition is reduced. Preventing a disease means reducing the risk of developing the disease.

As used herein, "pharmaceutically effective agent" includes, but is not limited to, conventional therapeutic agents, including, for example, anesthetics, vasoconstrictors, dispersants, and small molecule drugs and therapeutic proteins. including, but not limited to, any therapeutic or bioactive agent.

As used herein, "therapeutic effect" means an effect resulting from treatment of a subject that alters, typically ameliorates or ameliorates, or cures the symptoms of a disease or condition. do.

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an agent, compound, material, or composition containing at least enough of the compound to produce a therapeutic effect after administration to a subject. refers to the amount of Therefore, it is the amount necessary to prevent, cure, ameliorate, halt, or partially halt the symptoms of a disease or disorder.

As used herein, "therapeutic effect" means a composition comprising an agent, compound, material, or compound that produces a therapeutic effect in a subject to whom the agent, compound, material, or composition comprising the compound is administered. Refers to the ability of things.

As used herein, a "prophylactically effective amount" or "prophylactically effective dose" means that when administered to a subject, the intended prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or condition. , an amount of an agent, compound, material, or composition comprising a compound that has a reduced likelihood of onset or recurrence of a disease or condition, or a reduced incidence of viral infection. A complete prophylactic effect does not necessarily occur with a single administration, but may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in a single dose or in multiple doses.

As used herein, amelioration of symptoms of a particular disease or disorder by treatment, such as administration of a pharmaceutical composition or other therapeutic agent, may result from or relate to the administration of the composition or therapeutic agent. Refers to the alleviation of symptoms, whether permanent or temporary, permanent or temporary.

As used herein, a "prodrug" is a drug that has less cytotoxicity toward tumor cells compared to the parent drug and can be enzymatically activated or converted to a more active parent form. , a precursor or derivative form of a pharmaceutically active substance (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 al., (ed.), pp. 247-267, Humana Press, 1985).

As used herein, "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 "angiogenesis inhibitor" or "angiogenesis inhibitor" is a compound that blocks or interferes with the development of blood vessels.

As used herein, TNF-related or TNF-mediated disease refers to a disease, condition, or disorder in which TNFR1 or TNFR1 signaling plays a role in pathogenesis. This includes diseases, disorders, and conditions where inhibition of TNFR1 signaling may ameliorate the symptoms of the disease, condition, or disorder.

As used herein, "TNFR2 agonist" or "anti-TNFR2 agonist" refers to small molecules and TNFR2 antibodies or antigen-binding fragments thereof, and those that initiate, promote, or increase activation of TNFR2, and/or or other polypeptides that enhance one or more signaling pathways mediated by TNFR2. For example, TNFR2 agonists can promote or increase the proliferation of Treg cell populations. TNFR2 agonists can promote or increase TNFR2 activation by binding to TNFR2, eg, by inducing conformational changes that render the receptor biologically active. For example, TNFR2 agonists nucleate trimerization of TNFR2 in a manner that resembles or mimics the interaction between TNFR2 and its cognate ligand TNF (TNFα), thus inducing TNFR2-mediated signaling. It is possible. TNFR2 agonists can also induce proliferation of CD4+, CD25+, FOXP3+ Treg cells. TNFR2 agonists also inhibit cytotoxic T lymphocytes (e.g., through activation of immunoregulatory Treg cells or by directly binding to TNFR2 on the surface of autoreactive cytotoxic T cells and inducing apoptosis). For example, the proliferation of CD8+ T cells) can be suppressed. TNFR2 agonist antibodies or fragments thereof for use in the methods herein are capable of specifically binding to TNFR2 and are generally specific to another receptor of the tumor necrosis factor receptor (TNFR) superfamily member, e.g., TNFR1. be specific enough so that it does not bind to

As used herein, a TNFR2 selective agonist is a TNFR2 agonist that does not produce or substantially produces no TNFR1 signaling activity.

As used herein, Treg expanders are small molecules and A molecule that includes a polypeptide.

As used herein, the terms "pan-growth factor trap construct", "pan-EGFR ligand trap construct", "growth factor trap", "multispecific growth factor trap construct", "bispecific growth factor trap construct" , "EGFR ligand trap construct," "pan-HER ligand trap construct," "pan-HER therapeutic agent," "EGFR ligand trap construct," "HER ligand trap construct," and "growth factor trap construct" refer to HER or ErbB receptor used interchangeably to refer to pan-cell surface receptor molecules that include peptide-based compounds that modulate the activity of two or more human epidermal growth factor receptors (EGFRs), also called human epidermal growth factor receptors (EGFRs). Generally, pan-growth factor traps target at least two different HER receptors, such as through ligand binding and/or interaction with the receptors.

As used herein, the "extracellular domain" or "ECD" is the portion of a cell surface receptor that is present on the surface of the receptor and contains the ligand binding site. For purposes herein, reference to an "ECD polypeptide" means that the ECD polypeptide comprises a contiguous sequence that is related to another domain (e.g., a transmembrane domain, a protein kinase domain, or other domain) of the cognate receptor. Includes any ECD-containing molecule or portion thereof, unless otherwise specified.

As used herein, "knob-into-hole" or "knob-in-hole" (KIH) refers to a multimerization domain, such as an immunoglobulin Fc domain, that The steric interactions within the domains are engineered to promote stable interactions, resulting in heterodimers (or heteromultimers) compared to homodimers (or homomultimers) from mixtures of monomers. refers to a multimerization domain that is engineered to promote the formation of multimers). This can be accomplished, for example, by constructing knobs or projections and holes or cavities in complementary multimerization domains. A "knob" is created by replacing a small amino acid side chain from the interface of the first multimerization domain polypeptide (e.g., first Fc monomer) with a larger side chain (e.g., tyrosine or tryptophan). can be constructed. A compensatory "hole" of the same or similar size as a knob is inserted into a second complementary multimerizing polypeptide (e.g., a second 2) on the interface of the Fc monomer).

As used herein, "tethering" refers to the interaction between two domains of a receptor monomer, whereby the monomer has a conformation that makes the interaction less accessible. Refers to the interaction that occurs in For example, subdomain II of HER1, HER3, and HER4 can interact with subdomain IV to form a tethered, inactive structure. When in the tethered state, the receptor or isoform thereof is less available or unavailable for dimerization and/or ligand binding. The monomeric forms of ECD of HER1, HER3 and HER4 occur in tethered forms that exhibit lower ligand affinity than the unlinked forms. HER2 lacking certain residues of subdomain IV exists in an unrestricted form and is available for dimerization with HER1, HER3, and HER4. When the ligand binds to the tethered (monomeric) form, the tethering interaction is released and the ECD (or receptor) becomes available for dimerization, which involves the interaction between domain II of the two ECDs. It has a three-dimensional structure.

As used herein, a HER (ErbB)-related disease, a HER-related disease, or a HER-mediated disease is a disease in which the epidermal growth factor receptor (HER) and/or its ligands are responsible for the etiology, progression of the disease state, or symptoms thereof. Any disease, condition or disorder that is implicated in any aspect of. Engagement includes, for example, expression, overexpression, or activity of a HER family member or ligand. Diseases include, but are not limited to, cancer, such as, but not limited to, glioma, and pancreatic cancer, gastric cancer, head and neck cancer, cervical cancer, lung cancer, colorectal cancer, endometrial cancer. proliferative diseases including, but not limited to cancer, prostate cancer, esophageal cancer, ovarian cancer, uterine cancer, bladder cancer or breast cancer. Other conditions include rheumatoid arthritis (RA), non-malignant hyperproliferative diseases, eye conditions, skin conditions (e.g. psoriasis), conditions due to smooth muscle cell proliferation and/or migration, e.g. cell proliferation, including conditions associated with pathological inflammatory and/or autoimmune responses, such as strictures, atherosclerosis, muscular thickening of the bladder, heart or other muscles, or endometriosis; and/or conditions related to movement.

The term "subject" as used herein refers to animals, including mammals such as humans.

"Patient" as used herein 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; chickens, etc. Poultry; ruminants such as goats, cows, deer, and sheep; pigs; and other animals. Non-human animals exclude humans as possible animals. The polypeptides provided herein are derived from any animal, plant, prokaryotic, and fungal source. Most polypeptides are of animal origin, including mammalian origin, and are generally human or humanized for therapeutic use.

"Composition" as used herein refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, "stabilizer" refers to the stability of either the antibody or the conjugate under the conditions (e.g., temperature) under which the formulations herein are stored or used. Refers to a compound added to a formulation to protect it. Therefore, agents that prevent protein degradation from other ingredients in the composition are included. Examples of such agents are amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, inhibitors, or substrates, as well as other agents described herein.

As used herein, "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 two or more items, or any variation thereof. The elements of a combination are generally functionally related or related, such as elements used in a certain method.

As used herein, "combination therapy" refers to two or more different therapeutic agents, such as anti-TNFR constructs or antibodies or antigen-binding fragments thereof, and one or more treatments provided herein. Refers to the administration of drugs or other treatments, such as radiation and surgery. Multiple therapeutic agents may be provided and may be administered separately, sequentially, intermittently, simultaneously, or in a single composition.

As used herein, a "kit" refers to a packaged combination for purposes including, but not limited to, activation, administration, diagnosis, and evaluation of biological activity or properties. refers to combinations that optionally include other elements, such as additional reagents and instructions, for the use of that combination or its elements.

As used herein, "unit dosage form" refers to individually packaged physically discrete units suitable for human and animal subjects, as known in the art.

As used herein, "single dose formulation" refers to a formulation for direct administration.

As used herein, "multidose formulation" refers to a formulation that contains multiple doses of a therapeutic agent and can be administered directly to provide several single doses of a therapeutic agent. Doses may be administered over minutes, hours, weeks, days, or months. Multiple dose formulations may allow for dose adjustment, dose pooling, and/or dose division. Because multi-dose formulations are used over long periods of time, they typically include one or more preservatives to prevent the growth of microorganisms.

As used herein, a "product" is a product that is manufactured and sold. The terms used throughout this application are intended to encompass any composition included in an article of packaging or provided herein for packaging.

As used herein, "fluid" refers to any composition that is capable of flowing. Thus, fluids include compositions that are in the form of semisolids, 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 a biologically active portion thereof (e.g., an isolated antigen-binding fragments) are substantially free of cellular material from the cell or tissue from which the protein is derived or other contaminant proteins, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. Not included in The physical and chemical properties of the material upon further purification are determined by standard analytical methods such as thin layer chromatography (TLC), gel electrophoresis, and high performance liquid chromatography (HPLC) used by those skilled in the art. Pure or substantially pure means that the preparation is pure enough that a property (such as enzymatic activity or biological activity) is not detectably altered; the preparation is free from readily detectable impurities. If it does not appear to contain, it may be determined that it does not contain substantially. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In such cases, further purification may increase the specific activity of the compound.

As used herein, "cell extract" or "lysate" refers to a preparation or fraction made from lysed or disrupted cells.

As used herein, "control" refers to a sample that is substantially identical to the test sample, except that it has not been treated with the test parameters, or, if a plasma sample, a sample of interest. It may be from a normal volunteer not affected by the condition. The control may also be an internal control.

As used herein, the singular forms "a," "an," and "the," mean Contains multiple referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide that includes an "immunoglobulin domain" includes polypeptides that have one or more immunoglobulin domains.

As used herein, the term "or" means "and/or" unless it is explicitly indicated that only the alternatives are referred to or the alternatives are mutually exclusive. used.

As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" includes a precise amount. Therefore, "about 5 amino acids" means "about 5 amino acids" and "5 amino acids." For a particular parameter, about is a range within experimental error or as acceptable to those skilled in the art for the particular parameter.

As used herein, "optional" or "optional" refers to the occurrence or non-occurrence of the subsequently described event or situation, and that the statement is in accordance with the preceding article. This means that an event or situation may or may not occur. For example, a variant portion, as appropriate, means that the portion is a variant or a non-variant.

As used herein, any protecting groups, amino acids, and other compound abbreviations refer to IUPAC for their common usage, approved abbreviations, or biochemical nomenclature, unless otherwise indicated. - in accordance with the IUB Committee (see Biochem. (1972) 11(9):1726-1732).

For clarity, and not limitation, the detailed description is divided into the following subsections.

B. Structure and Method Overview Autoimmune diseases occur 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 over 100 types of autoimmune diseases; overall, approximately 75% of patients with autoimmune diseases are women. Traditional drugs for autoimmune diseases have harmful side effects such as infections, heart problems, and other diseases and disorders.

TNF interacts with immune cells through two receptors: TNFR1, which is overactivated in autoimmune diseases, and TNFR2, which suppresses autoimmune diseases; however, when TNRF1 is overactivated, it is suppressed. TNF blockers, such as infliximab (sold as Remicade®), adalimumab (sold as Humira®), and etanercept (sold as Enbrel®), block TNFR1 and TNFR2 and can cause adverse side effects. bring about. The constructs provided herein address this issue. The constructs provided herein not only shut down TNFR1 leading to increased TNFR2 activity, thereby treating the symptoms of autoimmune diseases, but also provide improved therapy because TNFR2 activity is not blocked. provide reduced or no harmful side effects. Various constructs are provided herein that address the problems of prior art TNF blockers. The types of constructs detailed and provided herein, identified by their activity, are summarized in the table below:

Constructs are provided for the treatment of TNF-mediated diseases, disorders and conditions, or diseases, disorders and conditions in which TNF plays a role in the pathogenesis or in which interference with TNFR1 signaling has an ameliorative effect. For example, TNFR1 antagonists can be used to treat a variety of disorders, including autoimmune disorders, as well as endometriosis, brain fog (e.g., from chemotherapy and COVID-19), Alzheimer's disease, acute inflammation (e.g., lung, kidney and other diseases). It may also be used to treat diseases and conditions (resulting from infection with influenza virus and SARS-COV2) that result in long-term or permanent damage to the human body. Previous TNF blockers have side effects and associated safety issues that preclude their use for most of these indications. The TNFR1 antagonist constructs provided herein may be used. These constructs described herein are monovalent in that they inhibit only TNFR1 and do not cause receptor clustering, they are specific, non-immunogenic, and last for at least about 3-4 weeks. It has a half-life of 100%, allowing it to be used approximately once a month.

Thus, multispecificities are provided, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and bispecific constructs containing TNFR1 antagonist and TNFR2 agonist activities. The construct includes at least one moiety that specifically interacts with TNFR1 or TNFR2, and generally modulates the interaction directly or indirectly, or has a pharmacological (pharmacodynamic or pharmacokinetic or both) moiety in the construct. ) including further portions providing properties. Thus, the constructs provided herein include at least two moieties: a binding moiety that interacts with TNFR1 or TNFR2, and a second moiety that modulates or alters the pharmacological property or activity of the construct or binding moiety. .

Among the constructs provided herein are constructs that are antagonists of TNFR1 activity. TNFR1 antagonist constructs include a moiety that binds or interacts with TNFR1 and inhibits TNFR1-mediated signaling, as well as prolonging serum half-life, eliminating ADCC and/or CDC activity, and inhibiting interaction with specific receptors. and a second portion that provides additional properties such as accommodation. TNFR1 antagonists and constructs may also include modifications to eliminate or reduce immunogenicity, particularly in humans, and may also include modifications to eliminate or reduce binding to existing antibodies.

TNFR1 antagonist constructs are selected to specifically bind TNFR1 and have minimal or no binding to TNFR2 or have no TNFR2 antagonist activity. Therefore, this construct only modulates TNFR1. In some embodiments, the TNFR1 antagonist construct is selected to also have or be linked to a second domain or portion that has TNFR2 agonist activity. As a TNFR1 construct, with an affinity such as Kd<50nM or <10nM or <5nM, especially with higher affinity (Kd<1nM or <0.1nM or higher affinity) and/or with potent TNFR1 signaling. Constructs that are designed or selected to interact with TNFR1 with significant inhibition (eg, IC5050 nM or <10 nM or <5 nM or <3 nM or, alternatively, 1 nM or <0.5 nM).

Also provided are multispecific, including bispecific, constructs that include a TNFR1 antagonist moiety linked directly or via a linker to a TNFR2 agonist moiety. Linkers provide advantageous properties to molecules such as increased serum half-life, increased stability, appropriate three-dimensional structure and plasticity, 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). , these constructs increase the specificity of TNFR1 inflammatory blockade and inhibit the natural immunosuppressive factor TNFR2, at least in part by upregulation of immunosuppressive Tregs and induction of protective and anti-inflammatory signaling pathways. resulting in preservation or amplification of function. Furthermore, TNF blockade resulting in inhibition of TNFR2 function also reduces T cell-induced monocyte activation, which increases the likelihood of opportunistic infections (e.g., Rossel et al. (2007) J Immunol. 179:4239-48).

There are many differences between the activity of the exemplary TNFR1 antagonist constructs provided herein and existing approved TNF blockers: TNF blockers, such as etanercept, adalimumab, infiximab, that they are not specific for TNFR1. Other blockers such as IL6, IL17, IL23 block only parts of the cytokine cascade rather than the entire one. Existing TNF blockers have the same mechanism of action on TNFR1 and TNFR2 and therefore block the activity of both. JAK inhibitors pose similar problems; they have pro-inflammatory and anti-inflammatory activities. For example, the inflammatory cytokine Il1 is not blocked by JAK inhibitors, the inflammatory cytokine IL6 is blocked by JAK inhibitors (secondary use in rheumatoid arthritis treatment), and the anti-inflammatory IL10 is blocked by JAK inhibitors. Not blocked. In contrast, the constructs provided herein combine the efficacy of TNFR1 and TNF inhibitor therapy with the benefits of TNFR2 agonists that eliminate or reduce the adverse effects of anti-TNFR1/anti-TNF therapy, and also provide immunosuppression. This contributes to the benefits of additional therapeutic modalities, including upregulation of sexual Tregs and induction of protective and anti-inflammatory signaling pathways.

TNFR1 antagonist constructs include one or more TNFR1 inhibitors, one or more linkers, and one or more activity modulators. For example, the structure of a TNFR1 antagonist construct provided herein can be represented by Formula 1:
(TNFR1 inhibitor) n-linker p-(activity modifier) q, formula 1a, or (activity modifier) q-linker p-(TNFR1 inhibitor) n formula 1b, where:
n and q are each an integer and are each independently 1, 2, or 3; p is 0, 1, 2, or 3; the activity modifier increases the serum half-life of the TNFR1 inhibitor , a polypeptide such as albumin, or a moiety such as an Fc that has been modified to have reduced or no ADCC activity; a TNFR1 inhibitor binds to TNFR1 and inhibits its activity, such as signaling activity. a molecule such as a polypeptide or small drug molecule. The activity modifier is not a human serum albumin antibody or an unmodified single Fc. Activity modifiers include modified Fc regions, Fc dimers, and other antibody domains, such as Fc modified to eliminate ADCC and/or CDC activity. Linkers include chemical linkers, polypeptides such as GS linkers, and hinge regions such as those derived from antibodies, so that constructs include chemical conjugates, fusion proteins, and combinations of both.

Multispecific constructs are also provided. The structure of multispecific, such as bispecific, constructs provided herein is represented by the following formula (Formula 2):
(TNFR1 inhibitor) n-(activity modifier) r1-(linker (L) p-(activity modifier) r2-(TNFR2 agonist) q,
[where n=1, 2, or 3, p=1, 2, or 3, q=0, 1, or 2, and each of r1 and r2 is independently 0, 1 , or 2]. Similar to the structure of Formula 1, the order of the components may be changed, and linkers may be added as appropriate. The construct may include additional linkers necessary to impart properties such as plasticity. Each linker may include multiple components. Formula 2 may also include activity modifiers instead of or in addition to the linker. Activity modifiers and linkers include Fc or Fc with a hinge region, or Fc with a GS linker, or other combinations of components. The Fc of these constructs contains an unmodified Fc region. The linker is as described above and will be explained in detail below.

Also provided is a TNFR2 agonist construct having formula 3:
(TNFR2 agonist) n-linker p-(activity modifier) q, formula 3a, or (activity modifier) q-linker p-(TNFR2 agonist) n, formula 3b,
[where n, p, and q are as described in Formula 1, and the linker and activity modifier are as described in Formula 1].

The components of Formulas 1-3, discussed in detail in the following sections, can be polypeptides or other molecules, such as small drugs, that specifically bind or interact with a target receptor. Each component of the constructs/molecules provided herein is described in turn in the sections below.

The properties of each component of the constructs provided herein are discussed in detail in the following sections. Accordingly, the components of the construct include, but are not limited to, the following components, which are described in detail in the following sections:
1. TNFR1 antagonist 2. TNFR2 agonist 3. Linker a. Glycine-serine linker b. Hinge area c. Chemical linker 4. Activity Modifiers a. Modified FC
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 are also provided, which are detailed in the following sections.

The constructs include TNF in a pathological modifier of the disease, condition, or disorder, such that inhibition of TNFR1 signaling is reduced or inhibited; The conditions may be suppressed or regressed, and/or inhibition is used in methods of treatment of diseases, disorders, and conditions to ameliorate symptoms of the disease, disorder, and/or condition. Such diseases, conditions, and disorders, including inflammatory diseases, including autoimmune diseases, are discussed in the sections below.

Also provided are pharmaceutical compositions for use in the methods and uses, and nucleic acids and vectors for producing constructs, including those that are polypeptides and fusion proteins. The following sections discuss diseases, disorders, and conditions, TNFR1/TNFR2 activity and their role in diseases, disorders, and conditions, existing treatments for diseases, disorders, and conditions, and their constructs provided herein. and components, methods of making the constructs, pharmaceutical compositions comprising the constructs and/or encoding the nucleic acids, and methods of treatment.

C. Tumor necrosis factor (TNF) and chronic inflammatory and autoimmune diseases and disorders details of the disease, and problems with existing treatments, and demonstrate 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 TNFα, TNF-α, or TNFα) is involved in the regulation of host defense against tumorigenesis/cancer, pathogenic infection, apoptosis, autoimmunity, and septic shock. It is a pleiotropic pro-inflammatory cytokine associated with inflammatory and immunomodulatory activities, including, especially in the organogenesis of lymphoid organs, as well as in the modulation of innate and adaptive immune responses. In humans, TNF is produced primarily by macrophages, but also by monocytes, dendritic cells (DCs), B cells, T cells, fibroblasts, and other cell types. It is 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 containing 157 amino acids (17 kDa). membrane-bound and soluble forms of TNF are biologically active. Transmembrane human TNF contains 233 amino acids, with respect to SEQ ID NO: 1, a cytoplasmic domain corresponding to residues 1-35, a transmembrane domain corresponding to residues 36-56, and a transmembrane domain corresponding to residues 57-233. Contains an extracellular domain. The soluble form of TNF corresponds to amino acid residues 77-233 as shown 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 Associated with autoimmune diseases and conditions. Overexpression of TNF has also been associated with neurodegenerative diseases and conditions such as Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. Additionally, TNF promotes osteoclast formation and overproduction of TNF is associated with bone loss. In rheumatoid arthritis (RA), TNF is overexpressed in the synovial fluid and synovium, where the expression of TNF receptors (TNFR) is upregulated. For example, overexpression of human TNF in mice results in the formation of hyperplastic synovium and the development of spontaneous RA-like lesions in joints with cartilage and bone destruction (e.g., Blueml et al. (2010) Arthritis & Rheumatism 62(6):1608-1619; Keffer et al. (1991) EMBO J.10(13):4025-4031; Esperito Santo et al. (2015) Biochem. Biophys. Res. Commun. 464:1145-1150; Blueml et al. (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 deleterious inflammatory processes, whereas TNFR2 is associated with beneficial inflammatory processes. It is associated with immunomodulatory processes. Membrane-bound TNF has been shown to primarily activate TNFR2, whereas soluble TNF has been shown to primarily activate TNFR1 (Blueml et al., (2010) Arthritis & Rheumatism 62(6):1608-1619) . Soluble TNF (solTNF; corresponding to residues 77-233 of SEQ ID NO: 1; see also the sequence shown in SEQ ID NO: 2) is involved in paracrine signaling (mainly mediated through TNFR1) and is associated with chronic diseases. However, transmembrane TNF (tmTNF) acts through cell-cell contacts to induce juxtacrine signaling (mainly through TNFR2), leading to the resolution of inflammation and to Listeria monocytogenes and Micobacterium tuberculosis. It is associated with the induction of immunity against pathogens such as (Zalevsky et al. (2007) J. Immunol. 179:1872-1883). Therefore, TNF signaling through TNFR1 and TNFR2 has different outcomes depending on the receptor type.

Due to the association between TNF overexpression and the development of inflammatory and autoimmune diseases and conditions, blockade of TNF can be used to treat, but are not limited to, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, ankylosing arthritis. It has been used to treat a variety of such diseases and conditions, including spondylitis, juvenile idiopathic arthritis (JIA), and inflammatory bowel disease (IBD; eg, Crohn's disease, ulcerative colitis, etc.). The use of TNF blockers, which block TNF and prevent signaling through 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 harmful inflammatory signaling through TNFR1, but also beneficial immunomodulatory signaling through TNFR2. As a result, the use of TNF blockers can be limited, especially in cases of chronic diseases/conditions that require long-term administration, such as arthritis or IBD. Approximately one-third of RA patients do not respond or do not have sustained treatment effects using anti-TNF therapy. Therefore, there is a need for therapies with improved therapeutic efficacy and safety, particularly those that block the inflammatory effects of TNFR1 signaling but maintain or promote the beneficial anti-inflammatory effects of TNFR2 signaling. . Such treatment methods are provided herein.

2. Tumor necrosis factor receptor (TNFR)
The homotrimer of TNF has two specific high-affinity homotrimeric receptors, TNFR1 (TNF receptor type 1; TNFRI, p55, p60, CD120a, TNF receptor superfamily member 1A, and TNFRSF1A). TNFR2 (also known as TNF receptor type 2; TNFRII, p75, p80, CD120b, TNF receptor superfamily member 1B, and TNFRSF1B) binds and signals through TNFR2 (also known as TNF receptor type 2; TNFRII, p75, p80, CD120b, TNF receptor superfamily member 1B, and TNFRSF1B). TNFR1 is expressed on all nucleated cell types; TNFR2 expression is expressed on immune cells such as monocytes, macrophages, activated T cells, regulatory T cells (Tregs), B cells, and natural killer (NK) cells. ) cells), endothelial cells, certain central nervous system (CNS) cells, and certain cardiac cells. TNFR2 expression on Tregs is induced by T cell receptor activation.

In vivo, TNFR1 and TNFR2 exist as membrane-bound receptors and as soluble "decoy" (ie, non-signaling) receptors after shedding from the cell surface. Soluble TNF binds preferentially/selectively to TNFR1; however, binding of membrane-bound and soluble forms of TNF activates TNFR1. The major ligand for TNFR2 is membrane-bound TNF. Although soluble TNF does not fully activate TNFR2, the soluble form of TNFR2 (after TNFR2 shedding) has high binding affinity for TNF, and TNF binds to membrane-bound signaling receptors. scavenges and inhibits TNFR2, contributing to the anti-inflammatory effect of TNFR2. Membrane-bound TNFR2 binds TNF with rapid on/off kinetics, allowing TNFR2 to concentrate TNF on the cell surface and pass the ligand to TNFR1, which mediates TNFR1 signaling. Each of TNFR1 and TNFR2 contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extracellular domains of TNFR1 and TNFR2 contain four cysteine-rich domains (CRDs) required for ligand binding. The intracellular domains of TNFR1 and TNFR2 initiate distinct signaling cascades and mediate distinct effector functions in response to TNF ligand binding.

Abnormalities in TNFR signaling are associated with several autoimmune diseases, and administration of TNF can be used as a therapeutic strategy for such diseases. For example, low doses of TNF selectively destroy autoreactive T cells in blood samples of type I diabetes and scleroderma patients, as well as in animal models of Sjögren's syndrome. Administration of TNF can lead to systemic toxicity, for example in cancer patients with high TNF levels. As described herein, toxicity is due to ubiquitous cellular expression of TNFR1; as described herein, agonization of TNFR2 results in more restricted cellular expression of TNF. Due to its availability, it is a safer treatment option than the administration of TNF. Enhancement of TNF signaling through TNFR2 can be achieved by administering a TNFR1 antagonist (see, eg, Faustman et al., (2013) Front. Immunol. 4:478).

a. TNFR1
Human TNFR1 (see SEQ ID NO: 3) is a major inflammatory receptor and is responsible for most of the pro-inflammatory, cytotoxic and apoptotic effects caused by TNF. Human TNFR1 is a homotrimeric receptor, and its binding by TNF induces a pro-inflammatory response (for a description of TNFR1 signaling see Morton et al. (2019) Sci Signal. 12(592): (See e.g. eaaw2418). TNFR1 contains 455 amino acid residues; residues 1-29 correspond to the signal peptide, residues 30-211 correspond to the extracellular domain, and residues 212-232 correspond to the membrane Corresponds to the transmembrane domain, and residues 233-455 correspond to the cytoplasmic domain. Within the extracellular domain, TNFR1 contains cysteine-rich domains (CRD) 1-4 corresponding to amino acid residues 43-82, 83-125, 126-166 and 167-196 of SEQ ID NO:3, respectively. CRD2 and 3 contact bound TNF, and CRD1, specifically amino acid residues 30-82 of SEQ ID NO: 3, contains the pre-ligand binding assembly domain, a hemorrhagic interaction motif that is essential for ligand binding and receptor function. PLAD). The cytoplasmic domain binds to the death domain (TRADD) and Fas-associated death domain (FADD) following binding of TNF to TNFR1, resulting in a signaling pathway that activates caspases and induces apoptosis. corresponding to residues 356-441 of SEQ ID NO: 3). TNF binding to TNFR1 also triggers a proinflammatory cascade through MAPK (mitogen-activated protein kinases; e.g., p38, JNK, ERK) and NF-κB (nuclear factor kappa light chain enhancer of activated B cells) signaling pathways. Start. TNFR1 plays a role in lymphoid organogenesis and in the immune response to pathogens, and is a major receptor involved in host antiviral 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 is primarily involved in pro-inflammatory signaling, is the driving force in the development of arthritis. For example, knockout of TNFR1 in mice and silencing of TNFR1 expression by RNA interference results in attenuation of collagen-induced arthritis (CIA), an animal model of arthritis. TNFR1-deficient mice that overexpress TNF are protected from the development of arthritis, and reintroduction of TNFR1 into mesenchymal cells results in the development of TNF-dependent arthritis. Furthermore, TNFR1 has been shown to promote osteoclast production and cause localized bone destruction, and the absence of TNFR1 on hematopoietic cells reduces bone destruction in a model of erosive arthritis. TNFR1 is also associated with cardiotoxic effects in TNF-induced models of heart failure and myocardial infarction, and has been shown to promote neurodegeneration in animal models of retinal ischemia (e.g., Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065; McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Ruspi et al (2014) Cellular Signaling 26:683-690; Faustman and Davis (2013) Front.Immunol. 4:478; Blueml et al. (2012) International Immunology 24(5):275-281; Dong et al. (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. Unlike TNFR1, TNFR2, which lacks a death domain, has a TNF receptor-associated factor 2 (TRAF2) binding site. TNFR2 signaling through TRAF2 promotes cell survival and proliferation through activation of NF-κB and activator protein 1 (AP1) and is associated with PI3K-PKB/Akt-mediated repair and migration. As discussed elsewhere herein, TNF signaling through TNFR2 also stimulates regulatory T cells (Treg), which play an important role in suppressing inflammatory and autoimmune diseases and disorders. Promotes proliferation and activation. TNFR2 signaling is involved in wound healing and repair and regeneration in models of myocardial infarction, whereas knockout of TNFR2 in mouse models of erosive arthritis results in joint inflammation and bone destruction.

TNFR2, which is primarily involved in anti-inflammatory signaling, is associated with protective effects on nerves, heart, gut, and bone. TNFR2 exhibits anti-inflammatory and protective effects; these effects are associated with, for example, experimental autoimmune encephalomyelitis (EAE), experimental colitis, heart failure/heart disease, myocardial infarction, inflammatory arthritis, and demyelination. demonstrated in sexual and neurodegenerative diseases, as well as infectious diseases. For example, activation of TNFR2 by TNF suppresses seizures, reduces cognitive dysfunction after brain injury, promotes survival after myocardial infarction, protects against myocardial ischemia/reperfusion injury, and after heart failure in mice. reduces remodeling and hypertrophy. TNFR2 agonism is also associated with pancreatic regeneration, remyelination, neuronal subtype survival, and stem cell proliferation. In blood samples from patients with type I diabetes, multiple sclerosis, Graves' disease, and Sjögren's syndrome, TNFR2 agonism selectively destroys autoreactive T cells, but not healthy cells. In animal models of type I diabetes, pancreatic tissue regenerates when autoreactive T cells are removed using low doses of TNF. TNF signaling through TNFR2 has been shown to induce the regeneration of oligodendrocyte precursors of myelin and therefore may be used to treat demyelinating disorders such as multiple sclerosis (MS). TNFR2 has also been shown to promote neuroprotection in animal models of retinal ischemia.

TNFR2 also regulates osteoclastogenesis. Osteoclasts are a type of bone cell that destroy bone tissue. Regulation of osteoclast formation is important for maintaining bone mass and protecting against joint inflammation and erosive destruction. Mice lacking TNFR2 exhibit enhanced osteoclastogenesis, TNF-induced worsening of arthritis, and focal bone destruction. Absence of TNFR2 in animal models of erosive arthritis results in disease progression, and TNFR2-deficient mice that overexpress TNF develop worsened arthritis and joint destruction compared to control mice. Expression of TNFR2 in hematopoietic cells attenuates TNF-induced arthritis, whereas loss of TNFR2 in hematopoietic cells increases recruitment of inflammatory cells to the synovium. In experimental colitis, lack of TNFR2 expression on CD4+ T cells accelerates disease onset and increases inflammation severity, whereas in experimental autoimmune encephalitis (EAE), symptoms are exacerbated in TNFR2-deficient mice ( For example, Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065; McCann et al. (2014) Arthritis & Rheumatology 66(10 ):2728-2738; Ruspi et al. (2014) Cellular Signaling 26:683-690; Faustman and Davis (2013) Front. Immunol. 4:478; Blueml et al. (2012) International Immunology 24(5):275 -281; see Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43):12304-12309). Polymorphisms in the TNFR2 gene have been correlated with various autoimmune diseases such as RA, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, and scleroderma. ; the polymorphism interferes with the binding of TNF to TNFR2, which limits the activation of NF-κB and interferes with the Treg TNFR2 signaling pathway (e.g., Yang et al. (2018) Front. Immunol. 9:784 checking).

TNFR1 contains an intracellular death domain and can activate apoptotic and/or inflammatory pathways, whereas TNFR2 binds TRAF and activates canonical and noncanonical NF-κB pathways to promote cell survival and proliferation. can be controlled. Generally, cells that express TNFR2 also express TNFR1 in varying proportions depending on cell type and function. Since TNFR1 signaling generally induces cell death, whereas TNFR2 signaling generally induces cell survival, the ratio of their co-expression in cells shifts the balance towards apoptosis or cell survival. As discussed above and elsewhere herein, TNFR1 is the major TNF receptor involved in the pathogenesis of RA, while TNFR2 has been shown to play an immunomodulatory role. However, both receptors are involved in mediating the antiviral activity of TNF. For example, animal disease models have shown that TNFR1 is associated with inflammatory neurodegeneration, whereas TNFR2 is associated with neuroprotection.

Selective inhibition of TNFR1 or selective activation of TNFR2 can be achieved using the TNFR1-selective antagonizing IgG1 antibody ATROSAB (Antagonistic TNF Receptor One-Specific Antibody) or the agonist TNFR2-selective TNF mutein (i.e., mutein protein). )EHD2 -scTNFR2 was demonstrated in a mouse model of NMDA-induced acute neurodegeneration. EHD2-scTNFR2 is derived from the IgE heavy chain CH2 domain and is derived from the mutant D143N/A145R (SEQ ID NO: 2 Residue numbering for soluble TNF as shown in and corresponding to D219N and A221R, respectively with reference to SEQ ID NO: 1; these mutations abolish affinity for TNFR1). Contains human TNFR2-selective single-chain TNF trimer. In an in vivo mouse model, co-injection of NMDA and ATROSAB or NMDA and EHD2-scTNFR2 into the basal ganglia produces a significant but incomplete neuroprotective effect compared to controls. The incomplete nature of these responses was attributed to the agonistic activity of ATROSAB, a byproduct of bivalent antibodies that induces aberrant receptor clustering and activation (Richter et al. (2013) PLoS One 8(8):e72156. Similarly, EHD2-scTNFR2 is immunogenic in humans due to its multiple fusion partners, and toxicology studies have shown that immune responses to IgE fragments cause autoimmune reactions (e.g., Weeratna (2016) Immun. Inflamm. Dis. 4(2):135-147). Therefore, there is a need for improved TNFR1 antagonists and improved TNFR2 agonists that overcome these limitations. Ru.

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 that have immunosuppressive properties through the production of cytokines. These include transforming growth factor beta, interleukin 35, and interleukin 10. Induction of Treg function may suppress several disease states. Induction may enhance transplant success, suppress allergies, and control responses to infections and autoimmunity such as severe acute respiratory syndrome. Tregs suppress and/or downregulate the induction and proliferation of effector T cells (Teffs), regulate the immune system, maintain immune homeostasis and tolerance to self-antigens, and prevent the development of autoimmune diseases and tissue destruction. can be prevented. Tregs express CD4, CTLA-4, CD25 (also known as IL-2 receptor alpha chain or IL2RA), and FOXP3 (transcription factor forkhead box P3), among other markers, and express TNFR1. also express TNFR2 at a 10-fold higher density. TNFR2 is expressed only by a subpopulation of Tregs, a maximally suppressed subset; this subset includes TNFR2-expressing CD4+FoxP3+ Tregs. TNF promotes Treg cell proliferation, upregulation of FoxP3 expression, and Treg cell suppressive activity/function through TNFR2 signaling. The autoimmune microenvironment contains more autoreactive CD8+ effector T cells than immunosuppressive CD4+ Tregs, leading to tissue destruction. As a result, immune balance is restored through preserved or enhanced TNFR2 function to expand Tregs and eliminate autoreactive T cells (see Sharma et al. (2018) Front Immunol. 9:883 ). For these reasons, and others discussed below, pharmacological preservation of Treg function by selective inhibition of TNFR1, along with TNFR2 stimulation (agonism), is likely to be useful in many acute and chronic inflammatory conditions (severe acute respiratory syndromes, autoimmune diseases).

In addition to upregulating the expression of TNFR2 on Tregs, TNF also upregulates the Treg surface expression of other costimulatory members of the TNF receptor superfamily (TNFRSF), such as 4-1BB and OX40; resulting in optimal activation and proliferation of Tregs and attenuation of excessive inflammatory responses. Neutralization of TNF (blocking TNFR2) blocks in vivo proliferation of Tregs (eg, Hamano et al. (2011) Eur. J. Immunol. 41:2010-2020).

Compared to conventional T cells CD4+FoxP3-, CD4+FoxP3+ Tregs constitutively express TNFR2, promoting Treg cell activation, proliferation, and survival. TNF signaling through TNFR2 (ie, TNFR2 agonism) promotes Treg activation and proliferation, whereas TNFR2 antagonism results in Treg shrinkage. For example, TNFR2 agonism selectively kills autoreactive T cells and expands suppressive Tregs in humans and animal models of autoimmunity. TNFR2 signaling promotes Treg cell proliferation and suppressive activity in experimental autoimmune encephalomyelitis (EAE; an animal model of inflammatory CNS demyelinating diseases such as multiple sclerosis) and mouse models of diabetes; Human antigen-specific Treg cells are induced by tolerogenic dendritic cells. TNFR2-deficient Tregs have a reduced ability to prevent experimental colitis in vivo, and TNFR2 is required for sustained expression of FoxP3 in Tregs, resulting in phenotypic and functional stability of Tregs. TNFR2 has been shown to be required for the in vivo immunosuppressive function of Tregs (e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728 -2738; Faustman and Davis (2013) Front. Immunol. 4:478; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Vanamee et al. (2017) Trends in Molecular Medicine 23(11 ):P1037-P1046; see Chen et al. (2013) J. Immunol. 190(3):1076-1084). In one study, antigen-specific Tregs produced in vitro were used in an established antigen-induced arthritis (AIA) model in which mice were immunized with methylated bovine serum albumin (mBSA) to induce T cell-mediated tissue damage. has been shown to inhibit disease and reduce joint inflammation and bone destruction (see, e.g., Wright et al. (2009) Proc. Natl. Acad. Sci. USA 106(45):19078-19083). thing). Although the use of Tregs in cell therapy is promising, due to manufacturing and other complications, conventional biological treatments that provide the benefits of Tregs without complications are needed.

As described and provided herein, TNFR2, and its expression by Tregs, is required for the suppression of inflammatory and autoimmune diseases and conditions. For example, Mycobacterium bovis Bacillus Calmetter-Guerin (BCG) induces transient proliferation of Tregs. In clinical trials, BCG has been shown to induce Treg production in type I diabetic patients, resulting in disease suppression and temporary recovery of islet cell function, and as a modulator to enhance Treg and/or Treg function in the treatment of type I diabetes. (see, e.g., Spence et al. (2016) Curr Diab Rep 16(11):110. doi: 10.1007/s11892-016-0807-6).

It is described and established herein that modulation of Treg function provides a therapeutic approach for the prevention or treatment of inflammatory and autoimmune diseases and conditions. However, Tregs constitute only about 1-5% of all CD4+ T cells in the blood. Their low number precludes clinical use. Ex-vivo production of Tregs and/or stimulation of their production in-vivo are factors that limit their therapeutic use. For example, in vivo stimulation with IL-2, anti-CD3, or anti-CD28 is too toxic, whereas ex vivo stimulation with these agents releases proinflammatory cytokines and may have heterogeneous and antagonizing properties. Generate a unique CD4+ population. Another approach has used the naturally occurring TNF receptor superfamily agonist, TL1A-Ig, or a TNFR2 monoclonal antibody agonist to expand Tregs in vivo and ex vivo, respectively. The TNFR2 agonist constructs and multispecific constructs provided herein can preserve and/or expand Treg populations in vivo without interfering with the therapeutic activity of anti-TNFR1 activity. As described and provided herein, selectively inhibiting inflammatory TNFR1 activity while maintaining or increasing TNFR2-associated Treg suppressive activity is useful in the treatment of inflammatory and autoimmune diseases and conditions. Beneficial. These diseases and conditions include, but are not limited to, RA, type I diabetes, heart failure, and multiple sclerosis (e.g., Goodall et al. (2015) PLoS ONE 10( 9):e0137065).

In the tumor microenvironment (TME), tumors are infiltrated by large numbers of immunosuppressive TNFR2+ Tregs, also known as effector T cells (Teffs), in contrast to the autoimmune microenvironment where proliferation of TNFR2+ Tregs prevents tissue destruction. Prevents the proliferation of tumor-killing CD8+ cytotoxic T lymphocytes (CTLs), allowing tumor growth. The antagonizing action of TNFR2 on lymphocytes in the TME inhibits or eliminates Tregs and allows activation and proliferation of effector T cells, a condition in which tumor growth can be controlled or reversed. Restores the balance between types of T cells. To be useful as a therapeutic agent, a TNFR2 inhibitor must not have the ability to aggregate immune cells via ADCC for two reasons: 1) aggregation is temporary; 2) ultimately leads to systemic depletion of Tregs, which maintains basal levels of Treg activity to maintain immune homeostasis; Because it is essential to do so, it is harmful to the patient. Tumor cells and myeloid-derived suppressor cells (MDSCs) also express TNFR2, and inhibition of TNFR2 in MDSCs controls metastasis as shown in a mouse liver cancer model. Therefore, blockade of TNFR2, such as through the use of non-aggregating antagonistic antibodies or other therapeutic agents provided herein, provides a useful treatment for certain types of cancer through inhibition of immunosuppressive Tregs. However, TNFR2 antagonists should only be administered to patients whose tumors exhibit overexpression of TNFR2 compared to adjacent normal tissue, as judged by immunohistochemistry. Therefore, such treatments must be accompanied by diagnostics to confirm overexpression (e.g., see Zhang et al. (2019) Thorac Cancer 10(3):437-444 for an exemplary assay. doi:10.1111/1759-7714.12948; Yang et al. (2017) Oncol Lett.14(2):2393-2398. doi:10.3892/ol.2017.6410; and Yang et al. (2018) Oncol Lett. 16(3) :2971-2978. doi:10.3892/ol.2018.8998).

4. Autoimmune/Inflammatory Diseases Mediated or Involved by TNF Elevated levels or uncontrolled expression of TNF and deregulation of TNF signaling can lead to chronic inflammation, resulting in autoimmune disease. Disease and tissue damage may occur. TNF-α is implicated in many diseases, disorders, and conditions. The constructs provided herein can be used to treat such diseases, disorders, and conditions. The following discussion describes some exemplary diseases, disorders, and conditions in which blockade of TNF may have therapeutic effect. TNF blockers such as etanercept, infliximab, adalimumab, certolizumab, and golimumab have adverse side effects that can limit their use in the treatment of such diseases, disorders, and conditions. Constructs provided herein that avoid some or all of these side effects can be used to treat these diseases, disorders, and conditions (e.g., the role of TNF in diseases and their treatment). For a review of the use of TNF blockers for the treatment of cancer, see Lis et al. (2014) Arch Med Sci. 10(6):1175-1185).

Inflammatory diseases include a range of disorders and conditions 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 mount a reaction against the body's own (or "self") cells; autoimmune diseases occur when the immune system attacks healthy, uninvaded self, cells, and tissues. do. 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)). including ulcerative colitis and ulcerative colitis), uveitis, fibrotic diseases, endometriosis, lupus, ankylosing spondylitis, psoriasis, multiple sclerosis (MS), Parkinson's disease, and Alzheimer's disease. Exemplary autoimmune and inflammatory diseases and disorders that can be treated with the constructs provided herein are discussed below.

a. Rheumatoid Arthritis and Other Types of Arthritis Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. The inflammation associated with rheumatoid arthritis affects the lining of the joints (ie, the synovial lining) and may also affect and inflame the membranes lining blood vessels and the heart. RA is characterized by infiltration of immune cells (eg, activated B cells) into the synovium and proliferation of synovial cells (thickening of the synovial lining). The growth mass, known as pannus, invades and destroys cartilage and bone, irreversibly destroying joint structure and function. This is mediated by the induction of inflammatory cytokines such as TNF, IL-1, IL-6. Tumor necrosis factor alpha (TNFα) is an important modulator of the induction and perpetuation of pro-inflammatory activities associated with RA. TNF is overexpressed in the synovial fluid and synovium, and TNFR expression is upregulated in the synovium (e.g. Blueml et al. (2012) International Immunology 24(5):275-281; Schmidt et al (2013) Arthritis & Rheumatism 65(9):2262-2273; see Keffer et al. (1991) EMBO J. 10(13):4025-4031). Other types of arthritis that can be treated with the constructs herein include, for example, psoriatic arthritis, juvenile idiopathic arthritis, and spondyloarthritis.

b. Inflammatory Bowel Disease (IBD) and Uveitis Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis, which are inflammatory diseases of the intestine and colon. Mice that overexpress TNF develop intestinal inflammation similar to Crohn's disease, whereas TNFR1 deficiency protects against Crohn's disease. (See, e.g., Fischer et al. (2015) Antibodies 4:48-70).

Uveitis is a type of eye inflammation that affects the wall of the eye (uvea), the middle layer of the eye between the retina and the sclera (white of the eye), and can lead to blindness. TNF-α is involved in its pathophysiology, and TNF blockers are used for treatment.

c. Fibrotic Diseases The constructs herein can be used to treat fibrotic diseases. Dupuytren's disease is typical of such diseases. Dupuytren's disease (DD) is a common fibrotic disease of the hands characterized by irreversible flexion contractures of the fingers; the condition is confined to the palms and causes irreversible curling of the fingers, impairing hand function. significantly impairs There is no approved treatment for early-stage disease, which manifests as nodules that remain quiescent for a while, then becomes active and progresses to chordal and flexural deformities of the fingers and loss of hand function. Treatments include surgical removal of the diseased tissue or cords (fasciotomy) or destruction of the cords using collagenase or needle fasciotomy. Surgical and nonsurgical treatments are associated with high recurrence and complication rates. Therapeutic intervention at early stages of the disease is advantageous to prevent progression to cord development and subsequent digital flexion contracture (see, e.g., Nanchahal et al. (2018) EBioMedicine 33:282-288 ).

Myofibroblasts express the contractile protein α-smooth muscle actin (α-SMA), aggregate into nodules, deposit excessive collagen extracellular matrix, and undergo their remodeling and Responsible for contraction. TNF converts palmar fibroblasts of DD patients into myofibroblasts through the Wnt signaling pathway, and DD myofibroblasts exhibit a dose-dependent decrease in contractility as well as α after treatment with anti-TNF therapy. - Shows decreased expression of SMA and procollagen. Treatment with the fully humanized IgG mAbs adalimumab and golimumab was most effective. However, the use of anti-TNF therapies such as adalimumab is associated with an increased risk of infection, and in a phase 2a trial evaluating the therapeutic efficacy of adalimumab in DD, only 1 patient (out of 21 who received adalimumab) ) developed a wound infection requiring hospitalization (see, e.g., Nanchahal et al. (2018) EBioMedicine 33:282-288). Therefore, other treatments 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 autoinflammatory disease and is caused by mutations in the TNFRSF1A gene, which encodes TNFR1. TRAPS is characterized by periodic, long-lasting unprovoked fever, systemic inflammation, abdominal pain, skin lesions, conjunctivitis, myalgia, and pericarditis, with inflammatory attacks lasting up to several weeks. A complication associated with the more severe clinical phenotype of TRAPS is type AA serum amyloidosis, which can lead to renal dysfunction and failure. Although disease onset usually occurs in early childhood, TRAPS can also occur in adults. The majority of TRAPS-associated mutations occur in the extracellular domain of TNFR1, which is involved in ligand binding. Highly penetrant mutations associated with the most severe clinical phenotypes occur in the extracellular cysteine-rich domain (CRD). Mutations can affect the folding and secondary structure of TNFR1, leading to defects in TNFR1 trafficking, altered ligand binding affinity, decreased activation-induced shedding, and impaired cell signaling. For example, ligand-independent gain-of-function of TNFR1 induces TRAPS pathophysiology, and certain mutations result in constitutive activity of TNFR1, NF-κB, and caspase-1. Conventional anti-TNF therapies, including etanercept, infliximab, etc., are only partially effective in treating TRAPS (see, e.g., Greco et al. (2015) Arthritis Research & Therapy 17:93), and others. treatment is needed.

e. Other diseases mediated by or involving TNFi. Neurodegenerative Diseases Aging and several neurodegenerative diseases are associated with elevated TNF levels in the central nervous system (CNS). TNF is involved in the initiation and maintenance of neuroinflammation and the regulation of other neurological processes such as synaptic function and plasticity. The level of TNFR1 in the hippocampus of old rats is approximately three times higher compared to the level of TNFR2. In animal models of disease, TNF, through its effects on TNFR1, is involved in chronic glial cell activation and impaired neuronal cell viability. In old animals, neurological changes include synaptic dysfunction and Ca2+ dysregulation, which can be reproduced in healthy young animals and in neuronal cultures using artificial elevation of TNF. TNF also enhances the activity of L-type voltage-sensitive Ca2+ channels (L-VSCC); similar effects are observed in hippocampal neurons of old rats with memory impairment. Studies in rats have shown that TNF blockade in the cerebellum accelerates learning in an eyeblink delay task. Selective blockade of TNFR1 signaling using XPro1595, a soluble dominant-negative TNF (DN-TNF) that preferentially inhibits TNFR1 signaling, improved behavioral performance in the Morris swimming task and suppressed microglial activation. This resulted in a reduction, prevention of hippocampal long-term depression (LTD), and a reduction in the activity of L-VSCC of CA1 neurons. These results indicate that TNF signaling through TNFR1 is involved in altering the neurological phenotype of aged animals and can cause pathological changes associated with neurological diseases (e.g. (See Sama et al. (2012) PLoS ONE 7(5):e38170).

a) Alzheimer's Disease TNF plays a central role in the inflammatory response; TNF protein levels are low in the healthy brain and 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, plaque and tangle accumulation, 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 are elevated in the entorhinal cortex, coinciding with the early appearance of pathology. (e.g., McCoy et al. (2006) J. Neurosci. 26(37):9365-9375). TNF-mediated processes are involved in the pathogenesis of AD, contributing to cognitive dysfunction and accelerated progression of AD. The bacterial endotoxin lipopolysaccharide (LPS), which induces inflammation and TNF production, accelerates the appearance and severity of AD pathology in several animal models of AD. Chronic activation of microglia, which are often in close physical association with amyloid plaques in AD brains, results in overproduction of pro-inflammatory mediators, including TNF, in the brain. Elevated TNF levels inhibit the phagocytosis of amyloid beta (Aβ) in the brains of AD patients and prevent efficient plaque clearance by microglia. Chronic inhibition of solTNF by administering DN-TNF, such as XENP345, or a lentivirus encoding DN-TNF inhibits AD-like symptoms induced by chronic systemic inflammation in an animal model of AD (3xTgAD mice). It prevented accelerated pathology and reduced LPS-induced intraneuronal accumulation of 6E10 immunoreactive proteins, particularly C-terminal amyloid precursor protein (APP) fragment (β-CTF), in the hippocampus, cortex, and amygdala. Genetic deletion of TNFR1 in 3xTgAD mice also prevents LPS-induced accumulation of β-CTF, which is neurotoxic. Neurons with familial AD (FAD) mutations accumulate β-CTF intracellularly, suggesting involvement in the pathogenesis of AD. These results demonstrate that soluble TNF is a mediator of the effects of neuroinflammation on early preplaque pathology in 3xTgAD mice and that targeted inhibition of solTNF in the central nervous system (CNS) may lead to amyloid-related pathology, cognitive deficits, and It has been shown that the onset of progressive loss of neurons in AD can be delayed (see, eg, McAlpine et al. (2009) Neurobiol. Dis.34(1):163-177).

b) Parkinson's Disease Parkinson's disease (PD) is the second most common neurodegenerative disease in the United States, with an incidence of 5% in individuals over the age of 65. Clinical symptoms of Parkinson's disease result from selective loss of dopaminergic neurons in the ventral substantia nigra pars compacta (SNpc), resulting in decreased striatal dopamine. Cerebrospinal fluid (CSF) and postmortem brains of PD patients and animal models of PD show elevated TNF levels. A cohort of early-onset PD patients in Japan showed an increased frequency of the polymorphic allele (-1031C) in the TNF gene promoter, resulting in higher transcriptional activity and elevated TNF levels. TNFR1 is highly expressed in nigrostriatal dopaminergic neurons, increasing their vulnerability to TNF-induced neuroinflammation and dopaminergic neuron toxicity. In vivo neutralization of soluble TNF (solTNF) by a dominant-negative TNF mutein (XENP345) is neuroprotective and retrograde induced by intrastriatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA). A 50% reduction in substantia nigra degeneration and attenuation of amphetamine-induced rotational behavior in rats indicates preservation of striatal dopamine levels. Delayed administration of XENP345 in embryonic rat mesencephalic neuron/glial cell cultures exposed to lipopolysaccharide (LPS) inhibits dopaminergic neuron degeneration despite sustained microglial activation and solTNF secretion. hindered. XENP345 also attenuated 6-OHDA-induced dopaminergic neuronal toxicity in vitro. Therefore, TNF is involved in the development of Parkinson's disease, and the progressive degeneration of the nigrostriatal pathway in humans can be slowed down by using TNF-blocking therapeutics, especially in the early stages of Parkinson's disease. (see, eg, McCoy et al. (2006) J. Neurosci. 26(37):9365-9375).

c) Multiple sclerosis (MS)
CNS-specific overexpression of TNF in transgenic mice causes spontaneous demyelination, indicating a role for TNF in multiple sclerosis (MS). Polymorphisms in the gene encoding TNFR1 are associated with increased susceptibility to developing MS. TNFR1 is required for disease induction in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and TNFR2 deficiency exacerbates the disease. Mice expressing non-cleavable, membrane-bound TNF are protected from EAE, indicating that the interaction of soluble TNF with TNFR1 is relevant to disease pathology (e.g., Fischer et al. (2015) Antibodies 4:48-70).

ii. Endometriosis TNF-α is involved in the pathophysiology of endometriosis. TNF-α levels are increased in the ascites of women with endometriosis, and their levels correlate with disease severity (see, eg, Koninckx (2008) Hum Reprod. 23:2017-2023). Peritoneal fluid TNF-α is produced locally by activated peritoneal macrophages, and TNF-α induces IL-8 secretion by peritoneal mesothelial cells. Peritoneal fluid concentrations of TNF-α and IL-8 correlate with the size and number of active peritoneal lesions (Bullimore, (2003) Med Hypotheses. 60:84-88). Serum TNF-α levels are elevated, and monocytes from endometriosis patients release more TNF-α in vitro compared to control monocytes. Ascites levels of MCP-1 are increased in endometriosis patients. TNF-α, IL-8, and MCP-1 cause an inflammatory Th-1 type response in ascites of endometriosis patients. TNF-α-mediated inflammation may be responsible for the pain associated with endometriosis. Blocking TNF-α appears to reduce disease development in animal models and may also be effective in humans. Due to the harmful side effects of existing TNF blockers, treatment of endometriosis with such blockers is not recommended (see Koninckx (2008) Hum Reprod. 23:2017-2023). However, the constructs provided herein are designed to avoid deleterious effects and may be considered for treating TNF-α mediated inflammation in endometriosis.

iii. Cardiovascular Disease TNFα was the first cytokine identified in human atherosclerotic plaques; TNFα is involved in endothelial activation and adhesion molecule upregulation that occurs early in the development of atherosclerosis. are doing. TNF has also been implicated in the pathogenesis of atherosclerosis by affecting lipid metabolism and inducing vascular inflammation. Blocking TNFα by TNF-binding proteins or blocking IL-1 by IL-1 receptor antagonists partially protects apoE knockout mice from atherosclerosis. Atherogenesis is primarily a result of the production of TNFα by bone marrow cells. The plaque area of apoE-/- and TNF-/- mice on a high-fat diet is half the size of the plaque area of mice that are apoE-/-. Transplantation of bone marrow from apoE-/- and TNF-/- mice into apoE-/- mice reduced the size of atherosclerotic lesions by 83%. The size of atherosclerotic lesions was also reduced after treatment of apoE-/- mice with a recombinant soluble p55 (TNFR1) TNF blocker, demonstrating the role that TNF plays in atherosclerosis. NF-κB signaling is involved in the production of TNF-α in human atherosclerotic plaques. Peripheral blood levels of TNFα in patients with cardiovascular disease have also been correlated with the development of myocardial infarction. Cardiotoxicity is primarily due to TNF-induced apoptosis of cardiomyocytes. The use of anti-TNF therapies such as infliximab and etanercept in clinical trials for the treatment of heart failure has resulted in failure and increased mortality; therefore, anti-TNF therapies have not been tested for the treatment of cardiovascular disease (e.g., Udalova et al. (2016) Microbial Spectrum 4(4):MCHD-0022-2015; see Kalliolias and Ivashkiv (2016) Nat.Rev.Rheumatol. 12(1):49-62). As a result, alternative treatments are required.

iv. Acute respiratory distress syndrome (ARDS)
Acute respiratory distress syndrome (ARDS) affects approximately 190,000 patients annually in the United States, with a mortality rate of up to 40%. There are no effective treatments that target the pathophysiological mechanisms underlying ARDS. ARDS is characterized by immune cell-mediated lung damage associated with the release of inflammatory cytokines and proteases. Uncontrolled local inflammatory responses in ARDS cause 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 pulmonary microvascular endothelium, and the alveolar capillaries are triggered by the action of proinflammatory mediators. Increased by damage to the barrier. TNF-α has been identified as an important effector molecule in ARDS and sepsis, a common cause of ARDS. For example, TNF-α contributes to increased endothelial permeability. Clinical trials involving the administration of non-selective anti-TNF antibodies for the treatment of sepsis have shown no survival benefit, and one trial showed that high doses were harmful.

TNFR1-deficient mice are protected from lung injury, sepsis, and other acute organ damage, whereas TNFR2-deficient mice are more susceptible to injury in these models, suggesting that selective antagonism of TNFR1 may not be therapeutically effective. It shows that it is possible. GSK1995057 is a short-acting, fully human domain antibody (dAb) fragment that selectively antagonizes TNFR1 but not TNFR2, reducing disease severity in mouse models of acute respiratory distress syndrome and in nonhuman Reduced signs of inflammation and lung damage in primates. In a randomized, placebo-controlled trial of nebulized GSK1995057 in 37 healthy humans challenged with low doses of inhaled endotoxin, treatment with GSK1995057 significantly reduced pulmonary neutrophilia and inflammatory cytokines in bronchoalveolar lavage fluid and serum samples. release, and reduced signs of endothelial damage. These results demonstrate the potential for pulmonary delivery of selective TNFR1 antagonists for the prevention and treatment of ARDS (see, eg, Proudfoot et al. (2018) Thorax 73:723-730).

v. Severe Acute Respiratory Syndrome (SARS) and COVID-19
Subjects infected with Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) exhibit fever and respiratory illness, general malaise, and lower respiratory symptoms including cough and shortness of breath, with an overall case fatality rate of approximately 10%. . TNF signaling promotes the pathogenesis of SARS by inducing excessive inflammation that causes significant tissue damage. The development of cytokine release syndrome (CRS) has been implicated in severe COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sustained viral stimulation increases levels of circulating cytokines such as IL-6 and TNFα, decreases lymphocyte counts, and causes inflammatory organ damage, particularly in the lungs and blood vessels, in some subjects. SARS-CoV-2 shares some similarities with SARS-CoV, the coronavirus strain that caused the 2002 SARS pandemic. SARS-CoV and SARS-CoV-2 use the spike (S) protein to engage the cellular receptor ACE2 (angiotensin converting enzyme 2) to enter cells. ACE2 receptor expression is upregulated by SARS-CoV-2 infection and inflammatory cytokine stimulation. In SARS-CoV infection, the S protein induces TNF-α converting enzyme (TACE)-dependent shedding of the ACE2 ectodomain, a process strictly associated with TNFα production. Loss of ACE2 activity due to shedding is associated with lung damage due to increased activity of the renin-angiotensin system. ACE2 knockout mice are prone to severe respiratory failure after chemical challenge, and ACE2 has been shown to alleviate ACE-induced intracellular inflammation. Downregulation of ACE2 is associated with severe respiratory distress associated with SARS-CoV infection. Increased TNFα production can therefore promote viral infection and cause organ damage such as lung damage.

As discussed, regulatory T cells (Tregs) are a type of immunosuppressive cell that exhibit diverse clinical applications in transplantation, allergy, infectious diseases, GVHD, autoimmunity, and cancer. Tregs co-express CD4+ and interleukin-2 receptor alpha chain CD25hi and are characterized by the level of induction of the intracellular transcription 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 Treg(100) and induces proliferation, and TNFR2 expression represents maximal suppression of Treg. Thus, if TNF is overproduced in response to infection (influenza, SARS-type virus, endotoxemia), Tregs may prevent an overreaction to inflammatory stimuli.

Anti-TNF can be a treatment for SARS and COVID-19. Adalimumab has been used in the treatment of COVID-19 (clinical trial conducted in China in February 2020 (ChiCTR2000030089); for example, Lucchino et al. (2020) Rheumatology (Oxford) 59(6):1200- 1203; Haga et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105:7809-7814). Knockout of TNFR2 in mice infected with SARS-CoV provides no protective effect; double knockout of TNFR1 and TNFR2 protected infected mice from infection-associated weight loss (e.g., McDermott et al. (2016) BMC (See Systems Biology 10:93). These results demonstrate that TNF signaling through TNFR1 primarily contributes to the pathogenesis of SARS-CoV infection by increasing pro-inflammatory processes and that selective inhibition of TNFR1 over inhibition of TNF This suggests a better therapeutic approach. The constructs provided herein can be used to treat acute inflammatory aspects of SARS and COVID-19. The construct is used in combination with an anti-infective agent; the construct is used to suppress or ameliorate the acute effects of a cytokine storm.

TNFα inhibition reduces the severity of virus-induced lung diseases, such as those caused by respiratory syncytial virus (RSV) or influenza virus in mice. Depletion of TNF using anti-TNF antibodies in these mouse models reduces lung recruitment of inflammatory cells and inhibits pro-inflammatory cytokine (e.g., IFNγ, IL-4, IL-5, TNF) production by T cells. production was reduced, reducing disease severity without interfering with viral clearance (see, eg, Hussell et al. (2001) Eur. J. Immunol. 31:2566-2573). These results demonstrate that TNF inhibitors and TNF receptor antagonists can prevent or attenuate TNF-induced immune activation and lung damage in human viruses such as those caused by SARS-CoV and SARS-CoV2. It has been shown that it may be beneficial in the treatment of sexual lung disease.

Allogeneic hematopoietic stem cell transplantation is complicated by the development of noninfectious idiopathic pneumonia syndrome (IPS), an acute lung dysfunction that resembles SARS pneumonia. Elevated TNFα levels have been found in the serum of patients who developed lung injury after allogeneic stem cell transplantation (SCT), indicating that donor-derived alloreactive T cells are involved in 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 subject to the immunodestructive effects of SCT conditioning regimens, the requirement for long-term use of immunosuppressive drugs to prevent or treat graft-versus-host disease (GvHD), and others that may impair host defenses. SCT complications (including acute GvHD) result in an increased risk of developing bacterial and fungal infections (see, e.g., Yanik et al. (2002) Biol. Blood Marrow Transplant. 8:395-400 ). Other indications that may be treated by the constructs provided herein include chemobrain, a condition experienced during and after chemotherapy, particularly women undergoing treatment for breast cancer. The treatments and constructs herein can also be used to treat long COVID.

As a result, the use of selective TNFR1 antagonists that retain protective TNF signaling through TNFR2 and, unlike anti-TNF therapy, do not increase the risk of serious infections may be of benefit to both viral and non-viral-induced lung infections. Providing safer and more effective treatment options for treating, preventing, or ameliorating injuries. The constructs provided herein are therefore ideal therapeutics for these indications.

D. Treatment of Rheumatoid Arthritis and Other Chronic Inflammatory and Autoimmune Diseases and Disorders Although there is no cure for rheumatoid arthritis (RA), for example, it can minimize pain and swelling, prevent bone deformity, and improve daily functioning. By maintaining the condition, treatment can improve symptoms and slow disease progression. The main treatment for RA is disease-modifying antirheumatic drugs (DMARDs), which are also used to treat other chronic inflammatory and autoimmune diseases and disorders, such as psoriasis, plaque psoriasis, psoriatic arthritis, and For the treatment of idiopathic arthritis, ankylosing spondylitis, Behcet's disease, inflammatory bowel disease (IBD; e.g., Crohn's disease and ulcerative colitis), multiple sclerosis, and lupus, and for the treatment of some cancers. used.

DMARDs are immunosuppressive and immunomodulatory agents and are classified as either conventional synthetic DMARDs (csDMARDs) or biological DMARDs (bDMARDs; eg, antibodies and fusion proteins). Conventional synthetic DMARDs include, for example, methotrexate (MTX), chemotherapeutic and immunosuppressive agents; hydroxychloroquine (HCQ; Plaquenil®), antimalarials; sulfasalazine (Azulfidine®), anti-inflammatory drugs. ; and leflunomide (Arava®), an immunosuppressant that inhibits dihydroorotate dehydrogenase. Biological DMARDs include, for example, abatacept (Orencia®), a fusion protein that prevents T cell activation and contains the Fc region of IgG1 fused to the extracellular domain of CTLA-4; anakinra (e.g., Kineret®), a recombinant human IL-1 receptor antagonist; Rituximab (e.g. sold under the Rituxan®, Truxima®, MabThera® trademarks), a recombinant human IL-1 receptor antagonist; Chimeric monoclonal antibody against CD20 that induces apoptosis in CD20+ cells such as B cells; tocilizumab (atlizumab, Actemra®, RoActemra®), a humanized monoclonal antibody against the IL-6 receptor (IL-6R) corticosteroids; tofacitinib (Xeljanz®), a small molecule inhibitor of Janus kinase (JAK), a protein tyrosine kinase involved in mediating cytokine signaling; and TNF inhibitors/anti-TNF agents, e.g. Certolizumab pegol (Cimzia®), infliximab (Remicade®), adalimumab (Humira®), golimumab (Simponi®), and etanercept (Enbrel®), etc. can be mentioned. In particular, combination therapy of methotrexate and biologic DMARDs is more effective than either monotherapy. Combination therapy can include multiple csDMARDs and multiple csDMARDs and one biological DMARD. Biological DMARDs, especially anti-TNF DMARDs, are not typically used in combination therapy because of the risk of serious side effects, including serious infections.

The following sections describe existing treatments and the problems associated with each and highlight how the treatments provided herein solve the problems.

1. Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs)
Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) are typically first-line treatments for RA and other autoimmune and chronic inflammatory diseases and disorders. csDMARDS include drugs such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine; methotrexate is the drug most commonly used for initial treatment and its mechanism of action involves the release of adenosine from fibroblasts. stimulation of shedding, reduction of neutrophil adhesion, inhibition of leukotriene B4 synthesis by neutrophils, inhibition of local IL-1 production, reduction of IL-6 and IL-8 levels, suppression of cell-mediated immunity, and Includes inhibition of synovial collagenase gene expression. Other conventional synthetic DMARDs act by inhibiting lymphocyte proliferation or causing lymphocyte dysfunction. For example, leflunomide inhibits dihydroorotate dehydrogenase, inhibits pyrimidine synthesis, and blocks lymphocyte proliferation. Sulfasalazine mediates its anti-inflammatory effects by preventing oxidative, nitrating and nitrosative damage, and hydroxychloroquine is a mild immunomodulatory agent that inhibits intracellular toll-like receptor 9 (TLR9). .

Hydroxychloroquine, which has the safest profile of traditional DMARDs, does not increase the risk of infection and does not cause liver toxicity or kidney dysfunction; common side effects of hydroxychloroquine include rash and diarrhea. . Retinopathy/maculopathy is a rare but serious side effect of hydroxychloroquine therapy and is associated with doses greater than 5 mg/kg/day, long-term use (>5 years of treatment), older age, and chronic kidney disease. . Other rare side effects of hydroxychloroquine include anemia, leukopenia, myopathy, and cardiomyopathy. Treatment with methotrexate, leflunomide, and sulfasalazine is associated with increased incidence of nausea, abdominal pain, diarrhea, rash/allergic reactions, bone marrow suppression, hepatotoxicity, and common, sometimes serious infections. Methotrexate and leflunomide also cause alopecia. Other side effects associated with methotrexate therapy include interstitial lung disease, folate deficiency, and liver cirrhosis. Leflunomide has also been associated with hypertension, peripheral neuropathy, and weight loss. Sulfasalazine has a very high risk of gastrointestinal disorders and can rarely cause DRESS syndrome (drug reaction with eosinophilia and systemic symptoms) (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. Source: see URL: ncbi.nlm.nih.gov/books/NBK507863/). These drugs are effective because they have immunosuppressive effects. Constructs provided herein that are selective anti-TNFR1 antagonists that advantageously maintain TNFR2 immunosuppressive activity may obviate the need for these immunosuppressive drugs.

2. Anti-TNF therapy/TNF blockers Anti-TNF therapy/TNF blockers (a type of biological DMARD) are typically prescribed after failure of conventional DMARDs, and include monoclonal antibodies (mAbs), such as the chimeric mAb infliximab ( (containing mouse variable regions and human IgG1 constant regions); and the fully humanized mAbs (IgG1) adalimumab (Humira®) and golimumab (Simponi®); TNF A pegylated humanized Fab' fragment of a mAb targeting certolizumab pegol (Cimzia®); and an extracellular receptor region containing a binding site for human TNFR2 fused to the Fc of human IgG1 TNFR2 fusion proteins, including the TNFR2-Fc fusion protein etanercept (Enbrel®). 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 that sequester TNF are used to treat, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (JIA) and/or inflammatory bowel disease (IBD; e.g., Crohn's disease and ulcers). It is used to treat a variety of diseases and conditions, including genital colitis).

Due to the immunosuppressive effects of TNF-targeted treatments, such treatments may be effective against, for example, listeriosis, tuberculosis reactivation, hepatitis B/C reactivation, herpes zoster reactivation, as well as invasive It is associated with severe side effects, including an increased risk of sepsis and serious infections such as fungal and other opportunistic infections. TNF is an important cytokine in the inflammatory and immune response to infection, and the use of drugs that remove TNF impairs host immunity to microorganisms and increases the risk of infection. For example, TNF blockers include M. associated with reactivation of S. tuberculosis infection. TNF plays an important role in resistance to Mycobacterium tuberculosis, and adalimumab therapy in RA patients has been shown to be effective against M. tuberculosis. significantly reduces reactivity to C. tuberculosis. As described herein, decreased immune reactivity may be associated with activation of Tregs and induction of apoptosis in effector lymphocytes. Anti-TNF therapy has been shown to induce macrophage apoptosis in rheumatoid synovium. Infliximab is associated with increased apoptosis in inflammatory cell infiltrates in the intestines of patients with Crohn's disease. Other antirheumatic drugs such as methotrexate and glucocorticoids can also induce apoptosis of immune cells (see, e.g., Vigna-Perez et al. (2005) Clin.Exp. Immunol. 141(2):372-380). thing). Adalimumab and infliximab, but not the TNFR2-Fc fusion protein etanercept, induce caspase-dependent apoptosis in cultured monocytes and downregulate the production of IL-10 and IL-12 by monocytes (e.g., Shen et al. (2005) Ailment Pharmacol. Ther. 21:251-258). The most common fungal infections associated with TNF blockers are histoplasmosis, candidiasis, and aspergillosis. Anti-TNF agents can also cause severe congestive heart failure, drug-induced lupus, and demyelinating central nervous system (CNS) disease, as well as worsening of lymphoma and non-melanoma skin cancers (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. Source: See ncbi.nlm.nih.gov/books/NBK507863/ thing).

Infliximab has also been associated with the development of leukopenia, neutropenia, thrombocytopenia, and pancytopenia (sometimes fatal). Etanercept is associated with increased incidence of opportunistic bacterial and viral infections in RA patients. Etanercept is also used to treat severe and refractory graft-versus-host disease (GvHD). Severe GvHD subjects treated with etanercept are at very high risk of developing invasive aspergillosis (IA), a life-threatening mold (i.e., fungal) infection caused by Aspergillus fumigatus (one study %, see Zoran et al. (2019) Sci. Rep. 9:17231). Treatment with etanercept results in downregulation of genes involved in immune response and TNF signaling. This includes genes involved in NF-κB signaling, antimicrobial humoral responses and apoptotic processes, as well as decreased secretion of chemokines such as CXCL10 from immune cells (e.g., Zoran et al. (2019) Sci. Rep 9:17231).

Other side effects associated with the use of TNF inhibitor therapy include increased susceptibility to the development of congestive heart failure, liver damage, demyelinating diseases/CNS disorders, lupus, psoriasis, sarcoidosis, and other autoimmune diseases; Cancers including lymphoma and solid malignancies (e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43):12304-12309; Zalevsky et al. (2007) J. Immunol. 179:1872-1883; see Zoran et al. (2019) Sci. Rep. 9:17231). Therefore, abrogating all TNF-mediated signaling by sequestering TNF is not an ideal therapeutic strategy. This is because it results in severe immunosuppression that can lead to serious, sometimes fatal infections and other dangerous side effects.

Anti-TNF therapy improves RA, but is not curative and requires years of ongoing and costly treatment. 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 is limited, especially in chronic diseases/conditions that require long-term administration, such as arthritis and IBD. Approximately 30% of RA patients do not respond or do not have a sustained response to anti-TNF therapy (see, e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738). ). Nonresponsiveness also occurs in non-RA patients receiving anti-TNF therapy. Depending on the anti-TNF agent, 13-33% of treated patients do not respond to treatment, and up to 46% stop responding, leading to discontinuation or dose escalation (e.g. Richter et al. (2019) MABS 11 (4):653-665). Therefore, there is a need for treatments with improved therapeutic efficacy and safety.

Anti-TNF therapeutics block/sequester TNF and inhibit soluble TNF (solTNF) and transmembrane TNF (tmTNF) signaling through TNFR1 and TNFR2, respectively; solTNF signaling is associated with chronic inflammation. On the other hand, tmTNF signaling is associated with resolution of inflammation and induction of immunity against pathogens such as Listeria monocytogenes and Mycobacterium tuberculosis. The main anti-inflammatory effect of anti-TNF therapy is achieved by blocking TNFR1, whereas blocking TNFR2 inhibits Treg cell activity. As discussed elsewhere herein, TNFR1 signaling is primarily inflammatory and is associated with inflammatory and autoimmune diseases and conditions, such as neurodegenerative disorders such as RA, psoriasis, IBD, and MS. on the other hand, TNFR2 signaling has anti-inflammatory and protective effects in various cell and organ types, such as nervous tissue, heart tissue, intestine and bone tissue, and plays a role in host defense against infection by pathogens. It is also involved in the mechanism. Therefore, as described herein, selective blockade of TNFR1, while preserving the beneficial effects of TNFR2 signaling, may reduce the undesirable effects associated with RA and other autoimmune and inflammatory diseases and conditions. Eliminating pro-inflammatory signaling improves therapeutic efficacy compared to anti-TNF therapy (e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738; Schmidt et al. See 2013) Arthritis & Rheumatism 65(9):2262-2273; Bluml et al. (2012) International Immunology 24(5):275-281; Zalevsky et al. (2007) J. Immunol. 179:1872-1883 ).

Anti-TNF therapies have failed to treat neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, stroke, and multiple sclerosis (MS), which are associated with overexpression of TNF. For example, in a phase II trial for the treatment of relapsing-remitting MS, lenercept (Ro45-2081), a TNFR1 receptor-Fc IgG1 fusion protein anti-TNF treatment, failed to improve symptoms compared to patients who received a placebo. neuropathy is increased/worsened and neuropathy is more severe in patients treated with lenercept. These results indicate that anti-TNF therapy can exacerbate demyelinating disease. TNFR1 has been shown to mediate inflammatory neurodegeneration, whereas TNFR2 induces neuroprotection, so blocking signaling through both receptors with anti-TNF therapy may inhibit TNFR2 signaling. abrogate neuroprotective effects. Blockade of TNFR1 with ATROSAB, a humanized monoclonal antibody that blocks TNFR1, or activation of TNFR2 with EHD2-scTNFR2, an agonist TNFR2-selective TNF mutein, confers protection of cholinergic neurons against cell death and NMDA-induced acute Recovering memory deficits associated with neurodegeneration in a mouse model of neurodegeneration. This is likely to be immunogenic. ATROSAB is a partial TNFR1 agonist. One of ordinary skill in the art would not administer a TNFR1 agonist. However, blockade of TNFR1 and TNFR2 abolished the therapeutic effect, demonstrating that TNFR2 plays an important role in neuroprotection and that selective blockade of TNFR1 can be used to treat neurodegenerative diseases in which anti-TNF therapy has failed. (e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. USA 113(43):12304-12309).

Due to side effects associated with the use of anti-TNF agents, nonresponse in some patients, lack of sustained response in patients who have an initial response, and treatment failure and/or worsening of neurodegenerative diseases such as MS. Therefore, other treatments are needed. Such treatment methods are provided herein.

E. Therapies that target TNFR1/TNFR2 The following sections discuss exemplary therapies that target TNFR1/TNFR2 and describe some of the issues and limitations associated with these therapies. Existing therapeutic agents may be modified as described in Section F and the Examples, or used, in whole or in part, in the constructs provided herein. modified to improve their properties.

1. TNFR1 Selective Antagonists As discussed and provided herein, treatment with TNF blockers, such as etanercept, infliximab, adalimumab, abrogates TNF signaling through TNFR1 and TNFR2. TNFR1 signaling causes inflammation, cytotoxicity, and apoptosis, whereas TNFR2 signaling contributes to the proliferation and activation of immunosuppressive Tregs that destroy effector T cells in autoimmune settings and prevent tissue destruction and disease progression. Partly due to its protective and anti-inflammatory properties. Treatment with TNF blockers through inhibition of TNFR2 signaling and the consequent depletion of immunosuppressive Tregs results in a pro-inflammatory microenvironment, failure to treat autoimmune and inflammatory diseases and disorders, and/or or may worsen. Dual blockade of TNFR1 and TNFR2 can also lead to opportunistic infections and cancer.

As provided herein, specific inhibition of TNFR1 signaling disrupts the balance between pro- and anti-inflammatory activities through the production of both regulatory and cytotoxic T cell subsets. Maintain normal TNFR2 function necessary to maintain normal TNFR2 function. Selective TNFR1 inhibition retains the potent anti-inflammatory activity of TNFR2 signaling, reduces opportunistic infections and cancer, and preserves TNF-induced Treg function.

a. TNFR1 Antagonizing Antibody Among the TNFR1 antagonist antibodies is ATROSAB (Antagonistic TNFReceptor One-Specific Antibody). The first TNFR1 blocking antibody, ATROSAB, is a full-length IgG1 humanized version of the neutralizing mouse anti-human TNFR1 monoclonal antibody H398. It was abandoned as a therapeutic agent because it has partial agonist activity in activating TNFR1, thereby mimicking the toxic pathway TNF activity. ATROSAB maintains the conformation of TNFR1 in an inactive state and prevents TNF binding. The Fc region of ATROSAB has been mutated to eliminate FCγR receptor binding and complement fixation, thereby avoiding unwanted immune system activation (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 are susceptible to receptor cross-linking, which tends to agonize TNFR1 instead of antagonizing it, as discussed elsewhere herein. Therefore, it is not suitable for the development of TNFR1 antagonists. This was not due to Fc cross-linking since the Fc-interacting portion of the antibody was removed by mutation. For example, IgG ATROSAB exhibited some TNFR1 agonist activity in the absence of TNF, which was observed to a limited extent over a narrow concentration range due to its bivalent molecular structure. Cross-linking of TNFR1 can occur due to secondary events such as interaction with FcγRs or anti-drug antibodies (ADA) and needs to be avoided to maintain antagonism of TNFR1 inhibitors. be. ADAs have been observed at a rate of 50% and 31% in patients treated with infliximab or adalimumab, respectively (e.g. Richter et al. (2019) mAbs 11(4):653-665; Richter et al., (2019)) mAb 11(1):166-177).

b. Monovalent TNFR1 Antagonizing Antibodies/Antibody Fragments Small antibody fragments have been developed, such as domain antibodies and derivatives and modified forms thereof, and exemplary antibody fragments and modified forms are discussed in the following sections. However, small antibody fragments have not been successfully developed into pharmaceuticals. Their use as therapeutic agents is limited; due to their small size, they have a short serum half-life and rapid peripheral clearance. For example, molecules less than 50-60 kDa in size undergo renal filtration; dAbs and other antibody fragments less than 50-60 kDa in size are rapidly cleared by the kidneys. For example, the dAb designated DMS5541 and similar molecules exhibit selectivity for TNFR1 and can potentially inhibit the deleterious effects of TNFR1 signaling. DMS5541, formed from two dAbs (anti-TNFR1 and anti-human serum albumin), is only about 25 kDa in size, too small to have desirable pharmacokinetics for therapeutic purposes. The association with HSA to stabilize the half-life is only 34 nM, meaning that it is often in a dissociated state with respect to HSA. The single domain antibodies (sdAbs) tested to date are E. It is expressed in E. coli and is prone to aggregation (unfolding) during manufacturing. Furthermore, sdAbs prepared in the cytoplasm (from direct expression in E. coli) often contain conserved molecules found in the variable heavy chain domain, both of which may lower the melting point and reduce refolding ability. Lacks disulfide bonds. The rapid clearance and short elimination half-life (less than a few hours) of small antibody fragments can reduce in vivo efficacy, require frequent dosing and/or continuous infusion, and reduce patient compliance. These molecules (see, eg, Holland et al. (2013) J Clin Immunol 33(7):1192-203) are derived from E. coli and are often misfolded, resulting in reduced solubility and immunogenicity, resulting in clinical failure (see, e.g., adisinsight.springer.com/drugs/800037882) .

Constructs such as the TNFR1 antagonist constructs provided herein address this issue as well as other issues such as immunogenicity and reaction with pre-existing antibodies. Provided herein are constructs comprising small antibody fragments, such as dAbs, with specificity for TNFR1 and/or TNFR2, which have a longer serum half-life, stability compared to prior art dAbs. The construct exhibits improved pharmacology, such as increased drug efficacy, reduced/delayed peripheral clearance, and drug irradiance, including reduced immunogenicity.

Therapeutic antibodies of various structures have the potential to become potent and well-tolerated therapeutic agents. The antibodies may be used in a variety of diseases and conditions, including, for example, rheumatoid arthritis (e.g., adalimumab sold under the trademark Humira®); cancer, e.g., non-Hodgkin's lymphoma (e.g., rituximab and ibritumomab tiuxetan, respectively); Rituxan® and Zevalin®), and breast and gastric cancers (e.g., trastuzumab, sold under the Herceptin® trademark); and respiratory syncytial virus infections (e.g., Palivizumab (sold under the trademark Synagis®) is used for treatment. Production of intact antibodies has several limitations, including dependence on mammalian cell expression. As a result, antigen-binding fragments of antibodies, such as Fabs (~57 kDa) and single chain Fv fragments (scFv, ~27 kDa), and can be selected in vitro, such as by phage display (evading animal immunity), and bacterial or yeast Other structures have been developed that can be manufactured in large quantities using cell cultures. Fab fragments contain a VH-CH1 polypeptide linked to a VL-CL polypeptide via a disulfide bond; scFvs are fusion proteins that contain a VH domain and a VL domain connected 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 the heavy chain (VH) of the antibody. or a light chain (VL) variable domain. dAbs are the smallest antigen-binding fragments of antibodies; their size is approximately 11-15 kDa, approximately one-tenth the size of a complete monoclonal antibody (mAb) (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 lack light chains. Nanobodies are small (approximately 15 kDa), have low immunogenicity, high affinity, are soluble and stable, and are encoded by a single gene/exon (VHH), making them modular and capable of being used in bacteria. and can be produced in high yields using yeast. (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, ATROSAB, a humanized semi-agonist/antagonist TNFR1-specific antibody, inhibits TNFR1-mediated cellular responses. ATROSAB exhibits some degree of TNFR1 agonist activity in the absence of TNF, possibly due to its bivalent molecular structure or binding to TNFR1. The parent mouse antibody H398 has a stronger inhibitory ability due to faster dissociation of ATROSAB (incidentally, higher koff value) than H398. This was determined using quartz crystal microbalance (QCM) measurements, where the antigen density on the chip is reduced in favor of monovalent interactions; slower dissociation of monovalent bound H398 from TNFR1; and the resulting longer receptor occupancy contributes to improved blockade of TNFR1. Therefore, monovalent derivatives of ATROSAB were developed to eliminate ATROSAB's TNFR1 agonist activity and improve its TNFR1 antagonist activity.

To increase the affinity and antagonistic activity of ATROSAB, single chain variable fragments (scFv) of ATROSAB were subjected to initial affinity maturation by site-directed mutagenesis of exposed residues within individual CDRs or combinations of CDRs. , and subjected to phage display selection against human TNFR1-Fc. The ATROSAB scFv consists of an ATROSAB heavy chain (see SEQ ID NO: 31) connected by a short peptide linker to the VL domain, corresponding to residues 1-113 of the ATROSAB light chain (see SEQ ID NO: 32). ) contains the VH domain corresponding to residues 1 to 115 of . The clone, scFv IG11 (see SEQ ID NO: 674), has six mutations in CDR-H2 of ATROSAB heavy chain with respect to SEQ ID NO: 31, Y52V, Y54T, S55Q, H57E, Y59K, and E62D; It exhibited slower receptor dissociation, improved equilibrium binding to human TNFR1-Fc, and improved inhibition of TNF-induced TNFR1 activation. This clone was further subjected to random mutagenesis to generate mutations Q1H, Y52V, Y54S, S55Q, H57E, Y59K, and E62D (see SEQ ID NO: 31) in the VH domain, and S96G (SEQ ID NO: 31) in the VL domain. A clone scFv T12B (see SEQ ID NO: 675) was generated containing the clone scFv T12B (see SEQ ID NO: 675). scFv T12B showed decreased dissociation from immobilized TNFR1-Fc and increased TNFR1 inhibitory activity compared to ATROSAB scFv and scFv IG11 (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; cited from pdfs.semanticscholar.org/d8e7/8b87d76dce36225c1d497939ef37445cfa8a. pdf (see also).

Humanization of H398 was redesigned by replacing the VH and VL framework regions of H398 with alternative germline genes to optimize CDR placement. scFv 13.7, which contains the VH domain of scFV T12B linked by a short peptide linker to the newly humanized VL domain of H398, has similar binding to human TNFR1-Fc in ELISA and QCM and TNF induction Improved inhibition of TNFR1 activity and improved thermal stability with a 10°C higher melting temperature compared to scFv T12B. Based on scFv 13.7, we generated IgG and Fab with constant regions identical to ATROSAB (IgG 13.7 and Fab 13.7, respectively), ATROSAB (1.4x), and Fab of ATROSAB, respectively. (Fab ATR; 8.7 times) increased binding to TNFR1. Fab 13.7 also had reduced dissociation from immobilized TNFR1-Fc compared to Fab ATR, exhibiting an 18.8-fold improved monovalent affinity. Therefore, affinity maturation and framework replacement improved the binding of Fab 13.7 to TNFR1. Fab 13.7 and IgG 13.7 showed selectivity for TNFR1-Fc and did not bind to TNFR2-Fc fusion protein; Fab 13.7 bound to human and rhesus TNFR1-Fc but , did not bind to mouse or rat TNFR1-Fc, showing a binding pattern similar to ATROSAB. In vitro, monovalent Fabs ATR and Fab 13.7 did not activate TNFR1, whereas ATROSAB showed slight activation of TNFR1 activity and IgG 13.7 strongly activated TNFR1. The agonist activity of IgG 13.7 may be due to increased affinity and slower dissociation from TNFR1, resulting in the formation of stable signaling competent receptor-antibody complexes. Fab 13.7 showed improved inhibition of TNFR1 activity and lacked agonist activity compared to Fabs ATR and ATROSAB. Incubation of Fab 13.7 or ATROSAB with cross-linked anti-human Fab serum revealed that Fab 13.7 does not activate TNFR-1, whereas ATROSAB does (e.g., Richter et al. (2019) mAbs 11(1):166-177).

Fab 13.7 (molecular weight approximately 47 kDa) 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 μg/ml×h. Fab 13.7 exhibited an initial half-life of 0.08 hours, a terminal half-life of 1.4 hours, and an AUC of 4.2 μg/ml×hour, similar to the values obtained with Fab ATR. To extend the half-life, we generated the Fab' fragment Fab'13.7 by introducing a free cysteine residue at the C-terminus of the CH1 domain, which was chemically conjugated to a branched PEG40 kDa moiety to create Fab' 13.7 PEG was produced. Fab 13.7 was also fused to the N-terminus of mouse serum albumin (MSA) via its Fd and a short flexible linker to generate Fab 13.7-MSA. Monovalent Fab-Fc fusion proteins are generated by fusing Fab 13.7 to a modified Fc that lacks cysteine residues in the hinge region and lacks the ability to dimerize through the CH3 domain, producing a one-arm, half-IgG molecule (IgG1half13.7) was produced. Monovalent Fv-Fc molecules also fuse the VH and VL domains into a heterodimeric knob-in-two-hole (kih) Fc chain (Fv13.7-Fckih) that lacks cysteine residues in the hinge region. generated by. None of the derivatives showed agonistic TNFR1 activity, and slightly reduced binding to human TNFR1-Fc was observed for Fab13.7 PEG, Fab13.7-MSA and IgG1half13.7 compared to Fab13.7; Fv13.7-Fckih binding 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 functional impairment and Fv13.7-Fckih showed the least change in biological activity. IgGhalf13.7 showed a similar half-life to Fab 13.7, and the AUC value was increased by 7.1 times. Fab13.7PEG, Fab13.7-MSA, and Fv13.7-Fckih extended the terminal half-life and increased AUC values with values of 14.4 hours, 9.7 hours, and 10.5 hours, respectively. . Therefore, the fusion protein Fv13.7-Fckih, designed for heterodimeric assembly of two peptide chains using knob-in-to-hole technology, has improved pharmacokinetic properties and TNFR1 antagonizing activity. showed the best combination (see e.g. Richter et al. (2019) mAb 11(1):166-177; also Richter, F. Thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One- Specific Antibody ATROSAB," Universitat Stuttgart, 2015; see also pdfs.semanticscholar.org/d8e7/8b87d76dce36225c1d497939ef37445cfa8a.pdf).

In another study, to improve pharmacokinetic properties such as serum half-life of Fab 13.7, an IgG-like Fc was incorporated into the molecule while retaining Fab-like heterodimerization of the polypeptide chains. To accomplish this, the heavy and light chain variable domains of Fab 13.7 were inserted into the N-terminus of a newly generated heterodimerized Fc chain, known as Fc-one/κ (Fc1κ). fused. The Fc heterodimerization approach is based on interspersed Ig domains, which are derived from the heterodimerized IgG1 heavy chain constant domain CH1 and kappa light chain constant domain CLκ, and contain sections of IgG1 CH3 sequences. , mediates FcRn binding and enables FcRn-mediated drug recycling in vivo. Interspersed Ig domains include "CH31," which includes an amino acid sequence fragment of CH1 and CH3, and "CH3κ" (CH3κ), which includes an amino acid sequence fragment of CLK and CH3. The IgG1 CH2 domain was also fused to the N-terminus of the CH31 and CH3κ domains to include the entire FcRn binding region of the IgG molecule. Addition of an IgG1 hinge region to the N-terminus of the CH2 domain results in a covalently linked, heterodimerized Fc moiety known as Fc1κ. In contrast to other Fc heterodimerization techniques, such as knob-into-hole, which involve substitution of one or more amino acids at the CH3-CH3 interface, Fc heterodimerization This was achieved by exchanging the resulting larger stretch of amino acid sequence. When asymmetric scFv-Fc1κ fusion proteins were prepared and compared to scFv fusions with Fc containing knob-into-holes, heterodimer formation was similar or improved compared to fusions containing knob-into-hole technology. (see e.g. Richter et al. (2019) mAb 11(4):653-665).

The variable domain of the TNFR1-specific Fab 13.7 molecule fuses the VH to the CH2-CH3κ chain and the VL to the CH2-CH31 chain (VL13.7-CH2-CH31/VH13.7-CH2-CH3κ; VL1C/VHκC) A monovalent TNFR1-specific antagonistic antibody-derived molecule known as atrocimab (size 72 kDa) ( Fv-Fc1κ fusion protein). Atrocimab lacks the ability to mediate Fc effector function due to mutations introduced in Fc1κ. The lack of binding to effector molecules of the immune system prevents activation of TNFR1 due to secondary cross-linking of atrocimab bound to cells expressing FcγR. Atrocimab binds to TNFR1 with high affinity (KD 2.7 nM) and has demonstrated TNF-inducing activity of TNFR1 in various in vitro assays and in the presence of anti-human IgG antibodies (i.e., cross-linking antibodies) with IC50 values of 16-55 nM. showed improved pharmacokinetic properties. Compared to the parental Fab 13.7 molecule, binding and inhibition of TNFR1 was slightly reduced, which may be due to changes in VH and VL pairing after fusion to the CH2 domain. The initial and terminal half-lives of atrocimab were determined to be 2.2+/-1.2 hours and 41.7+/-18.1 hours, respectively, and the AUC was 5856+/-1369.9 μg/ml x hours. Ta. The terminal half-life of atrocimab was extended nearly 40-fold compared to Fab 13.7 and 1.3-fold compared to ATROSAB; however, the lower injected dose of Fab 13.7 and ATROSAB These values may be inaccurate as they may affect kinetic properties (see, eg, Richter et al. (2019) mAbs 11(4):653-665).

ii. Domain Antibody (dAb)-Based TNFR1 Antagonists Another class of therapeutic small fragments of antibodies are domain antibodies (dAbs; also known as single domain antibodies, or sdAbs), which are monomeric, antibody-based antibodies. Contains heavy chain (VH) or light chain (VL) variable domains. dAbs are the smallest antigen-binding fragments of antibodies; they are approximately 11-15 kDa in size, approximately one-tenth the size of a complete monoclonal antibody (mAb). Similar to dAbs, Nanobodies found in camelids produce antibodies that contain only a heavy chain whose antigen-binding site is a single unpaired variable domain known as a VHH. In a dAb, there are three complementarity determining regions (CDRs) in each VH and each VL. Thus, each dAb contains three of the six CDRs from the antibody's VH-VL pair. These are highly diversified loop regions that bind target antigens.

Due to their small size, dAbs are produced in higher yields from bacterial cultures and are more suitable for phage display since only a single polypeptide chain is produced. Specific dAbs with high affinity and potency can be rapidly generated by protein engineering. The small size of dAbs also improves tissue penetration, stability, and delivery formulation selection. Due to their small size, it is possible to create molecules containing linked dAbs specific for different antigens/targets, which is not possible with conventional antibodies and is achievable with other antibody fragments such as Fabs and scFvs. is difficult. Due to their monomeric and monovalent binding modality, dAbs are suitable for use when targets are not amenable to intervention by monoclonal antibodies. TNFR1 is one such target; TNFR1 is activated/agonized by antibody-induced receptor cross-linking (e.g. Holt et al. (2003) Trends in Biotechnology 21(11):484-490 ; Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273; Goodall et al. (2015) PLoS ONE 10(9):e0137065).

dAbs, scFvs, Fvs, Disulfide Bonds Small size antibody fragments, such as Fvs and Fabs, are easier to produce and handle and are rapidly distributed throughout the body compared to larger molecules; however, their in vivo halving The period is short, limiting therapeutic efficacy. As with other antibody fragments, increasing the serum half-life of dAbs increases therapeutic efficacy and reduces dosing frequency, particularly in applications that require binding to antigen in the bloodstream, such as in the treatment of rheumatoid arthritis or cancer. . This can be accomplished by pegylation, conjugation to serum albumin, fusion with a second dAb that specifically binds serum albumin, or fusion to an Fc fragment or complete antibody constant region. Fusion with the Fc region also allows recruitment of Fc effector functions, including complement activation, antibody-dependent cytotoxicity, or Fc-mediated clearance of immune complexes (e.g., Holt et al. (2003) Trends in Biotechnology 21(11):484-490; see Goodall et al. (2015) PLoS ONE 10(9):e0137065).

a) Anti-TNFR1 dAb-anti-albumin dAb fusion construct DMS5540 is a 25 kDa murine TNFR1 antagonist, a non-competitive (does not interfere with TNF binding) anti-antibody fused to an albumin-binding dAb (AlbudAb; to prolong serum half-life). Bispecific single variable domain antibody comprising TNFR1 dAb. DMS5540, which does not bind to human TNFR1, was found to inhibit TNFα-mediated cytotoxicity in the mouse fibroblast cell line L929, which is highly sensitive to TNFα-mediated cytotoxicity. DMS5540 was administered intravenously to mice and 4 hours later an intravenous bolus injection of TNFα was given and serum IL-6 levels were assessed. DMS5540 induced a decrease in IL-6 responses when compared to mice receiving a control dAb lacking specific antigen binding but fused to AlbudAb (DMS5538) or to mice receiving no dAb. demonstrated a dose-dependent inhibition of TNFα-mediated signaling effects in vivo (see, eg, Goodall et al. (2015) PLoS ONE 10(9):e0137065).

In another study, mice with collagen-induced arthritis (CIA) were treated with DMS5540, an isotype (negative) control dAb (DMS5538), or a mouse IgG1 Fc domain (mTNFRII-Fc; mTNFR2.Fc) for 10 days from the date of onset of arthritis. treated with murine TNFR2, which blocks both receptors (TNFR1 and TNFR2), inhibits murine TNF, and disease progression was monitored. Systemic cytokine concentrations were measured, lymph node and spleen T cell subset numbers were assessed, and intrinsic Treg cell function was assessed. Compared to negative controls, blockade of TNFR1 with DMS5540 and blockade of TNFR1/2 with mTNFRII-Fc similarly inhibited disease progression, indicating that blockade of TNFR1 or TNF was effective in inhibiting arthritic joints. It has been shown to protect joints from damaging inflammatory mediators. Effector T cell activity, measured in terms of expression levels of pro-inflammatory cytokines (e.g., IFNγ, IL-10, RANTES), increased after blockade of TNFR1/2 with mTNFRII-Fc, but not after selective blockade of TNFR1 with DMS5540. TNFR2 signaling does not increase, indicating an immunomodulatory role for TNFR2 signaling (eg, suppression of T cell effector function). Furthermore, blockade of TNFR1, but not TNFR1/2, resulted in Treg cell proliferation and activation, while increased expression of FoxP3 and TNFR2, both expressed by Tregs, was observed in joints undergoing remission; demonstrating their role in resolving inflammation. These results demonstrate that inhibiting TNFR1, but not TNFR2, signaling suppresses inflammation, promotes Treg cell suppressive activity, and increases therapeutic efficacy compared to traditional TNF inhibition methods (e.g., McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738).

DMS5540 also showed that mTNFR2. Prevents inflammation-induced osteoclast formation and bone loss more effectively than Fc (anti-TNF). TNFR2-deficient mice showed increased LPS-induced bone destruction. In vitro, DMS5541, the human equivalent of DMS5540 containing an anti-human TNFR1 dAb, reduced human osteoclast formation more effectively than etanercept in the presence and absence of low doses of TNF. These results indicate an osteoprotective role of TNFR2 signaling. As a result, selective inhibition of TNFR1 may also be used for therapeutic intervention in inflammatory bone loss disorders such as osteomyelitis and periprosthetic osteolysis and aseptic loosening (e.g. Esperito Santo et al. Biochem. Biophys. Res. Commun. 464:1145-1150).

DMS5541 (also known as TNFRI-AlbudAb), which contains a non-competitive human TNFR1-specific dAb fused to AlbudAb, was evaluated for selective blockade of TNFR1-mediated TNF signaling in ex vivo cultured human rheumatoid arthritis ( RA) Synovial mononuclear cells (MNCs) express TNFR1 and TNFR2 and spontaneously produce inflammatory cytokines and chemokines in the absence of exogenous stimuli. DMS5541 inhibits the production of proinflammatory cytokines GM-CSF, IL-10, IL-1β, IL-6, and chemokines IL-8, RANTES (CCL5), and MCP-1 (CCL2) by etanercept. inhibition at a level comparable to TNF ligand blockade. This inhibition was not due to cytotoxicity, as DMS5541 inhibited TNFα-induced cytotoxicity of human rhabdomyosarcoma KYM-1D4 cells in a dose-dependent manner, similar to TNF blockade by etanercept. Furthermore, DMS5541 inhibited the production of soluble TNFR1 but did not inhibit the production of soluble TNFR2, indicating selectivity for TNFR1. These results indicate that the TNFR1 pathway is the major inflammatory pathway involved in the TNF response observed in ex vivo cultured RA synovial MNC disease models (e.g., Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262-2273).

b) Domain antibody fragment named GSK1995057 and GSK2862277 The domain antibody fragment designated GSK1995057 (see SEQ ID NO: 55) is a short-acting, short-acting antibody fragment that selectively antagonizes TNF signaling through TNFR1 rather than TNFR2. A type of fully human domain antibody (dAb) fragment (including the VH chain). Due to its small size, GSK1995057 may be sprayed directly into the lungs and has been investigated in the treatment of animal and human models of acute respiratory distress syndrome (ARDS) by inhalation. GSK1995057 reduces lung inflammation in non-human primates (cynomolgus monkeys) and human models of ARDS. Lung neutrophil infiltration is central to the pathogenesis of ARDS and is augmented by damage to the alveolar-capillary barrier caused by the action of inflammatory mediators. TNF-α contributes to increased endothelial permeability, and GSK1995057 prevents this increase, indicating that TNFR1 signaling mediates TNF-induced endothelial permeability (e.g., Proudfoot et al. (2018) ) See Thorax 73:723-730). The trial failed due to its inherently short half-life and neutralization by autoantibodies. The immunogenicity of GSK1995057 is more likely due to the failure to properly humanize the dAb than to E. This may be due to improper folding of the protein made in E. coli; it is derived from a human antibody fragment and only the hypervariable sequences have been modified to adapt its specificity for TNFR1 (e.g. (See International PCT Publication No. WO2008/149148A2).

In monkeys exposed to a single inhaled lipopolysaccharide (LPS) challenge, a well-established model that elicits a clinically relevant inflammatory response that models subclinical tissue damage, pretreatment with GSK1995057 inhibits pulmonary Neutrophil infiltration, levels of pro-inflammatory chemokines, markers of endothelial damage and alveolar capillary leak are reduced in a dose-dependent manner. The results show that inhaled GSK1995057 can produce the same results as high doses of parenterally administered antibody. 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, pretreated subjects had less systemic inflammation compared to subjects who received a placebo. There was also less evidence of neutrophilic lung inflammation and endothelial damage in response to LPS challenge. Despite these results, clinical translation is unlikely. In the trial, GSK1995057 was administered before LPS challenge, but ARDS patients generally require treatment after (rather than before) the initial injury (e.g., Proudfoot et al. (2018) Thorax 73:723 -730).

Another issue is the deleterious effect of anti-drug antibodies (ADA) on anti-TNFR1 agents, which was observed in the clinical phase I trial of GSK1995057, in which cytokines were tested at doses of 2-10 μg/kg. Release infusion reactions are observed due to high levels of pre-existing naturally occurring anti-immunoglobulin autoantibodies (ie, ADA) present in approximately 50% of drug-naive healthy subjects. Specifically, ADA is a human anti-VH (HAVH) autoantibody, and the complex between the HAVH autoantibody and the framework sequence of GSK1995057 activates TNFR1 signaling and targets subjects with high HAVH autoantibody titers. mild to moderate infusion reactions occurred (see, e.g., Cordy et al. (2015) Clin. Exp. Immunol. 182:139-148).

Binding of HAVH autoantibodies to the framework region of dAb GSK1995057 induces cytokine release in vitro. The epitope on autoantibody GSK1995057 was characterized. Existing anti-drug antibodies (ADA) bind to epitopes close to the C-terminal region of VH dAbs, including dAb GSK1995057. To counter this, a modified dAb named GSK2862277 (see SEQ ID NO: 56) was generated by adding a single alanine residue to the C-terminus of the modified dAb. This modification reduced binding to HAVH autoantibodies. In serum samples from healthy subjects screened positive for HAVH autoantibodies that bind to GSK1995057, the frequency of pre-existing autoantibodies ranged from 51% for GSK1995057-specific HAVH autoantibodies to 7% for GSK2862277-specific autoantibodies. %. Human in vitro systems and animal in vivo experiments show that GSK2862277 does not induce TNFR1 activation, even in the presence of GSK2862277-specific autoantibodies, and that the pharmacological and biophysical properties of GSK2862277 (target affinity, in vitro The efficacy and in vivo pharmacokinetics and pharmacodynamics) were shown to be comparable to the properties of the parent dAb (GSK1995057).

In a Phase I clinical trial to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple doses of inhaled (i.h.) and intravenous (i.v.) GSK2862277, GSK2862277 was , was found to be generally well tolerated when administered by inhalation or intravenously. However, one subject had a mild infusion reaction with cytokine release after repeated IV administration. This subject had high serum levels of pre-existing antibodies to GSK2862277, and serum antibodies from this subject were shown to activate TNFR1 signaling in in vitro assays. The interaction between GSK2862277 and autoantibodies results in antibody-mediated GSK2862277-dependent cross-linking of cellular TNFR1, agonizing the receptor and releasing cytokines. Therefore, despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, deleterious effects were still associated with the presence of new pre-existing antibody responses specific for the modified dAb framework. These results highlight the challenges in developing biological antagonists to TNFR1 (see, eg, Cordy et al. (2015) Clin. Exp. Immunol. 182:139-148). Therefore, there remains a need for improved TNFR1 antagonists.

iii. Nanobody (Nb)
Similar to dAbs, nanobodies (Nbs) are small antigen-binding fragments derived from camelid heavy chain antibodies that lack light chains. They are small (15 kDa), have low immunogenicity, high affinity, soluble and stable, and are encoded by a single gene/exon (VHH), making them modular and useful in bacteria or yeast. High yield production is possible.

iv. Anti-TNFR1 Nanobody-Anti-Albumin Nanobody Fusion Construct TROS (TNF Receptor One-Silencer; also called Nb Alb-70-96) is a trivalent high-affinity nanobody of human TNFR1 that competes with TNF for binding to TNFR1. selective inhibitor. To generate TROS, two anti-human TNFR1 nanobodies (Nb70 and Nb96; see SEQ ID NOs: 683 and 684, respectively) were generated from a VHH library constructed by immunizing alpacas with recombinant human soluble TNFR1. ) was linked to an anti-albumin nanobody (Nb Alb) via a (Gly4Ser)3 linker to generate trivalent TROS and increase serum half-life. The serum half-life of the resulting TROS is about 24 hours; the serum half-life of monovalent Nb is only about 1.5 hours. Treatment with TROS delays the onset of murine experimental autoimmune encephalomyelitis (EAE; a model for MS) and prevents established disease. The therapeutic effect is due to the diversion of TNF to signals through TNFR2 and the effects of such signaling. TROS also suppresses 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 (e.g., Steeland et al. (2015) J. Biol Chem. 290(7):4022-4037; see Steeland et al. (2017) Sci. Reports 7:13646).

c. Dominant-Negative Inhibitors of TNF (DN-TNF)/TNF Muteins Another class of TNF inhibitors are dominant-negative inhibitors of TNF (DN-TNF), also known as TNF muteins, which lack signaling capacity. . DN-TNF is an engineered variant of TNF with mutations that abolish binding to and signaling through TNFR1 and TNFR2. DN-TNF generates soluble TNF (sTNF or solTNF) by rapidly exchanging subunits with native TNF homotrimers to form inactive mixed TNF heterotrimers with disrupted receptor-binding surfaces. ) and prevent interaction with TNF receptors. DN-TNF does not affect transmembrane TNF (tmTNF), maintaining the protective role of TNF signaling through TNFR2. DN-TNF inhibits TNF-induced NF-κB activity and caspase-mediated apoptosis and reduces disease severity in animal models of arthritis and Parkinson's disease. These molecules may be immunogenic due to their structure.

DN-TNF, a selective inhibitor of soluble TNF, does not inhibit tmTNF signaling, unlike anti-TNF therapies that bind to solTNF and tmTNF, and L. does not suppress the resistance of mice to infection by P. monocytogenes. An example of DN-TNF is shown in SEQ ID NO: 1 (corresponding to residues L57Y, S86Q, Y87H, I97T, Y115Q, and A145R with respect to the sequence of solTNF, shown in SEQ ID NO: 2), which impairs binding to TNFR. A TNF mutant containing one or more of the following substitutions: L133Y, S162Q, Y163H, I173T, Y191Q and A221R. Additional modifications may also be included, for example to improve expression and allow site-specific pegylation (see, e.g., Zalevsky et al. (2007) J. Immunol. 179:1872-1883). .

For example, the TNF mutations R32W and S86T, see SEQ ID NO: 2, have a hundreds-fold loss in affinity for TNFR2, but do not affect binding to TNFR1. The R32W/S86T double mutant abolishes all binding to TNFR2 without losing binding to TNFR1. Mutual L29S, L29G, L29Y, R31E, R31N, R32Y, R32Y, S86T, L29S / R32W, L329S / S86T, R32W / S86T, L29S / R32W / S86T, R31N / R32T , R31E / S86T, R31N / R32T / S86T, and E146R (see SEQ ID NO: 2) also confers selectivity for TNFR1. Mutations D143N, D143Y, A145R and D143N/A145R (see SEQ ID NO: 2) render the TNF mutant selective for TNFR2 (e.g. Loetscher et al. (1993) J. Biol. Chem. 268(35):26350-26357; see US Pat. No. 5,422,104).

Modified TNF, called XPro1595 (INmuneBio; see SEQ ID NO: 701), is a pegylated soluble DN-TNF mutein that preferentially inhibits TNFR1 signaling, with mutations V1M, R31C, C69V, Y87H, C101A , and A1456R (see SEQ ID NO: 2) (see, eg, US Publication No. 2015/0239951). XPro1595 reduces neuroinflammation and is being studied in the treatment of Alzheimer's disease (see, eg, Clinical Trial Identifier Number NCT03943264). XPro1595 blocks the development of amyloid pathology in a mouse model of Alzheimer's disease (3xTgAD), prevents loss of neuronal communication and cognitive impairment in a different (tgCRND8) mouse model of Alzheimer's disease, and reverses neuronal communication dysfunction and normal aging. Reduces cognitive impairment in rats and, in a third model of Alzheimer's disease (5xFAD), prevents young mice from developing amyloid pathology, cognitive impairment, and dysfunction of neurotransmission. In older mice with pathology similar to Alzheimer's disease, XPro1595 reduced amyloid, improved cognition, rescued neuronal communication, and normalized innate and adaptive immune responses.

Levels of TNFR1 are elevated in the hippocampus in young (6 months) but not old (22 months) Fischer 344 rats compared to TNFR2. When treated with XPro1595, aged rats showed improved Morris water maze performance, decreased microglial activation, decreased susceptibility to long-term depression in the hippocampus, increased levels of GluR1-type glutamate receptors, and L-type voltage-sensitive Ca2+ channels. (L-VSCC) activity was demonstrated in hippocampal CA1 neurons, indicating that functional changes associated with brain aging may arise from selective changes in TNF signaling. In animal models of Parkinson's disease and aging, XPro1595 suppresses neuroinflammation and microglial activation. In EAE (a model of MS), XPro1595 ameliorates disease, improves remyelination, and reduces CNS pathology and neuroinflammation. XPro1595 also improves inflammatory arthritis and reduces susceptibility to infection in treated animals. Compared with etanercept, which has no therapeutic effect, treatment with XPro1595 delayed the onset of EAE and improved symptoms more efficiently. Administration of XPro1595 increased the expression level of TNR2 in the lesion area of EAE, indicating that tmTNF signaling through TNFR2 is involved in nerve regeneration (e.g., Yang et al. (2018) Front. Immunol 9:784; see Sama et al. (2012) PLoS ONE 7(5):e38170). XPro1595 does not inhibit the activity of transmembrane TNF (which activates TNFR1 and TNFR2) and therefore cannot block the inflammatory effects of TNFR1. This also applies to other dominant-negative TNF reagents discussed below.

XENP345 (see SEQ ID NO: 702) is a pegylated DN-TNF mutein containing the mutation I97T/A145R with respect to SEQ ID NO: 2. In vivo neutralization of soluble TNF (solTNF) by XENP345 in animal models of Parkinson's and Alzheimer's disease is neuroprotective, reduces neurodegenerative and cognitive dysfunction, and slows neurodegenerative disease progression (e.g., McCoy (2006) J. Neurosci. 26(37):9365-9375; see McAlpine et al. (2009) Neurobiol. Dis. 34(1):163-177).

R1antTNF (see SEQ ID NO: 703) is a structural human TNF variant in which each of the six amino acid residues in the receptor binding site, corresponding to residues 84-89 of SEQ ID NO: 2, are mutated. TNFR1 selective antagonist mutant TNF identified from the phage library shown. R1antTNF containing the A84S, V85T, S86T, Y87H, Q88N, and T89Q mutations has affinity for TNFR1 similar to wild-type human TNF and does not interfere with TNFR2 activity. R1antTNF ameliorated liver injury in two models of acute hepatitis, as evidenced by reduced serum levels of alanine aminotransferase and the proinflammatory cytokines IL-2 and IL-6. However, the plasma half-life of R1antTNF is very short (12 minutes), similar to wild-type TNF. To increase the in vivo half-life of R1antTNF, a PEGylated version of PEG-R1antTNF was generated in which PEG was attached to the N-terminal site of R1antTNF. PEG-R1antTNF reduces morbidity, improves disease symptoms, ameliorates demyelination in EAE mouse models, and suppresses Th1 and Th17 cell activation and inflammatory T cell infiltration in the spinal cord. PEG-R1antTNF also inhibited NF-κB, suppressed smooth muscle cell proliferation, and decreased the expression of chemokines and adhesion molecules, thereby inhibiting IL-1 after inducing femoral artery injury in the external vascular cuff model. Reducing intimal hyperplasia and arterial inflammation in receptor antagonist-deficient mice. Comparing the effects of PEG-R1antTNF and etanercept on antiviral immunity using recombinant adenoviral vectors, PEG-R1antTMF did not reactivate viral infection and did not affect the clearance of injected adenovirus. However, viral load increased after treatment with etanercept. PEG-R1antTNF treatment also slowed and ameliorated CIA symptoms in preventive and therapeutic settings and was more effective than etanercept when used to treat pre-existing CIA (e.g., Yang et al. (2018) Front. Immunol. 9:784; Shibata et al. (2008) J. Biol. Chem. 283(2):998-1007; Kitagaki et al. (2012) J. Atheroscler. Thromb. 19(1):36-46; Fischer et al. (2015)) Antibodies 4:48-70; Horiuchi et al. (2010) Rheumatology (Oxford) 49:1215-1228).

Soluble TNFR1 is also associated with an increased risk of developing MS; therefore, neutralization of soluble TNFR1, which cannot be achieved with DN-TNF/TNF muteins, may be beneficial. In contrast to inhibitors of solTNF, such as DN-TNF, TNFR1 antagonists can block the binding of lymphotoxin alpha (LT-α), another member of the TNF superfamily, to TNFR1. LT-α may have a proinflammatory role in RA and in animal disease models such as CIA and EAE. Therefore, simultaneous blockade of TNF and LT-α binding to TNFR1 by TNFR1 antagonists may have additional benefits in acute and chronic inflammatory diseases and disorders compared to solTNF inhibition (e.g., Fischer et al. (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 anti-tumor immune responses and enable tumor immune evasion. Tregs are therefore therapeutic targets in the treatment of, for example, autoimmune and chronic inflammatory diseases and conditions, graft-versus-host disease (GvHD), transplant rejection, and cancer. TNF signaling through TNFR2 regulates Treg function and activity. TNFR2 agonists upregulate Treg activity, while TNFR2 antagonists downregulate Treg activity. The Treg stimulatory effects of the TNF-TNFR2 signaling pathway can be exploited for the treatment of several human diseases and disorders, such as autoimmune and chronic inflammatory diseases through agonism, and cancer through antagonism (e.g., Zou et al. (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 proliferation and activation of Tregs that regulate the immune system, reduces the activity of tissue-damaging autoreactive CD8+ T cells, and provides anti-inflammatory , and induce signal transduction pathways with cell survival, regenerative and protective effects, such as neuroprotective, cardioprotective, enteroprotective, and osteoprotective effects. Therefore, enhancement of TNFR2 signaling by TNFR2-selective agonists may be useful, especially in the treatment of autoimmune and chronic inflammatory diseases and disorders, including neurodegenerative diseases where anti-TNF therapy/TNF blockers have failed. Can be used to enhance the therapeutic effects of antagonism.

a. TNFR2 Agonist Antibodies Human TNFR2 selective agonist antibodies include commercially available MR2-1 (monoclonal mouse IgG1 that binds to human, cynomolgus and rhesus monkey TNFR2; Hycult Biotech), and clone MAB2261 (monoclonal mouse IgG2A that binds to human TNFR2; R&D Systems). ). TNFR2 agonists, such as antibodies, potently stimulate the proliferation of a homogeneous population 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 were cultured using standard in vitro human Treg expansion protocols (i.e. using anti-CD3 antibodies, anti-CD28 antibodies, IL-2 and rapamycin) and Proliferating Tregs with higher levels of FoxP3 (and other characteristic Treg markers), and when expanded in the presence of TNFR2 agonist antibodies, compared to the absence of TNFR2 agonist antibodies. If grown in Tregs isolated from type 1 diabetic patients exhibiting a resting phenotype are activated and expanded upon in vitro treatment with TNFR2 agonist antibodies. Such Tregs are more potent in inhibiting autologous CD8+ T cells (see, eg, Zou et al. (2018) Front. Immunol. 9:594).

Treatment of isolated Tregs expanded using standard in vitro protocols using MR2-1, a commercially available agonistic human TNFR2 monoclonal antibody (mAb) containing mouse IgG1, resulted in a homogeneous population of FoxP3+Helios+CD127low Tregs. are generated; these Tregs maintain their phenotype and high suppressive activity in humanized mouse models. Therefore, TNFR2 agonists may enhance ex vivo expansion of Treg cells from impure cell populations for use in Treg-based immunotherapy (e.g., Zou et al. (2018) Front. Immunol. 9:594). (see ).

b. TNFR2-selective TNF muteins and fusions thereof As described herein, TNF can be engineered to selectively bind to TNFR1 or TNFR2; A variant of TNF that contains one or more mutations that increase and/or reduce or eliminate binding to TNFR1. TNFR2 selective mutations include non-conservative substitutions of the Asp residue at position 143 of soluble TNFs such as D143Y, D143F or D143N (see SEQ ID NO: 2) or 145 of soluble TNFs such as A145R. (See, eg, US Pat. No. 9,081,017). Other mutations in TNF that confer selectivity for TNFR2 include, but are not limited to, K65W, D143E, D143W, D143V, 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/S147 D, 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, (according to SEQ ID NO: 2) (see, eg, US Patent Application Publication No. 2020/0102362).

Trimerization of TNF ligands is essential for TNFR-mediated signal transduction. At low concentrations, such as in serum, the trimers dissociate and they are degraded. To generate functionally active receptor-specific TNF muteins, it is necessary to create stable trimers. TNF07 is a soluble TNF (sTNF or solTNF) mutein containing the mutation S95C/G148C (according to the sequence of residues shown in SEQ ID NO: 2) that forms stable TNF trimers and functions as a TNFR2 agonist. be. The S95C/G148C mutation results in the formation of an intermolecular Cys-Cys covalent bond; thus, a stable trimer is formed as a result of the covalent internal disulfide bridges of sTNF at strategic positions between the TNF monomers. is formed. Despite lacking a TNFR2 selective mutation, TNF07 functions as a TNFR2 agonist. TNF07 induces strong TNFR2 signaling, proliferates FoxP3+ Treg cells, and selectively induces the death of autoreactive CD8+ T cells isolated from type 1 diabetic patients (e.g., Ban et al. (2015) Molecular and Cellular Therapies 3:7; see Zou et al. (2018) Front. Immunol. 9:594).

Several TNFR2 agonists have been generated that include fusions of single chain TNFR2-selective TNF mutein trimers and multimerization domains. 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 a multimerization domain, such as a dimerization domain or a trimerization domain, generates hexameric or nonamer molecules, respectively, for TNF subunits; these hexameric and nonamer molecules of TNF The trimeric protein mimics membrane-bound TNF trimers and therefore can effectively activate 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 may also include Fc domains, such as IgG1- and IgG4-derived domains, optionally containing modifications that alter immune effector function. Commonly used trimerization domains include chicken tenascin C (TNC) and human TNC. Dimerization and trimerization enhance TNFR2 signaling, e.g. by increasing the molecular weight of the molecule and/or introducing FcRn recycling, e.g. if the dimerization domain is Fc. thereby improving the half-life of the fusion protein.

STAR2 (also known as TNC-sc-mTNF (221N/223R)) is a nanomeric agonist TNFR2-specific murine TNF variant that does not bind to TNFR1, and is a single-chain murine TNF trimer that binds to each TNF subunit. The unit 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 murine 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 It is linked to the N-terminus of the first TNF subunit of the chain trimer. The specificity of STAR2 for TNFR2 is due to the mutations D221N and A223R (see the sequence of mouse TNF shown in SEQ ID NO: 5) within the individual TNF subunits, which indicate the difference between STAR2 and mouse TNFR1. Causes steric collisions. Fusion to the TNC trimerization domain causes spontaneous oligomerization, creating three covalently linked TNF trimers, mimicking membrane-bound TNF. STAR2 stimulates Treg proliferation in vitro and in vivo through a TNFR2-dependent, IL-2-independent mechanism. Pretreatment of allogeneic hematopoietic stem cells with STAR2 before transplantation into mice prolonged survival and reduced the severity of GvHD in a TNFR-2 and Treg-dependent manner. The human equivalent of the TNFR2-specific STAR2 agonist, TNC-scTNF (143N/145R), consists of residues 9-157 of soluble TNF (see SEQ ID NO: 2) and is according to SEQ ID NO: 2 (solTNF). containing the mutations D143N/A145R and potently stimulated proliferation of CD4+FoxP3+ Tregs in vitro from CD4+ T cells isolated from healthy donors (e.g., Chopra et al. (2016) J. Exp. Med. 213(9): 1881-1900; see Zou et al. (2018) Front. Immunol. 9:594).

TNC-scTNFR2 is a soluble human TNFR2 agonist that targets the N-terminus of human tenascin C (hTNC), comprising residues 110-139 of SEQ ID NO: 806, of a TNFR2-selective single-chain TNF variant (scTNFR2; SEQ ID NO: 803). It is a fusion of trimerization domains (see also SEQ ID NO: 807) and contains three TNF domains connected by two short peptide linkers (GGGGS). The TNFR2-selective TNF molecule, scTNFR2, resembles soluble trimeric TNF, with each TNF subunit having mutations D143N/A145R (according to SEQ ID NO: 2) that eliminate binding to TNFR1, SEQ ID NO: 1 (corresponding to residues 4-157 of SEQ ID NO: 2). Since TNFR2 is fully activated only by membrane-bound TNF and not by soluble TNF trimers, the trimerization domain of TNC is fused to the N-terminus of scTNFR2, generating TNC-scTNFR2. TNC-scTNFR2 exists in trimeric assemblies of single-chain fusion proteins, resembling nonameric TNF molecules; this oligomeric TNF mutein, due to its high avidity, is a protein that binds membrane-bound TNF ( memTNF) activity, induces clustering of TNFR2 and formation of TNFR2 signaling complexes, and efficiently activates TNFR2. TNC-scTNFR2 exhibits neuroprotective properties; it protects 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-OHDA. These results indicate that TNC-scTNFR2 can ameliorate neurodegenerative processes (see, eg, Fischer et al. (2011) PLoS ONE 6(11):e27621).

EHD2-scTNFR2 (see SEQ ID NO: 810) is an agonist TNFR2-selective TNF mutein fusion protein that is derived from the heavy chain CH2 domain of IgE and creates a disulfide-linked dimer containing a hexameric TNF domain. A covalently stabilized protein with mutations D143N/A145R (residue numbering for soluble TNF as set forth in SEQ ID NO: 2) that abolishes binding to TNFR1, fused to the mobilization domain EHD2 (SEQ ID NO: 808). contains a human TNFR2-selective single-chain TNF trimer (scTNFR2; SEQ ID NO: 803). Each TNF subunit within scTNFR2 contains residues 4-157 of SEQ ID NO:2. EHD2 is fused to the N-terminus of 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. ing. EHD2-scTNFR2 exhibits neuroprotective properties in mouse models of NMDA-induced acute neurodegeneration (e.g., Dong et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113(43):12304-12309; and U.S. Pat. (See Application Publication No. 2020/0102362).

The TNFR2 agonist fusion protein contains three TNF muteins with mutations D143N/A145R (see SEQ ID NO: 2) that abolish binding to TNFR1, fused to a dimerization domain that is Fc. It also contains a single chain TNFR2 agonist (scTNFR2), resulting in the production of a protein (scTNFR2-Fc) that is hexameric with respect to the TNF domain. The Fc may be an IgG4 or an IgG1 Fc, optionally containing mutations that eliminate Fc effector functions such as ADCC and CDC. Three TNF muteins containing residues 12-157 of SEQ ID NO: 2 are joined together by two short peptide linkers, and the dimerization domain is linked by a third short peptide linker to form a single-chain trimeric TNF molecule. (scTNFR2) is attached to the N-terminus or C-terminus. All three linkers may be the same or different and may include GS linkers such as (GGGGS) n residues 116-121 of SEQ ID NO: 707, and/or other combinations of Gly and Ser, where or all or a portion of the stalk region of TNF-α (GPQREEFPRDLSLISPLAQAVRSSSRTPSDK (SEQ ID NO: 812), corresponding to residues 57 to 87 of SEQ ID NO: 1, or at least 10, 15, or 20 Dimerization enhances signaling by TNFR2 agonists and also improves the half-life of the fusion protein. Other dimerization domains that can be used in the fusion protein include: Fc fusion proteins derived from other dimerizing molecules, such as IgE heavy chain domain 2 (EHD2; see SEQ ID NO: 808) and IgM heavy chain domain 2 (MHD2; see SEQ ID NO: 811) ( For example, see International Application Publication No. WO2019/226750).

3. Anti-TNFR2 Antagonizing Antibodies and Small Molecule Inhibitors TNFR2 antagonists inhibit the proliferation and induce death of Tregs, and can also inhibit the proliferation and induce death of TNFR2-expressing tumor cells. TNFR2 antagonists can reduce or inhibit the proliferation of myeloid-derived suppressor cells (MDSCs) and/or induce apoptosis in MDSCs by binding to TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment. TNFR2 antagonists also induce proliferation of T effector cells, including cytotoxic CD8+ T cells, through inhibition of Treg proliferation and activity. As a result, TNFR2 antagonists are effective against infectious diseases and certain cancers that express TNFR2, such as T-cell lymphoma (e.g., Hodgkin's lymphoma and cutaneous non-Hodgkin's lymphoma), ovarian cancer, colon cancer, multiple myeloma, renal cell It may be useful in treating cancers such as breast cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, and lung cancer. (See, e.g., US Patent Publication No. 2019/0144556; Torrey et al. (2017)) Sci. Signal. 10:eaaf8608).

As discussed herein, TNFR2 expression is restricted to certain immune cells, including Tregs and MDSCs, endothelial cells, and certain neurons and cardiac cells. The restricted expression of TNFR2 makes it an ideal drug target as systemic toxicity is less likely to occur with anti-TNFR2 therapeutics.

TNFR2 antagonist antibodies and antigen-binding fragments thereof are directed to epitopes within human TNFR2 that include one or more of residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4), or residues 130-149, 137-144, or 142-149, or larger epitopes comprising at least 5 contiguous or non-contiguous residues within these epitopes, e.g. is not combined with TNFR2 antagonists also include 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 residues 75-128 of SEQ ID NO: 4 , 86-103, 111-128, or 150-190 (see, eg, US Patent Publication No. 2019/0144556).

Generally, an antagonist TNFR2 antibody or antigen-binding fragment thereof has an affinity that is at least 10 times greater than the affinity of the same antibody or antigen-binding fragment for a peptide that includes the KCSPG sequence (SEQ ID NO: 839) of human TNFR2 (SEQ ID NO: 839). SEQ ID NO: 840). An antibody or antibody fragment that binds with similar affinity (e.g., less than a 10-fold difference in affinity) to an epitope that includes one or more residues of the KCRPG sequence, and to an epitope that includes a KCSPG motif, is an antagonizing TNFR2 antibody. isn't it. Antagonizing TNFR2 antibodies include TNFRAB1 (see SEQ ID NOs: 1213 and 1213, respectively, for the heavy and light chain sequences of TNFRAB1), TNFRAB2 and TNFR2A3 (e.g., U.S. Patent Publication No. 2019/0144556 for a description thereof). (see issue). TNFR2 antagonists also include 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 at least about 85% alignment Included are antibodies and antibody fragments that contain identical CDR-H3 sequences. For example, TNFRAB1 specifically binds residues 130-149, including residues KCSPG, of TNFR2 with a 40-fold higher affinity than residues 48-67, including residues KCRPG of TNFR2 (e.g., See U.S. Patent Application Publication No. 2019/0144556).

TNFRAB1 (see SEQ. No. 4) also binds to an epitope within residues 161-169 (CKPCAPGTF; SEQ ID NO: 1258). Another antagonizing TNFR2 antibody, TNFRAB2, has an epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851), as well as positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; No. 1248), and one or more residues 116-123 (RICTCRPG; SEQ ID No. 1249). TNFR2A3 is a mouse antagonizing human TNFR2 antibody discovered by immunization of mice with human TNFR2 and subsequent CDR mutagenesis, in which CDR-H3 of the precursor antibody generated is -H3 sequence ARDDGSYSPFDYFG (SEQ ID NO: 1223). TNFR2A3 binds to two different epitopes within human TNFR2; the first epitope comprises residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and contains the KCRPG motif; the second epitope comprises: The downstream sequence includes residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464). These data demonstrate that the CDR-H3 sequence of antagonizing TNFR2 antibodies largely determines antigen-binding properties, and that the CDR-H3 motif confers TNFR2-dominant antagonizing properties to such antibodies or antigen-binding fragments thereof. It has been shown that this is a modular domain that can be substituted with an anti-TNFR2 antibody that does not exhibit antagonizing activity. For example, replacing the CDR-H3 sequence of a neutral anti-TNFR2 antibody (i.e., an antibody that is neither an antagonist nor an agonist) with the CDR-H3 sequence of an antagonist TNFR2 antibody, such as the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, The type-neutral antibody is converted to an antagonistic TNFR2 antibody, such as a dominant antagonizing TNFR2 antibody, which is an antagonist that inhibits TNFR2 activation even in the presence of a TNFR2 agonist, such as TNF or IL-2 (e.g., U.S. Patent Publication No. 2019/2019). 0144556).

The TNFR2 antagonist antibody or antigen-binding fragment thereof has a CDR-H1 sequence shown in any of SEQ ID NOs: 1214, 1215, and 1231 to 1233; a CDR-H2 sequence shown in any of SEQ ID NOs: 1216, 1224, and 1230; The CDR-H3 sequence shown in any of SEQ ID NO: 1217, 1223, and 1225-1229, or the CDR-H3 of TNFRAB1 corresponding to residues 99-112 of SEQ ID NO: 1212; any of SEQ ID NO: 1218 and 1234-1236 or the CDR-L1 sequence of TNFRAB1 corresponding to residues 24 to 33 of SEQ ID NO: 1213; the CDR-L2 sequence illustrated in any of SEQ ID NOs: 1219, 1220, 1237, and 1238; CDR-L2 sequence of TNFRAB1 corresponding to residues 49-55 of number 1213; or CDR-L3 sequence shown in any of SEQ ID NO: 1221, 1222, and 1241-1244, or residues 88-96 of SEQ ID NO: 1213 may contain the CDR-L3 sequence of TNFRAB1 corresponding to . Exemplary framework regions that can be used to develop humanized anti-TNFR2 antibodies that include one or more of the above CDRs include, but are not limited to, U.S. Patent Nos. 7,732,578 and 8,093,068 and the framework areas described in International Application Publication No. WO 2003/105782. Another approach to engineering humanized anti-TNFR2 antagonist antibodies is to combine the heavy and light chain variable region sequences of an antagonist TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3, with the heavy and light chain variable regions of a consensus human antibody. It is to align with the variable region. Consensus human antibody heavy and light chain sequences are known in the art (e.g., the "VBASE" human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.). Department of Health and Human Services, NIH Publication No. 91-3242, (1991); Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836). In this way, variable domain framework residues and CDRs can be identified by sequence alignment. For example, CDR-H3 of a consensus human antibody may be replaced with CDR-H3 of an antagonistic TNFR2 antibody, such as CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, to produce a humanized TNFR2 antagonist antibody. Exemplary variable domains of consensus human antibodies 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, as described in U.S. Patent No. 6,054,297 (e.g. (see US Patent Application Publication No. 2019/0144556). The CDR-H1 and CDR-H2 sequences of the exemplary consensus sequence of the human antibody heavy chain variable domain of SEQ ID NO: 1245 may be replaced, for example, with the corresponding CDR sequences of a phenotype-neutral TNFR2-specific antibody, and By replacing the CDR-L1, CDR-L2 and CDR-L3 sequences of the exemplary consensus sequence of the human antibody light chain variable domain of SEQ ID NO: 1246 with the corresponding CDR sequences of a phenotype-neutral TNFR2-specific antibody, Antagonized TNFR2 antibodies may also be generated.

Other TNFR2 antagonists can be obtained using techniques known in the art, such as phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display, or as described in U.S. Patent Publication No. 2019/0144556. identified by screening for peptides that bind to epitopes within TNFR2, such as the peptides set forth in any one of SEQ ID NOs: 1247-1464, by using any other method known in the art, such as can be done.

Human TNFR2 antagonist mAbs inhibit Treg proliferation and reduce their suppressive activity when added to standard Treg proliferation culture conditions (see, e.g., Zou et al. (2018) Front. Immunol. 9:594). thing). Two potent and dominant anti-human TNFR2 antagonizing antibodies that outcompete TNF, the natural agonist of TNFR2, can inhibit TNF-induced in vitro proliferation of human Tregs and induce death of Tregs in vitro. TNFR2 antagonists bind TNFR2 specifically through the F(ab) region, independent of the Fc region or antibody cross-linking, and block TNF binding to TNFR2 by binding to the antiparallel dimer of TNFR2. do. As a result, TNF-induced activation of the NF-κB pathway in Tregs is inhibited, and the conversion of transmembrane TNFR2 (tmTNFR2) to soluble TNFR2 (sTNFR2) is suppressed. Tregs isolated from ovarian cancer tissues were found to be more sensitive to cell death by TNFR2 antagonist mAbs due to higher levels of TNFR2 expression in tumor-infiltrating Tregs. TNFR2 antagonists also induce the death of TNFR2+OVCAR3 (ovarian cancer) tumor cells, which also express TNFR2. These results demonstrate the therapeutic potential of TNFR2 antagonists in the treatment of tumors by targeting tumor-infiltrating Tregs and tumor cells (e.g., Zou et al. (2018) Front. Immunol. 9:594 ; see Torrey et al. (2017) Sci. Signal. 10:eaaf8608).

In addition to anti-TNFR2 antagonist 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-κB, destroying TNF mRNA, and targeting reactive oxygen species and α1-acid glycoprotein, and also inhibit intracellular TNFR2. The surface expression of TNFR2 on T cells is inhibited by inhibiting the transport of TNFR2 to the cell surface. Thalidomide reduces the number and function of Tregs in patients with chronic lymphocytic leukemia, and in patients with acute myeloid leukemia, combination therapy of lenalidomide and azacytidine downregulates TNFR2 expression on CD4+ T cells and reduces the number of TNFR2+ Tregs. , has been shown to enhance effector immune function. However, in patients with multiple myeloma, treatment with thalidomide and its analogues increases the number of Tregs, likely due to increased serum levels of TNF after treatment, thereby reducing the effect of thalidomide on TNFR2+ Tregs. It has been shown to be disease specific (see, for example, Zou et al. (2018) Front. Immunol. 9:594).

Another small molecule inhibitor of TNFR2 is panobinostat, a histone deacetylase inhibitor that can reduce the expression of FoxP3 and inhibit the suppressive activity of Tregs. Combination therapy with panobinostat and azacitidine reduces the number of TNFR2+ Tregs in the blood and bone marrow of patients with acute myeloid leukemia, and as a result, increased IFNγ and IL-2 production by effector T cells may result in a therapeutic effect in these patients. (e.g., Zou et al. (2018) Front. Immunol. 9:594). Cyclophosphamide, a DNA alkylating agent commonly used as a cytotoxic chemotherapeutic agent in the treatment of cancer, can inhibit the immunosuppressive function of Tregs at low doses and inhibits PROb colon cancer after a single dose. This maximizes the depletion of suppressive Tregs in mice with the tumor, leading to the activation of anti-tumor immune responses. In a mouse model of mesothelioma, cyclophosphamide treatment depleted TNFR2hiTreg. The combination of cyclophosphamide and etanercept inhibited the growth of established CT26 tumors in mice by blocking TNF-TNFR2 interaction and eliminating TNFR2-expressing Treg activity (e.g., Zou et al. (2018) ) Front. Immunol. 9:594). Triptolide, an immunosuppressive molecule isolated from the Chinese herbal medicine Tripterygium wilfordii, inhibits the expression of TNF and TNFR2 in the colon of a mouse colitis model, and also reduces the number of Tregs and inhibits tumor growth in melanoma-bearing mice. (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 that block TNF and inhibit its signaling through TNFR1 and TNFR2 have limited therapeutic efficacy, tolerability. There are limits to safety and safety. Anti-TNF therapy ameliorates RA and other autoimmune and inflammatory diseases and conditions by interfering with TNF signaling through TNFR1 and overriding apoptotic and inflammatory pathways. However, these anti-TNF therapies also block the beneficial effects of TNFR2 signaling, including protective, prosurvival, proregenerative, and anti-inflammatory signaling pathways, as well as proliferation of TNFR2-associated immunosuppressive Tregs. These include serious and sometimes fatal side effects, including serious infections. Other side effects associated with the use of TNF blockade therapy include congestive heart failure, liver damage, demyelinating diseases/CNS disorders, lupus, psoriasis, sarcoidosis, as well as increased susceptibility to the development of additional autoimmune diseases; Cancers include lymphoma and solid malignancies. Anti-TNF therapy has failed to treat demyelinating and neurodegenerative diseases and may worsen disease symptoms.

TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, and constructs comprising nucleic acids, and methods for selective inhibition of TNF signaling through TNFR1 are provided herein. (see, eg, Figure 2, which shows an exemplary bispecific construct). Also provided are constructs and methods for selective inhibition of TNF signaling through TNFR1, including maintaining or enhancing TNFR2 signaling. These constructs and methods provide improved therapeutic approaches for the treatment of diseases and disorders of the TNF/TNFR1 axis. These therapeutic approaches include, but are not limited to, the treatment of autoimmune, chronic inflammatory, neurodegenerative, and demyelinating diseases, disorders, and conditions, and cancers that also have an inflammatory component. As described herein, the simultaneous or sequential selective agonism of TNFR2 with TNFR1 antagonism has therapeutic effects and is effective in controlling cell survival and proliferation, including anti-inflammatory and NF-κB pathways. Selective TNFR1 antagonists by activating desired signaling pathways as well as by inducing the proliferation of immunosuppressive Tregs that remove excess autoreactive/effector T cells from the autoimmune microenvironment resulting in tissue destruction. can increase the therapeutic index of

Sections 1 and 2 describe methods for targeting TNRF1 and TNRF2, respectively. Section 3 provides an overview of the constructs provided herein that solve the problems of traditional approaches, particularly those targeting TNFR1; Section 4 provides an overview of the constructs provided herein Describe the structure and components.

1. Selective Blocking of TNFR1 by TNFR1 Antagonists However, the use of multivalent agents such as antibodies against TNFR1 is not feasible. TNF trimers bind to the three TNFR1 chains as a preligand assembly complex, mediated by the preligand assembly domain (plad) of each monomeric TNFR. This differs from most receptor systems, which require ligand binding before cluster formation on the surface of cells. TNF receptors are single transmembrane glycoproteins with approximately 28% homology, primarily in the extracellular domain, and both receptors contain four tandemly repeated cysteine-rich motifs. Their intracellular sequences have little homology to each other and are largely unrelated, and early studies delineated their signaling functions (Grell et al. (1994) J. Immol. 153(5 ):1963-72). They contain several motifs with known functional importance. TNFR1 and TNFR2 each contain an extracellular preligand binding assembly domain (PLAD) domain (distinct from the ligand binding region) that precomplexes the receptor. Binding of trimeric TNF ligands to TNFR trimers in the cell membrane induces conformational changes and signal activation (MacEwan (2002) Br J Pharmacol. 135(4):855-875; and Lo et al. (2019) Sci Signal. 12(592):eaav5637).

As a result, antibodies and other multivalent substances that bind TNFR1 may not be suitable for use as antagonists, as they can cause superclustering that leads to activation of TNFR signaling. Monovalent antagonists such as single-domain antibodies (dAbs or sdAbs), nanobodies (Nbs; camel single-domain antibodies), scFv fragments, and Fab fragments, on the other hand, bind to one TNFR1 molecule and cross-link as well. It also does not induce clustering of the above receptors, and activation of TNFR1 signaling is abolished. Monovalent antagonists may bind to domains 1, 2, 3, or 4 of the TNFR1 extracellular domain, or to epitopes spanning multiple domains (e.g., U.S. Patent Nos. 9,028,817 and 9,028,822 (see issue), however, these existing antagonists have been ineffective therapeutics. Among the various problems were short serum half-life and immunogenicity, as well as other problems. Selective blockade of TNFR1 can be achieved using TNFR1 antagonists having the properties described and provided herein.

2. Selective Activation of TNFR2 by TNFR2 Agonists As described herein, selective activation of TNFR2 includes, for example, TNFR2 agonist antibodies and antigen-binding fragments thereof, and TNFR2-selective TNF muteins and fusion proteins thereof. can be achieved using a TNFR2-specific agonist. Antigen-binding fragments of antibodies that bind to the first and/or second epitope of human TNFR2 may be used. The first epitope of TNFR2 comprises amino acid residues 48-67 of SEQ ID NO: 4, and the second epitope comprises amino acid residues 48-67 of SEQ ID NO: 4, e.g., residues 128-147 of SEQ ID NO: 4, 130-149, 135-147, or 135-153 (see, eg, International Application Publication No. WO 2014/124134; and US Patent No. 9,821,010). Other epitopes on TNFR2 have been identified and may be used to design antigen binding fragments with TNFR2 selectivity, as discussed below.

In contrast to the antagonizing action of TNFR1, dimeric and trimeric molecules are used to agonize TNFR2, which mimic the action of membrane-bound TNF, the main ligand that activates TNFR2. Accordingly, TNFR2 agonists include TNFR2-selective TNF muteins and antibody fragments. Exemplary are TNF muteins and antibody fragments fused to multimerization domains, particularly dimerization or trimerization domains, as discussed below. To extend the half-life of these molecules, they may be associated or conjugated with polyethylene glycol, with or without a cleavable linker (e.g., Santi et al. 2012) Proc. Natl. with or without PEGylation); and ADCC-inactivated/FcRn-optimized Fc domains of antibodies with or without PEGylation (e.g., reviewed in Strohl (2015) BioDrugs 29(4):215-239). Examples of half-life prolonging factors include PEGylation, modification of glycosylation, sialylation, PASylation (a polymer of PAS amino acids with a length of about 100 to 200 residues), and ELPylation (e.g., Floss et al. (2010)). J. Trends Biotechnol.28(1):37-45), Hapylation (glycine homopolymer), fusion to human serum albumin, fusion to GLK, fusion to CTP, GLP fusion, constant human immunoglobulin (IgG) fusions to fragment (Fc) domains, fusions to transferrin, fusions to nonstructural polypeptides such as XTEN (genetic (also referred to as fusion, see e.g. Schellenberger et al. (2009) Nat Biotechnol. 27(12):1186-90) and increasing size, increasing hydrodynamic radius, changing charge, or Other such modifications and fusions that target receptors for recycling rather than clearance, as well as combinations of such modifications, are included. Specific examples of half-life extenders are discussed and illustrated in detail below.

3. TNFR1 antagonist constructs, TNFR2 agonist constructs; multispecific, including bispecific, TNFR1 antagonist and TNFR2 agonist constructs; A construct is provided. Constructs provided herein include constructs discussed below that are multispecific, such as bispecific to inhibit TNFR1 signaling and agonize TNFR2. The design of these constructs because the bispecific antagonists TNFR1 or TNFR2 can inhibit the ability of TNF to induce conformational activation changes in the resting trimeric TNFR, preventing its signaling. Caution is required. Other multimeric molecules run the risk of receptor aggregation, thus forcing TNFR to signal cellular inflammation and apoptosis. Multispecific constructs herein generally target different receptors, such as TNFR1 and TNFR2, respectively. By inhibiting TNFR1 signaling and advantageously agonizing TNFR2 activity, this provides improved treatment of TNF-mediated diseases, conditions, and disorders.

Among the constructs provided herein are TNFR1 antagonist constructs. These include fusion protein constructs such as TNFR1 antagonist-Fc fusion constructs. specifically targets TNFR1 or contains TNFR2 agonist activity without antagonizing or substantially antagonizing TNFR2, as described herein and exemplified in the Examples. Alternatively, a TNFR1 antagonist may be selected, produced, or designed. TNFR1 antagonist constructs improve the therapeutic efficacy and safety of conventional TNFR1 antagonists, including monovalent antagonists such as dAbs, scFvs and Fabs.

Also provided are selective TNFR2 agonist constructs, such as TNFR2-Fc fusion constructs, that improve the therapeutic efficacy of traditional TNFR2 agonists. For example, as shown herein, the half-life of Fc fusion constructs increases the half-life of previous TNFR1 antagonists or TNFR2 agonists, which, for example, reduces the frequency of dosing and improves patient compliance. , improving the therapeutic index. Also provided are selective TNFR2 agonist constructs, such as TNFR2-Fc fusion constructs, that improve the therapeutic efficacy of traditional TNFR2 agonists. For example, as shown herein, the half-life of Fc fusion constructs increases the half-life of previous TNFR1 antagonists or TNFR2 agonists, which, for example, reduces the frequency of dosing and improves patient compliance. , improving the therapeutic index. Alternative candidate half-life extenders, including pegylation and fusion to peptides, are discussed above, and exemplary extenders are detailed below (Strohl (2015) BioDrugs 29(4): 215-239; see also Tan et al. (2018) Current Pharmaceutical Design 24:4932-4946), including PEGylation using linear or branched PEGs (e.g. Swierczewska et al. (2015) Expert Opin Emerg Drugs 20(4):531-536).

The TNFR1 agonist construct includes a suitable linker and a suitable activity modifier. They may be assembled in any order. The structure of the TNFR1 antagonist construct can be represented by Formula 1:
(TNFR1 inhibitor) n-linker p-(activity modifier) q, formula 1a, or (activity modifier) q-linker p-(TNFR1 inhibitor) n, formula 1b, where:
n and q are each an integer and are each independently 1, 2, or 3; p is 0, 1, 2, or 3; the activity modifier increases the serum half-life of the TNFR1 inhibitor , a polypeptide such as albumin, or a moiety such as an Fc that has been modified to have reduced or no ADCC activity; a TNFR1 inhibitor is a polypeptide or moiety that binds to and inhibits TNFR1 activity. Molecules such as small drug molecules. The activity modifier is not a human serum albumin antibody or unmodified Fc. Also provided are TNFR2 agonists of formula 3: (TNFR2 agonist) n-linker p-(activity modifier) q, where n, p and q, the linker, and the activity modifier are as shown for formula 1. .

Also provided are multispecific constructs, including bispecificities comprising a TNFR1 antagonist (TNFR1 inhibitor) and a TNFR2 agonist, linked directly or via a linker. Such a construct may include a TNFR1 antagonist of the formula above, or may have the structure shown in Formula 2 below. Bispecific and multispecific constructs selectively inhibit inflammatory and deleterious TNFR1 signaling and enhance protective and anti-inflammatory TNFR2 signaling. They contain moieties that provide advantageous pharmacokinetic properties, including increased serum half-life and stability, and decreased peripheral clearance, as compared to conventional TNFR1 antagonists and TNFR2 agonists.

The structure of multispecific, such as bispecific, molecules/constructs provided herein is represented by the following formula (Formula 2):
(TNFR1 inhibitor) n-(activity modifier) r1-linker (L) p-(activity modifier) r2-(TNFR2 agonist) q,
[wherein n = 1, 2, or 3, p = 1, 2, or 3, r1 and r2 are each independently = 0, 1, 2, and q = 0, 1, or 2 ].

Similar to Equation 1, the order of the components may vary. The linker may include multiple components, such as a GS linker, a polymeric moiety such as PEG, or other such linkers, or a hinge region, or a combination of other components, and the activity modifier can include an Fc region, or a modified Fc region, or a polypeptide that increases half-life, or a moiety that alters the activity of the construct, such as resistance to endogenous inhibitors. The components of Formulas 1 and 2 may be polypeptides or include other molecules such as small drugs or chemical linkers that specifically bind, or non-peptide activity modifiers. Examples of each component are described below.

A construct containing the following is also provided (Equation 5):
(TNFR2 agonist) n-linker p-(activity modifier) q, formula 5a, or (activity modifier) q-linker p-(TNFR2 agonist) n, formula 5b,
wherein each component is as defined above in Formula 1, and the TNFR2 agonist can be a small molecule or a polypeptide, such as a TNFR2 single chain antibody agonist or a portion thereof.

4. Components of TNFR1 Antagonist Constructs, TNFR2 Agonist Constructs, and Multispecific, Including Bispecific TNFR1 Antagonist/TNFR2 Agonist Constructs Descriptions and examples of constructs, as well as each component of the constructs provided herein, are described below. section. Exemplary forms of each construct are shown and described by Equations 1 and 2 above and Equations 3 and 4 below.

a. TNFR1 inhibitor moiety (TNFR1 antagonist)
The TNFR1 inhibitor moiety in Formula 1 above and the multispecific molecule/construct (Formula 2 above) provided herein is any molecule, including polypeptides or small molecules, that inhibits TNFR1 signaling. . This includes TNFR1 inhibitors that selectively inhibit TNFR1 signaling without inhibiting TNFR2 signaling.

To avoid receptor clustering that agonizes TNFR1, TNFR1 antagonist constructs are generally monomeric/monovalent. The TNFR1 antagonist inhibitor component of the construct may be one known to have TNFR1 antagonist activity or may be selected from a library, such as a phage library, an antibody library, or an aptamer library. Identification may be performed by, for example, Some TNFR1 inhibitor moieties are modified or selected to increase specificity or affinity for TNFR1 and have agonist activity toward TNFR1 (with adverse side effects from such activity being less than grade 2 and generally Some moieties have little or no (such as grade 1 or below based on the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system) and, where appropriate, also have agonist activity towards TNFR2. In those cases, the TNFR1 inhibitor moiety may be provided as a single chain antibody, or in any other form described herein, including, for example, a half-life extender, e.g. Any of those described, such as linked to a modified Fc region or Fc dimer, or to another moiety or moieties that increase serum half-life.

For example, as provided herein, the TNFR1 inhibitor component of a TNFR1 antagonist construct may be or include a human domain antibody (dAb) that specifically binds TNFR1. A dAb may include a variable region heavy chain (VH) or light chain (VL) domain. dAbs used herein include, for example, DOM1h-574-208 (SEQ ID NO: 54) (derived from DMS5541; see SEQ ID NO: 38), GSK1995057 (see SEQ ID NO: 55) and GSK2862277 (SEQ ID NO: U.S. Pat. No. 9,028,817; and U.S. Pat. No. 9,028,822. US Patent Application Publication Nos. 2006/0083747, 2010/0034831, and 2012/0107330; and International Application Publication Numbers: WO2004/058820, WO2004/081026, WO2005/035572, WO2006/038027, WO200 7 /049017, WO2008/149144, WO2008/149148, WO2010/094720, WO2011/051217, WO2011/006914, WO2012/172070, WO2012/104322, and WO2015/104322, and other related family member applications and patents; and See also Enever et al. (2015) Protein Engineering, Design & Selection 28(3):59-66 (provides sequences and discussion of various dAbs). Vhh dAbs are provided that include heavy chains. These dAbs may be linked directly or indirectly to moieties, such as Fc or HSA, that increase serum half-life and may also impart other properties or activities to the construct.

The anti-TNFR1 inhibitor component may be or include a Nanobody. Examples of these are (Nb)Nb70 and/or Nb96 (see SEQ ID NOs: 683 and 684, respectively). These dAbs and Nbs are investigated for immunogenicity and, if necessary, modified using molecular modeling and mutagenesis to remove predicted immunogenic sequences. Immunogenic sequences can be eliminated by standard methods known in the art. For example, potentially antigenic peptides are identified and conservative substitutions are made for each amino acid to identify those that are non-antigenic and retain activity. Other methods are known (see, eg, Schubert et al. (2018) PLoS Comput Biol.14(3):e1005983, which describes methods for deimmunizing proteins).

Thus, for example, the TNFR1 antagonist dAb moiety may be a dAb set forth in any of SEQ ID NOs: 54-672, or a dAb set forth in any of SEQ ID NOs: 54-672, or any TNFR1 antagonist dAb known to those skilled in the art. dAbs may have about or at least about 85%, 90%, 95%, 98%, 99% or more sequence identity to.

Other TNFR1 antagonists include, for example, antigen-binding antibody fragments. For example, TNFR1 antagonists can be Fab fragments, Fab' fragments, single chain Fv (scFv), disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single chain Fab (scFab), hsFv (helix-stabilized Fv). ), a free light chain, or an antigen-binding fragment of any of the above. A linker, such as a GS linker, may also be included within the construct, for example to increase plasticity.

For example, the TNFR1 inhibitor portion of the antagonist may include an antigen binding fragment derived from a TNFR1 antagonist antibody called ATROSAB. The fragment has at least 85%, 90%, 95% or more sequence identity to or to the heavy or light chain CDRs of ATROSAB (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity). The TNFR1 antagonist is at least 85%, 90% of the VH (residues 1-115 of SEQ ID NO: 31) and/or VL (residues 1-113 of SEQ ID NO: 32) of ATROSAB, or the VH or VL of ATROSAB , 95%, or more sequence identity. For example, it may include a dAb derived from ATROSAB. TNFR1 antagonists include other monovalent antibody fragments of ATROSAB, including, for example, Fab or scFv fragments such as ATROSAB Fab (FabATR) light and heavy chains set forth in SEQ ID NO: 679 and 680, respectively, or as set forth in SEQ ID NO: 673. ATROSAB scFv (scFv IZI06.1). For example, the scFv is linked to residues 1-113 of the ATROSAB light chain (see SEQ ID NO: 32) by a short peptide linker (e.g., GGGGSGGGGSGGSAQ, such as SEQ ID NO: 673, or a linker shown in any of SEQ ID NOs: 813-834). The VH domain corresponding to residues 1-115 of the ATROSAB heavy chain (see SEQ ID NO: 31) is linked to the VL domain corresponding to the ATROSAB heavy chain (see SEQ ID NO: 31). TNFR1 antagonists may include variants of ATROSAB scFV that include 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 comprising the sequence set forth in SEQ ID NO: 675, or scFv 13.7 comprising the sequence set forth in SEQ ID NO: 676, or at least 90% sequence identity to the sequences of scFv IG11, scFv T12B, and scFv 13.7. Including variants containing. The TNFR1 antagonist may also include the sequence of amino acid residues from Fab13.7 light chain and heavy chain (derived from scFV13.7) as shown in SEQ ID NOs: 681 and 682, respectively.

TNFR1 inhibitors in TNFR1 antagonist constructs also include TNF variants (mutes) that bind to TNFR1 and reduce or inhibit signal transduction. These include, but are not limited to, mutations L29S, L29G, L29Y, R31E, R31N, R32Y, R32W, S86T, L29S/R32W, L29S/S86T, R32W/S86T, which confer selectivity for TNFR1. , L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, and E146R (see SEQ ID NO: 2). included. TNFR1 antagonists can include, for example, the TNFR1 selective antagonist TNF mutein derived from the mutein designated XPro1595 (see SEQ ID NO: 701). XPro1595 contains mutations V1M, R31C, C69V, Y87H, C101A and A1456R with respect to SEQ ID NO:2. Other exemplary TNFR1 selective antagonizing TNF muteins are XENP345 (see SEQ ID NO: 702), which includes mutations I97T/A145R (with respect to SEQ ID NO: 2); and mutations A84S, V85T, S86T, Y87H, It is derived from a TNFR1 selective antagonizing TNF mutein called R1antTNF (see SEQ ID NO: 703), which includes Q88N and T89Q (with respect to SEQ ID NO: 2). TNFR1 inhibitors used in TNFR1 antagonists also include small molecule inhibitors that can be chemically attached to a linker.

As described herein, see, e.g., the Examples, a TNFR1 inhibitor (antagonist) moiety may be modified to improve its specificity/selectivity for TNFR1 and, if appropriate, TNFR2 It may also be modified to have agonist activity. TNF binds to TNFR1 with a low pM affinity (Kd19 pM); in general, the antagonists herein have at least the same affinity as TNF, but whose activity "locks" the receptor into an inactive conformation. This is not necessary as the receptor will be locked unless it is caused by TNFR1 antagonist constructs provided herein include KD values of less than or equal to 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 embodiments, the TNFR1 antagonist is 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, 8 30pM, 820pM , 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM, 700pM, 690pM, 680pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM , 560pM, 550pM, 540pM, 530pM, 520pM, 510pM, 500pM, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400pM, 390pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM , 310pM, 300pM, 290pM, 280pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, 160pM, 150pM, 140pM, 130pM, 120pM, 110pM, 100pM, 90pM, 80pM, 70pM , 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).

TNFR1 antagonist constructs provided herein are also selected or designed to lack or reduce binding to other TNFR superfamily members. For example, they are evaluated using any suitable in vitro binding assay to identify those that do not specifically bind to another TNFR superfamily member, such as TNFR2. Assays include, for example, ELISA-based methods. For example, a TNFR1 antagonist construct may specifically bind to human TNFR1 or a TNFR1-derived peptide with greater affinity than to another family member or its corresponding peptide. The increased affinity is at least or at least about 5 times greater (e.g., at least 5 times greater, 6 times greater, 7 times greater, 8 times larger, 9 times larger, 10 times larger, 20 times larger, 30 times larger, 40 times larger, 50 times larger, 60 times larger, 70 times larger, 80 times larger, 90 times larger, 100 times larger, 200 times larger. 300 times larger, 400 times larger, 500 times larger, 600 times larger, 700 times larger, 800 times larger, 900 times larger, 1,000 times larger, 2,000 times larger, 3,000 times larger, 4,000 times larger larger, 5,000 times larger, 6,000 times larger, 7,000 times larger, 8,000 times larger, 9,000 times larger, 10,000 times larger, or more).

Among the TNFR1 antagonist constructs provided herein are constructs 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 be used in the presence of TNFR1 of about 104 M-1 s-1 or more (e.g., 1.0 x 104 M-1 s-1, 1.5 x 104 M-1 s-1, 2 .0×104M-1s-1, 2.5×104M-1s-1, 3.0×104M-1s-1, 3.5×104M-1s-1, 4.0×104M-1s-1, 4 .5×104M-1s-1, 5.0×104M-1s-1, 5.5×104M-1s-1, 6.0×104M-1s-1, 6.5×104M-1s-1, 7 .0×104M-1s-1, 7.5×104M-1s-1, 8.0×104M-1s-1, 8.5×104M-1s-1, 9.0×104M-1s-1, 9 .5×104M-1s-1, 1.0×105M-1s-1, 1.5×105M-1s-1, 2.0×105M-1s-1, 2.5×105M-1s-1, 3 .0×105M-1s-1, 3.5×105M-1s-1, 4.0×105M-1s-1, 4.5×105M-1s-1, 5.0×105M-1s-1, 5 .5×105M-1s-1, 6.0×105M-1s-1, 6.5×105M-1s-1, 7.0×105M-1s-1, 7.5×105M-1s-1, 8 .0×105M-1s-1, 8.5×105M-1s-1, 9.0×105M-1s-1, 9.5×105M-1s-1, 1.0×106M-1s-1 or more) can indicate the kon value of For example, the TNFR1 antagonists provided herein may be less than or equal to about 10-3 s-1 (e.g., about 1.0 x 10-3 s-19.5 x 10-4 s-1) when complexed with TNFR1. , 9.0×10-4s-1, 8.5×10-4s-1, 8.0×10-4s-1, 7.5×10-4s-1, 7.0×10-4s-1 , 6.5×10-4s-1, 6.0×10-4s-1, 5.5×10-4s-1, 5.0×10-4s-1, 4.5×10-4s-1 , 4.0×10-4s-1, 3.5×10-4s-1, 3.0×10-4s-1, 2.5×10-4s-1, 2.0×10-4s-1 , 1.5×10-4s-1, 1.0×10-4s-1, 9.5×10-5s-1, 9.0×10-5s-1, 8.5×10-5s-1 , 8.0×10-5s-1, 7.5×10-5s-1, 7.0×10-5s-1, 6.5×10-5s-1, 6.0×10-5s-1 , 5.5×10-5s-1, 5.0×10-5s-1, 4.5×10-5s-1, 4.0×10-5s-1, 3.5×10-5s-1 , 3.0×10-5s-1, 2.5×10-5s-1, 2.0×10-5s-1, 1.5×10-5s-1, or 1.0×10-5s-1 1 or less).

The C-terminus of a TNFR1 antagonist (the TNFR1 inhibitor portion of the constructs of Formula 1 and Formula 2), such as any of the TNFR1 antagonist constructs described herein, is as discussed below and elsewhere herein. , to the activity modifier, or more commonly via a linker or combination of linker elements, or to the N-terminus of the TNFR2 agonist (or to the small molecule TNFR2 agonist) may also be fused. Alternatively, the N-terminus of the TNFR1 inhibitor moiety may be fused to the C-terminus of a TNFR2 agonist, or the C-terminus of the TNFR1 inhibitor moiety (or a small molecule TNFR2 agonist) can be linked directly or via a linker to an activity modifier. or to a linker.

The linker (L), discussed in more detail below, is any that improves pharmacological properties, including increasing stability and plasticity, and reducing steric hindrance, and optionally imparting additional properties to the construct. be. A linker may include multiple components, each component providing specific properties. For example, a TNFR1 antagonist may 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 region is modified, for example, to eliminate or reduce ADCC activity and/or to alter receptor binding and/or for other such activities and properties. Linkers also include chemical linkers, as discussed below. For example, in some embodiments, the linker is a poly(ethylene glycol) (PEG) molecule, or a branched PEG molecule, such as one with a molecular weight of about 30 kDa or greater.

b. TNFR2 Agonist and TNFR2 Antagonist Constructs TNFR2 agonist (regulatory T cell generating factor) constructs can also be used to treat other diseases, disorders, and conditions, inflammatory and autoimmune diseases, and also solid tumors. Regulatory T cells (Treg) suppress autoimmunity and exert immunosuppressive effects in the tumor microenvironment and the like. Treg proliferation is positively regulated by TNFR2, and lack of TNFR2 correlates with reduced Treg numbers and worsening of experimental arthritis. Accordingly, TNFR2 agonist constructs may be used to treat many autoimmune diseases, other chronic inflammation, and other acute inflammatory conditions (eg, SARS, COVID-19).

TNFR2 antagonist constructs suppress regulatory T cells and are used in the treatment of cancer and other hyperproliferative diseases (TNFR2 is a "checkpoint receptor"). Regulatory T cells accumulate in the tumor microenvironment and are involved in suppressing anti-tumor immune responses. TNFR2 antagonist constructs are for the treatment of cancer and other hyperproliferative diseases such as Dupuytren's contracture and idiopathic pulmonary fibrosis.

As discussed above, TNFR2 agonist constructs including TNFR2 agonists are also provided. These include TNFR2 agonists linked directly or via linkers to activity modifiers, such as bispecific constructs containing TNRF1 antagonists and TNFR2 agonists in various configurations with linkers having appropriate structure and properties. Also included are multispecific constructs. In some embodiments, the TNFR2 agonist is in a bispecific construct. The TNFR2 agonists provided herein do not activate, or do not substantially activate, TNFR1, particularly in multispecific, such as bispecific, molecules/constructs, and/or Selectively activate or agonize TNFR2 without interfering with inhibition of TNFR1 signaling through the TNFR1 antagonist portion of multispecific molecules such as bispecifics.

TNFR2 agonists can be any known to those skilled in the art, including agonist antibodies and antigen-binding portions thereof, as well as single chain and other constituent derivatives of antibodies, and can be small molecule agonists. TNFR2 agonists can also be generated by in silico design, and/or by candidate preparation and library screening, and the like. For example, phage libraries, or antibody libraries, or aptamer libraries can be screened to identify TNFR2 agonists. TNFR2 agonist antibodies, or antigen-binding fragments thereof, can be generated by screening libraries of antibodies and antigen-binding fragments thereof for functional molecules that bind to epitopes within TNFR2 and selectively promote receptor activation. . Examples of such methods and molecules are those described in International Application Publication No. WO2017/040312.

Development of TNFR2 selective agonists can include elucidation of epitopes within TNFR2 that promote agonist receptor binding. Epitope mapping analyzes using linear peptides and constrained cyclic and bicyclic peptides derived from different regions of TNFR2 show that agonistic TNFR2 antibodies are conformation-dependent to epitopes derived from different regions of the TNFR2 polypeptide. It shows that it is combined with For example, one identified epitope of TNFR2 comprises residues 56-60 of SEQ ID NO: 4 (KCSPG). Agonist TNFR2 antibody MR2-1 binds to this epitope; this antibody does not bind to the epitope comprising residues 142-146 of SEQ ID NO: 4 (KCRPG). Human TNFR2 can be selected to bind to an epitope, such as that comprising residues 56-60 of SEQ ID NO: 4. Generally, human TNFR2 agonists are selected to bind to an epitope within human TNFR2 that includes at least five discrete or contiguous residues within residues 96-154 of SEQ ID NO: 4 (CGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGT; SEQ ID NO: 841). been or may be designed and/or may bind to an epitope within residues 111-150 (TREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA; SEQ ID NO: 842) of SEQ ID NO: 4, to which MR2-1 further binds. Human TNFR2 agonists also include residues 115-142 of SEQ ID NO: 4 (NRICTCRPGWYCALSKQEGCRLCAPLRK; SEQ ID NO: 843), and residues 122-136 of SEQ ID NO: 4 (PGWYCALSKQEGCRL; SEQ ID NO: 844), and/or residues of SEQ ID NO: 4. an epitope within residues 96-122 (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 further binds), and/or An epitope within amino acids 48-67 of SEQ ID NO: 4 (QTAQMCCSKCSPGQHAKVFC; SEQ ID NO: 847), and/or residues 130-149 of SEQ ID NO: 4 (KQEGCRLCAPLRKCRPGFGV; SEQ ID NO: 848), and/or residues 110-1 of SEQ ID NO: 4. 147 (CTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGGF; SEQ ID NO: 849) and/or an epitope comprising positions 106 to 155 of SEQ ID NO: 4 (ETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGF) ; SEQ ID NO: 850) and/or the remainder of SEQ ID NO: 4. It may bind to an epitope comprising groups 137-144 (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 fragment thereof specifically binds to an epitope within or comprising any one amino acid residue of SEQ ID NOs: 853-1211, thereby causing this antibody or antigen-binding The fragment specifically binds human TNFR2, but not other TNFR superfamily members, particularly TNFR1. Human TNFR2 agonist antibodies or antigen-binding fragments thereof do not bind or have impaired/reduced binding to other members of the TNFR superfamily, including TNFR1 (see, e.g., International Application Publication No. WO 2017/040312). (see ).

Epitopes within TNFR2 that can be used to screen for TNFR2 agonists include peptides whose sequences are shown in any of SEQ ID NOs: 853-1211. These peptides can be converted into cyclic and polycyclic formats (e.g., cysteine residues at the N- and C-terminal positions or within the peptide chain) to restrict the peptide fragments to different three-dimensional conformations. (by incorporating into various internal positions), mimicking the structurally rigid framework of TNFR2 and the conformational constraints of peptide fragments within TNFR2. Cyclic and polycyclic peptide fragments can then be immobilized on a solid surface and ELISA used to screen for molecules that bind to, for example, the TNFR2 agonist antibody MR2-1. Using this assay, peptides containing residues within the epitope of TNFR2 that promote receptor activation are constructed by structurally prepositioning these amino acids to resemble the conformation of the corresponding peptide in the native protein. can be organized into Cyclic and polycyclic peptides thus obtained (e.g. peptides having a sequence of any one of SEQ ID NOs: 853-1194, in particular KCSPG motifs such as SEQ ID NOs: 905, 921, 927, 970, and 1085). can be used to screen libraries of antibodies and antigen-binding fragments thereof to identify TNFR2 agonists for use herein. The constrained peptide acts as a surrogate for the epitope within TNFR2 that promotes receptor activation, and therefore antibodies or antigen-binding fragments generated using this screening technique bind to the corresponding epitope within TNFR2. and is an agonist of receptor activity (see, for example, International Application Publication WO 2017/040312). Phage display is used to generate TNFR2 agonists. The phage display library is contacted under conditions where specific binding occurs. A TNFR2-derived peptide (eg, any peptide of SEQ ID NOs: 853-1194) is immobilized on a solid support or in a phage. Phage containing the TNFR2 binding moiety forms a complex with the target on the solid support, and unbound phage is washed away. Bound phages are then released from the target by changing the buffer to extreme pH (pH 2 or 10), changing the ionic strength of the bagger, adding denaturants, or other known means. be done. Protein elution may be performed to isolate bound phage (see, eg, International Application Publication No. WO 2017/040312).

MR2-1 is an example of an agonist TNFR2 antibody that binds to TNFR2 and enhances TNFR2-mediated Treg cell proliferation. MR2-1 binds to osteoprotegerin, but by modifying the variable region of the heavy chain and/or light chain of this antibody, or specifically the CDRs of the heavy chain and/or light chain of MR2-1, The resulting antibodies or fragments thereof may bind to TNFR superfamily members other than TNFR2, eliminating the ability to generate agonistic TNFR2 antibodies or antigen-binding fragments thereof. This can be achieved using genetic engineering and/or antibody library screening techniques, for example, as described in International Application Publication No. WO2017/040312.

As provided herein, TNFR2 agonists include antigen-binding fragments of agonist human anti-TNFR2 antibodies, such as MR2-1 and MAB2261, such as MR2-1 commercially available from Hycult Biotech; and MAB2261 from R&D Systems. may include. For example, the VH and VL domains of MR2-1 or MAB2261, or one or more of the CDRs contained therein, are used to generate a TNFR2 agonist. Such agonists may include human domain antibodies (dAbs) specific for TNFR2; may include a VH or VL having 85%, 90%, 95% or more sequence identity to a VH or VL of MR2-1 or MAB2261, provided that the resulting TNFR2 retains TNFR2 agonist activity. . TNFR2 agonists also include other antigen-binding fragments derived from the MR2-1 or MAB2261 antibodies, or sequences of amino acids having at least or at least about 85%, 90%, 95% or more sequence identity thereto, such as Fab fragments. , Fab' fragment, F(ab')2 fragment, Fv fragment, disulfide bonded Fv (dsFv), Fd fragment, Fd' fragment, single chain Fv (scFv), single chain Fab (scFab), hsFv (helix stabilized Fv) ), minibodies, diabodies, anti-idiotypic (anti-Id) antibodies, free light chains, or antigen-binding fragments of any of the foregoing. Antibody fragments include any of the above fragments, such as tandem scFv, Fab-scFv (HC C-terminus or LC C-terminus), Fab-(scFv)2 (C-terminus), scFv-Fab-scFv, Fab-CH2- Combinations of scFv, scFv fusions (C-terminus, or N-terminus), Fab-fusions (HC C-terminus, or LC C-terminus), scFv-scFv-dAb, scFv-dAb-scFv, dAb-scFv-scFv, and tribodies are Can be mentioned. A TNFR2 agonist, the sequence of which is provided herein or known in the art, has a sequence identity of about or at least about 85%, 90%, 95%, or more, and TNFR2 agonist activity. Including any dAb that has.

In some embodiments, the TNFR2 agonist is a TNFR2 agonist monoclonal antibody scFv (including those known in the art), or about or at least about 85%, 90%, 95% or 95% of such scFv. scFv with greater sequence identity, provided that the resulting construct retains TNFR2 agonist activity. In some embodiments, the TNFR2 agonist is a Fab fragment of a TNFR2 agonist monoclonal antibody or a Fab thereof, or has at least about 85%, 90%, 95% or more sequence identity and TNFR2 agonist activity. It may be Fab.

TNFR2 agonists may also be or include TNF muteins that have been modified to bind to TNFR2 and have agonist activity (see, eg, SEQ ID NOs: 765-800). Examples of such embodiments include TNFR2 selective TNF muteins, such as 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/E 146T/S147D, A145R/S147T, E146D/ TNF variants having one or more of S147D, D143V/F144L/A145S, and D143V/A145S, and combinations thereof, such as the combination of D143V/A145S and S95C/G148C (see SEQ ID NO: 2). It is a TNFR2 agonist containing. For example, a TNF variant with mutations D143N/A145R (SEQ ID NO: 781) binds and agonizes TNFR2 and can be used in the constructs provided herein. Combinations of TNF muteins with mutations S95C/G148C and any others listed with reference to SEQ ID NO: 2 or known or identified are also included in the constructs provided herein. TNFR2 selective agonist.

The TNFR2 agonist may comprise a fusion of a single chain TNFR2 selective TNF mutein trimer and a multimerization domain. 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 a multimerization domain, such as a dimerization domain or a trimerization domain, generates hexameric or nonamer molecules, respectively, for TNF subunits; The 3-mer mimics trimers of membrane-bound TNF, thereby activating TNFR2 signaling. Dimerization domains include, for example, EHD2 (SEQ ID NO: 808), discussed above. EHD2 is derived from the IgE heavy chain CH2 domain, and MHD2 (SEQ ID NO: 811) is derived from the IgM heavy chain CH2 domain. Dimerization domains also include Fc domains, such as those derived from IgG1 (see SEQ ID NO: 10) and IgG4 (see SEQ ID NO: 16), which alter immune effector functions and/or Alternatively, modifications such as ones that enhance FcRn recycling are included as appropriate. Trimerization domains include, for example, the trimerization domain of chicken tenascin C (TNC) (SEQ ID NO: 805) and the trimerization domain of human TNC (SEQ ID NO: 807). Dimerization and trimerization enhance TNFR2 signaling and improve the pharmacological properties of the construct. For example, the half-life of the fusion protein is increased by increasing the molecular weight of the molecule, eg, when the dimerization domain is Fc, and/or by introducing FcRn recycling.

As provided herein, a TNFR2 agonist has any of the mutations described herein that confer selectivity for TNFR2 and/or reduce or eliminate binding to TNFR1. It may also include a TNF mutein (TNFmut) trimeric chain. An example of such a mutation is the substitution D143N/A145R (see SEQ ID NO: 2), fused to a multimerization domain (MD), such as a dimerization domain or a trimerization domain. The multimerization domain may be fused to the N-terminus 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. It will be done. Such TNFR2 agonists have formulas 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 the multimerization domain (activity modifier); TNFmut is a TNFR2-selective TNF mutein, such as the mutein with mutations D143N/A145R; and L1, L2, and L3 are Gly-Ser A linker, such as a linker, described below, which may be the same or different.

In certain embodiments, the multimerization domain includes EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), trimerization domain of chicken TNC (SEQ ID NO: 805), trimerization domain of human TNC (SEQ ID NO: 805), 807), IgG1 Fc, or IgG4 Fc. If the dimerization domain is an IgG1 Fc or an IgG4 Fc, it is the same Fc and not an additional Fc that is used to link the TNFR1 antagonist to the TNFR2 agonist. IgG1 or IgG4 Fc can be modified to enhance or eliminate immune effector functions such as ADCC, ADCP and/or CDC activity and/or enhance FcRn binding. Multimerization domains, such as the Fc region, increase the in vivo stability and serum half-life of the construct. For purposes herein, the Fc region in the constructs of Formulas 1-5, or variations thereof, is generally modified to alter or modulate the pharmacological properties or activity of the construct. Fc modifications are discussed in detail below. Any multimerization domain known in the art is also contemplated for use in the TNFR2 agonists herein.

A TNF mutein can be a TNF variant with any one or more mutations that confer TNFR2 selectivity. Examples of mutations include K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S1. 47T, Q88N/T89S /A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E1 46S/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 (SEQ ID NO. 2). TNF variants with mutations D143N/A145R are contemplated for use herein. Any other mutations known in the art that confer TNFR2 selectivity are also contemplated for use herein. A TNF mutein may include the entire sequence of soluble TNF (i.e., residues 1-157 of SEQ ID NO: 2) or be of sufficient length to bind and/or agonize TNFR2, e.g., SEQ ID NO: 2. may include subsequences of soluble TNF, such as residues 4-157, 9-157, or 12-157 of .

The L1, L2, or L3 linkers may be the same or different. Specifically, the linker may include a short peptide linker, such as a GS linker. For example, the linker may include (GGGGS)n, where n=1-5 (SEQ ID NO: 1471). The linker also comprises the stalk region of TNF-α, which comprises 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) as shown in SEQ ID NO: 1. (at least 10, 15, or 20 contiguous residues). For example, a linker comprising all or a portion of the stalk region, including at least 10, 15, or 20 contiguous amino acid residues, can be present between the N-terminal or C-terminal TNF mutein and the multimerization domain. All three linkers may be (GGGGS)n, where n is generally 1-10 (SEQ ID NO: 1472), or other combinations of Gly-Ser, or of the stalk region of TNF. It may include mixtures of Gly-Ser residues, such as (GGGGS)n and all or part, comprising at least 10, 15, or 20 contiguous amino acid residues. Exemplary linkers are set forth in SEQ ID NOs: 813-834, 1471 and 1472.

TNFR2 agonists provided herein include about 100 nM or less (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 agonist is 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, 830pM. 30pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 520pM, 510pM, 500pM, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400pM, 390pM, 3 80pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM, 280pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, 160pM, 150pM, 140pM, 1 30pM, 120pM, 110pM, 100pM, 90pM, specifically binds with a KD value of 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).

TNFR2 agonists stimulate the proliferation of Tregs (e.g., CD4+, CD25+FOXP3+ Tregs), e.g., in vivo in a subject to whom the agonist is administered, or in vitro in a sample containing Treg that is contacted with the TNFR2 agonist for testing purposes. It is an inducible agonist. Proliferation of Treg is, for example, between about 0.00001% and 100.0% (eg, 0.00001% to 100.0%), as measured by, eg, FACS analysis, as compared to a subject or sample comprising a population of cells not treated with a TNFR2 agonist. 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%).

Accordingly, TNFR2 agonists may be used to promote Treg cell proliferation and may be administered to a mammalian subject, such as a human patient with an autoimmune or chronic inflammatory disease or disorder, to increase the immune response in the patient. (e.g., the amount of CD8+ cytotoxic T lymphocytes produced in vivo in response to self-antigens or non-threatening foreign antigens). For example, administration of a TNFR2 agonist to a human patient, or administration of a population of Treg cells expanded ex vivo by treatment with a TNFR2 agonist, results in a reduction of, for example, 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 .0mg/mL), or 0.001 to 1.0mg/mL (e.g., 0.001mg/mL, 0.005mg/mL, 0.010mg/mL, 0.050mg/mL, 0.10mg/mL, 0 .20mg/mL, 0.30mg/mL, 0.40mg/mL, 0.50mg/mL, 0.60mg/mL, 0.70mg/mL, 0.80mg/mL, 0.90mg/mL, or 1. 0 mg/mL) can cause a decrease in the amount of secreted immunoglobulin (eg, IgG) that cross-reacts with self-antigens or non-staggering antigens. Additionally or alternatively, the TNFR2 agonist increases the number of cytotoxic T cells (e.g., levels of CD8+ T cells) in a subject, as measured by, e.g., FACS analysis, compared to a subject not treated with a TNFR2 agonist. , for example, about 0.00001% to 100.0% (for example, 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%). For example, a TNFR2 agonist can be administered to a subject (eg, a mammalian subject, such as a human) to treat an autoimmune or chronic inflammatory disease or disorder such as those described herein. Treating a subject in this manner reduces the amount of autoreactive CD8+ T cells within the subject.

The TNFR2 agonists provided herein can be evaluated to identify agonists that lack specific binding to another TNFR superfamily member, particularly TNFR1. This can be accomplished using any of a variety of in vitro binding assays such as ELISA-based methods known to those skilled in the art. For example, as a TNFR2 agonist, human TNFR2 or a TNFR2-derived peptide, such as a peptide fragment comprising residues 48-67 of SEQ ID NO: 4 in human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 847), versus another TNFR2 such as TNFR1 For example, at least or at least about 2 times, 3 times, 4 times, or 5 times greater than the affinity of the agonist for the superfamily member (e.g., 5 times greater, 6 times greater, 7 times greater, 8 times greater, 9 times bigger, 10 times bigger, 20 times bigger, 30 times bigger, 40 times bigger, 50 times bigger, 60 times bigger, 70 times bigger, 80 times bigger, 90 times bigger, 100 times bigger, 200 times bigger, 300 times bigger , 400 times larger, 500 times larger, 600 times larger, 700 times larger, 800 times larger, 900 times larger, 1,000 times larger, 2,000 times larger, 3,000 times larger, 4,000 times larger, 5 ,000 times greater, 6,000 times greater, 7,000 times greater, 8,000 times greater, 9,000 times greater, 10,000 times greater, or more) Can be mentioned.

TNFR2 agonists provided herein include agonists that exhibit high kon and low koff values upon interaction with TNFR2, consistent with high affinity receptor binding. For example, the TNFR2 agonists provided herein may be more than about 104 M-1 s-1 (e.g., about 1.0 x 104 M-1 s-1, 1.5 x 104 M-1 s-1, 2.0×104M-1s-1, 2.5×104M-1s-1, 3.0×104M-1s-1, 3.5×104M-1s-1, 4.0×104M-1s-1, 4.5×104M-1s-1, 5.0×104M-1s-1, 5.5×104M-1s-1, 6.0×104M-1s-1, 6.5×104M-1s-1, 7.0×104M-1s-1, 7.5×104M-1s-1, 8.0×104M-1s-1, 8.5×104M-1s-1, 9.0×104M-1s-1, 9.5×104M-1s-1, 1.0×105M-1s-1, 1.5×105M-1s-1, 2.0×105M-1s-1, 2.5×105M-1s-1, 3.0×105M-1s-1, 3.5×105M-1s-1, 4.0×105M-1s-1, 4.5×105M-1s-1, 5.0×105M-1s-1, 5.5×105M-1s-1, 6.0×105M-1s-1, 6.5×105M-1s-1, 7.0×105M-1s-1, 7.5×105M-1s-1, 8.0×105M-1s-1, 8.5×105M-1s-1, 9.0×105M-1s-1, 9.5×105M-1s-1, or 1.0×106M-1s-1 may exhibit a kon value of greater than or about greater). For example, the TNFR2 agonists provided herein, when complexed with TNFR2, have less than or approximately less than about 10 -4s-1, 9.0×10-4s-1, 8.5×10-4s-1, 8.0×10-4s-1, 7.5×10-4s-1, 7.0×10 -4s-1, 6.5×10-4s-1, 6.0×10-4s-1, 5.5×10-4s-1, 5.0×10-4s-1, 4.5×10 -4s-1, 4.0×10-4s-1, 3.5×10-4s-1, 3.0×10-4s-1, 2.5×10-4s-1, 2.0×10 -4s-1, 1.5×10-4s-1, 1.0×10-4s-1, 9.5×10-5s-1, 9.0×10-5s-1, 8.5×10 -5s-1, 8.0×10-5s-1, 7.5×10-5s-1, 7.0×10-5s-1, 6.5×10-5s-1, 6.0×10 -5s-1, 5.5×10-5s-1, 5.0×10-5s-1, 4.5×10-5s-1, 4.0×10-5s-1, 3.5×10 -5s-1, 3.0×10-5s-1, 2.5×10-5s-1, 2.0×10-5s-1, 1.5×10-5s-1, or 1.0× 10-5s-1).

As provided herein, a TNFR2 agonist is linked directly or indirectly via a linker to a TNFR1 antagonist, such as any of those 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, may be connected to the C-terminus of a TNFR1 antagonist via one or more linkers, as discussed below and elsewhere herein. be fused. Alternatively, the C-terminus of a TNFR2 agonist can be fused to the N-terminus of a TNFR1 antagonist. When the TNFR2 agonist has the structure shown in Formula 3, the N-terminus of the multimerization domain is linked to the C-terminus of the TNFR1 antagonist, and when the TNFR2 agonist has the structure shown 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 be any linker, 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, etc. Appropriate linkers and combinations thereof may also be included. In some embodiments, the linker is a poly(ethylene glycol) (PEG) molecule of 30 kDa or greater or a branched PEG molecule. As mentioned above, if the TNFR2 agonist has the structure shown in Formula 3 or 4, then if the multimerization domain is an Fc, 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), multispecific TNFR1 antagonist-TNFR2 agonist constructs (such as Formula 2), and TNFR2 agonist constructs (such as Formulas 3-5) described above may optionally include a linker and an activity modifier. Also included. Linkers have a variety of functions, including providing additional or improved biological and pharmacological properties, and structural purposes for joining different molecules. An exemplary linker is the Gly-Ser polypeptide, hinge region (see, eg, Tables 1-4, above, showing various hinge region sequences, and combinations thereof).

Also included are polypeptide linkers and chemical linkers for chemical bonding. Linker peptides can be included as spaces between polypeptides to promote proper protein folding and stability of the polypeptide, improve protein expression, and increase biological activity of the components of the construct. Peptide linkers are designed to be primarily unstructured, flexible peptides. Linkers may be included as shown in exemplary formulas 1-4 above. For example, in the bispecific constructs provided, the components are fused via a linker (L) in an N-terminal to C-terminal or C-terminal to N-terminal configuration. Linkers generally link a polypeptide, such as an Fc region, alone or with one or more other linkers, including, for example, glycine-serine (GS) linkers and/or short peptide linkers such as immunoglobulin (Ig) hinge regions. In combination, it includes a peptide linker. In embodiments herein, for example, the C-terminus of a TNFR1 antagonist is fused to the N-terminus of a peptide linker, and the C-terminus of a peptide linker is fused to the N-terminus of a TNFR2 agonist. In other embodiments, the C-terminus of the TNFR2 agonist is fused to the N-terminus of a peptide linker, and the C-terminus of the peptide linker is fused to the N-terminus of a TNFR1 antagonist. Linkers increase molecular weight, increase stability and serum half-life, enhance tissue retention, and reduce or reduce peripheral elimination, thereby improving the therapeutic index of the molecule. The linker also increases the plasticity of the molecule, allowing each part of the molecule to interact with its target antigen/epitope, such as TNFR1 and TNFR2 provided herein. As discussed below and elsewhere herein, embodiments in which the linker comprises an immunoglobulin Fc region, generally a modified Fc region, confer additional properties, including, for example, neonatal Fc receptor (FcRn) recycling. may further improve serum stability and half-life and/or enhance or eliminate immune effector function.

i. Peptide Linkers Linkers for fusion proteins are well known to those skilled 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 may be designed from, or derived from, or based on linkers derived from naturally occurring multidomain proteins. Empirical linkers designed by researchers are generally classified according to their structure, e.g., flexible linkers, rigid linkers, used to deliver prodrugs activated by cleavage of the linker in situ, and in vivo. They fall into three categories: cleavable linkers.

In addition to the role of binding functional domains (like flexible and rigid linkers) or releasing free functional domains in vivo (like in vivo cleavable linkers), linkers may also improve the properties of the linking moiety. . These include, for example, improving biological activity, increasing expression yield, and achieving a desirable pharmacokinetic profile. Databases and methods for selecting linkers are known to those skilled in the art (e.g. George et al. (2002) "An analysis of protein domain linkers: their classification and role in protein folding," Protein Eng. 15:871 -879).

a) Flexible Linkers Flexible linkers are usually applied when the bound domains require some degree of movement or interaction. Flexible linkers are generally rich in small or polar amino acids, such as Gly and Ser, providing excellent plasticity and solubility. These are suitable choices when specific movements or interactions (eg scFv) are required for the fusion protein domain. Additionally, flexible linkers do not have a rigid structure, but can function as passive linkers to maintain the distance between functional domains. The length of the flexible linker may be adjusted to allow proper folding or achieve optimal biological activity of the fusion protein.

Flexible linkers are generally made from 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. configured. The small size of these amino acids provides plasticity and allows mobility of the connectivity functional domains. Incorporation of Ser or Thr may maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the protein moiety.

Exemplary flexible linkers are linkers that contain primarily or only stretches of Gly and Ser residues ("GS" linkers). An example is a flexible linker with the sequence (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number "n", the length of this GS linker may be selected or cherry-picked to achieve appropriate separation of functional domains and maintain the necessary interdomain interactions. . The flexible linker is also rich in small molecule or polar amino acids such as Gly and Ser, and may contain additional amino acids such as Thr and Ala to maintain plasticity, and may contain additional amino acids such as Lys and Glu to improve solubility. It may also contain polar amino acids.

The SCDKTH hinge sequence and other hinge sequences may be replaced or preceded by short polypeptide linkers to confer protease resistance and increase the flexibility of the fusion protein. An example of a polypeptide linker is a (Gly-Ser)n amino acid sequence (GS linker) with several Glu or Lys residues dispersed throughout to increase solubility. For example, polypeptide linkers include, but are not limited to, (GlySer)n (wherein n=1-10); (GlySer2); (Gly4Ser)n (wherein n=1-10); (Gly3Ser); ) n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG ; GGSSGGSGGSSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS (see SEQ ID NOs: 816-827 for GS linkers). The linker can be a polyGly peptide of at least 2 to 18 residues or more in length, or a similar linker of the same length and plasticity. Exemplary polypeptide linkers for molecules provided herein include, but are not limited to (see SEQ ID NOs: 816-827 for Gly-Ser linkers): for example, GSGS, GGGGS, or GGGGSGGGGSGGGGS. It will be done. Another linker that provides similar performance is the (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 containing only glycine may also 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 to proteolytic enzyme digestion during protein purification from expression organisms. There is.

Some other types of flexible linkers include KESGSVSSEQLAQFRSLD (SEQ ID NO: 829), and EGKSSGSGSESKST (SEQ ID NO: 830). Gly and Ser residues in the linker provide plasticity, and Glu and Lys improve solubility.

b) Rigid Linkers Although flexible linkers have the advantage of passively connecting functional domains and allowing some movement, the lack of rigidity of these linkers can be limiting. Rigid linkers are chosen when spatial separation of domains is required to maintain stability or biological activity of the fusion protein. Rigid linkers exhibit relatively rigid structures by adopting an α-helical structure or by containing multiple Pro residues. Linker length can be easily adjusted by changing copy number to achieve optimal distance between domains.

An alpha-helix-forming linker with the sequence (EAAAK)n (SEQ ID NO: 831) has been applied in the construction of many recombinant fusion proteins. The α-helical structure is rigid and stable, with intrasegmental hydrogen bonds and a compact backbone. Rigid alpha-helical linkers can function as rigid spacers between protein domains. An example of a rigid linker is A(EAAAK)nA (SEQ ID NO: 832), where n=2-5. This linker exhibits an α-helical structure stabilized by Glu-Lys+ salt bridges within the segment. Another type of rigid linker has a Pro-rich sequence (XP)n, where X represents any amino acid, usually Ala, Lys, or Glu. The presence of Pro in the non-helical linker increases rigidity and allows effective separation of protein domains. An example of such a linker is a 33 residue peptide containing repeats -Glu-Pro- and -Lys-Pro-.

A person skilled in the art may select from known linkers or design a linker. Desirable properties and requirements therefor are known. The following discussion summarizes some exemplary linkers (see Chen et al. (2013) Adv. Drug. Deliv. Rev. 65:1357-1369), which are flexible and rigid; and provide details of cleavable linkers and linkers that may be used). Flexible linkers are rich in small and/or hydrophilic amino acids, such as Gly or Ser, and have been used to connect functional domains that provide structural plasticity and facilitate interactions or movements between domains. . Rigid linkers may be used if sufficient separation of protein domains is required. Rigid linkers are designed or selected to adopt an α-helical structure or incorporate proline. Rigid linkers can keep protein moieties apart. Flexible and rigid linkers are stable in vivo and bound proteins cannot be separated. The cleavable linker allows release of the free functional domain in vivo via reductive or proteolytic cleavage. They are commonly used for delivery of prodrugs to target sites.

In Formula 2 above, additional linkers may be included, such as between the TNFR1 antagonist and/or TNFR2 agonist moiety and the activity-modifying moiety, such as the Fc moiety; such linkers may include, for example, at least the residues SCDKTH ( All or a portion of the hinge sequence of trastuzumab sufficient to provide plasticity, including the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) corresponding) or having a sequence having at least 98% or 99% sequence identity thereto, the hinge region of nivolumab, or any other suitable antibody hinge region or sequence known in the art. May include all or part, including sufficient portions.

In certain embodiments, only a GS linker is included. Other short peptide linkers known in the art are also contemplated for use in the bispecific molecules provided herein. For example, an N- or C-terminal extension from Fc can be used as a linker. C-terminal extension from human IgG, ELQLEESSAEAQDGELDG (SEQ ID NO: 833) or a sequence with at least 98% or 99% sequence identity thereto, or sequence ELQLEESSSAEAQGG (SEQ ID NO: 834) or with at least 98% or 99% sequence identity thereto Variants containing sequences with the same properties can also be used as linkers.

Whether or not the fusion protein may include a second Fc subunit (see, e.g., Figure 2) and may be modified to include a knob-in-hole (see below). (see description). This is assembled in a mammalian cell expression system to form a knob-in-hole mediated Fc dimer, creating an Fc dimer that further increases the serum half-life and stability of the molecule. In certain embodiments, a second Fc subunit is fused to a second TNFR2 agonist to generate a bivalent antibody-like structure. In other embodiments, only one Fc subunit is included (Fc monomer).

ii. Chemical Linkers In some embodiments, the linker is a chemical linker. These include polypeptide modifiers such as linkers that are non-cleavable moieties, chemical cross-linking reagents, and polymer molecules that include pegylated moieties. Chemical linkers are more suitable for creating branched structures and other structures that cannot be achieved with peptide linkers.

Exemplary linkers include non-cleavable linkers. Non-cleavable linkers include, for example, amide linkers and amide and ester bonds with succinic spacers (see, eg, Dosio et al., (2010) Toxins 3:848-883). Examples of chemical crosslinkers include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and SIAB (succinimidyl (4-iodoacetyl) aminobenzoate). . SMCC is an amine-to-sulfhydryl crosslinker that contains NHS ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane-stabilized spacer arm. SIAB is a short NHS ester and iodoacetyl crosslinker for conjugation of amines to sulfhydryls. Other exemplary crosslinking reagents include, but are not limited to, thioether linkers, chemically labile hydrazone linkers, 4-mercaptovaleric acid, BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS. , MPBH, SBAP, SIA, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfonyl) ) benzoate), and bismaleimide reagents such as DTME, BMB, BMDB, BMH, BMOE, BM(PEO)3, and BM(PEO)4, which are commercially available (Pierce Biotechnology, Inc.). Bismaleimide reagents allow free thiol groups of cysteine residues of antibodies to be coupled to thiol-containing targeting agents or linker intermediates either sequentially or simultaneously. Other thiol-reactive functional groups other than maleimide include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. Other exemplary linkers and methods of use are well known to those skilled in the art, such as those described in US Patent Publication No. 2005/0276812 and Ducry et al. (2010) Bioconjug. Chem. 21:5-13; It's a method.

The linker may be optionally substituted with groups that modulate properties such as solubility and reactivity. For example, a sulfonate substituent can increase the aqueous solubility of the reagent, facilitate the coupling reaction between the linker reagent and the antibody or drug moiety, and/or facilitate the coupling reaction. Linker reagents are also available from Molecular Biosciences Inc. (Boulder, CO.) or from commercial sources such as Toki et al. (2002) J. Org. Chem. 67:1866-1872; US Patent No. 6,214,345; Nos. 2003/130189 and 2003/096743; and International Application Publication Nos. WO02/088172, WO03/026577, WO03/043583 and WO04/032828. For example, a linker reagent such as DOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) is an isopropyl compound according to the procedure of Axworthy et al. (2000) Proc. Natl. Acad. Sci. It can be prepared by reaction of aminobenzyl-DOTA activated with chloroformate (Aldrich) and 4-maleimidobutyric acid (Fluka). The DOTA-maleimide reagent reacts with free cysteine amino acids of cysteine engineered antibodies and provides metal complex ligands on the antibody (Lewis et al. (1998) Bioconj. Chem. 9:72-86). Chelate linker labeling 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 linker for covalent attachment of two or more moieties to an antibody through a branched multifunctional linker moiety (e.g., Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King et al. (2002) Tetrahedron Letters 43:1987-1990). If an antibody has only one reactive cysteine thiol group, many other moieties can be attached via dendritic linkers. Exemplary dendritic linker reagents are known (see, eg, US Patent Publication No. 2005/0276812).

Another example of a chemical linker (which may also be an activity modifier for use in the constructs herein) are PEG molecules and branched PEG molecules, particularly those with a molecular weight of 30 kDa or greater. The PEG linker provides the introduction of multispecificity and bivalency (in the case of TNFR2 agonists where receptor clustering enhances signal transduction), increases the molecular weight of the molecule, and increases the in vivo serum half-life. PEG linkers also ameliorate difficulties in antibody redesign by, for example, avoiding the introduction of non-natural structures that are rapidly degraded and removed and/or cause immunogenicity.

d. Activity Modifiers Among the components of the construct are portions or regions that modulate or alter the activity and/or pharmacological properties of the construct (see Formulas 1 and 2 above). Such examples include Fc regions, modified Fc regions, other multimerization domains, Fc dimers and modified Fc, and other moieties, such as polymeric moieties, such as half-life extending polypeptides. Polypeptides, albumins such as human serum albumin (HSA), and transferrin, as well as polymers such as PEG, discussed elsewhere herein, that can increase serum half-life. Activity modifiers include, but are not limited to, prolonging plasma half-life by decreasing access to proteases, decreasing renal filtration, and/or altering intracellular pathways by receptor-mediated recycling; provide absorption across the epithelial bilayer by binding to receptors that receive it; targeting in vivo sites that overexpress or uniquely express particular receptors or antigens; and as exemplified in the discussion below. , other properties known in the art.

As provided herein, the constructs include, as an activity modifier, the Fc region of a human immunoglobulin such as IgG, e.g., IgG1 Fc (SEQ ID NO: 10), IgG2 Fc (SEQ ID NO: 12), IgG3 Fc (SEQ ID NO: 12), No. 14), or 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 κ Fc region, such as an IgG1 Fc from trastuzumab, including the CH2 and CH3 domains of the trastuzumab heavy chain (see, eg, residues 234-450 of SEQ ID NO: 26; also SEQ ID NO: 27). (see ). The Fc subunit of the bispecific molecules provided herein can also be an IgG4 Fc, such as, for example, the IgG4 Fc from nivolumab (Opdivo®), which includes the CH2 and CH3 domains of the nivolumab heavy chain. (see, eg, residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30).

The Fc region is used, for example, in antibody-dependent cellular cytotoxicity (ADCC; also known as antibody-dependent cytotoxicity), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cellular cytotoxicity, as discussed below. Mutations or modifications may be made to eliminate, reduce, or enhance immune effector functions, including any one or more of the disorders (CDC). In some embodiments herein, immune effector function is eliminated or reduced, for example, when the construct is a bispecific molecule used to treat inflammatory and autoimmune diseases and conditions. Ru. When the therapy is used to treat tumors or cancer, immune effector function may be enhanced to improve anti-tumor immune responses and therapeutic efficacy. Additionally or alternatively, the Fc region is modified to enhance FcRn recycling, increasing in vivo serum stability and half-life of the molecules provided herein.

For purposes herein, the Fc region or domain is modified, particularly to reduce or eliminate ADCC. Small molecule therapeutics such as antibody fragments (eg, 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), such as receptor clustering, activation of immune effector functions, poor tissue penetration and poor angiogenesis. Undesirable effects of the mAb, such as lack of access to the target in the region, may be prevented. However, small antibody fragments have poor pharmacokinetic properties. For example, due to their small size, dAbs and other antibody fragments are rapidly cleared by the kidneys, with molecules of size 50-60 kDa or less undergoing renal filtration. The rapid clearance and short elimination half-life (less than a few hours) of small antibody fragments reduces their efficacy in vivo and requires frequent administration and/or continuous infusion.

Several methods can be used to extend the retention and in vivo half-life of small antibody fragments such as dAbs. For example, as provided herein, TNFR1 antagonists, TNFR2 agonists and dAbs in combination/multispecific constructs are half-life prolonging agents or include linkers such as IgG1 and IgG4. It is fused to the Fc region of IgG. Fc may be monomeric or dimeric. Fusing small antibody fragments, such as dAbs, to the Fc region of an IgG molecule increases the size of the molecule, thereby preventing it from being cleared/excreted from the body and increasing the number of neonatal Fc receptors (FcRn) expressed on endothelial cells. This protects the antibody from lysosomal degradation and increases its half-life in vivo. However, addition of Fc can introduce undesirable properties such as complement activation, release of proinflammatory cytokines, and induction of immune effector functions that can lead to cytotoxicity. Because TNFR1 is almost ubiquitously expressed and TNFR2 is expressed in many tissues, the use of ADCC-enriched antibodies is generally undesirable and relies on the antibody's antagonistic activity for efficacy.

As described herein, the Fc region in multispecific constructs, such as TNFR1 antagonists, TNFR2 agonists, and bispecifics, has pharmacokinetic and pharmacodynamic (i.e., pharmacological) properties. Modified to improve and eliminate undesirable properties. For example, the Fc region may be modified to utilize/enhance neonatal FcR recycling to increase half-life in vivo, and/or antibody-dependent cellular cytotoxicity (ADCC; also known as antibody-dependent cell-mediated cytotoxicity). are mutated to eliminate Fc-related immune effector functions such as antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Additionally, in embodiments where the construct is multispecific, such as bispecific, e.g., it includes a TNFR1 antagonist and a TNFR2 agonist, and includes an Fc dimer, the dimer can be mutated to In-holes are introduced to prevent homodimerization. Numerous modifications to the Fc portion (or region) are known to those skilled in the art (see, eg, Li et al., (2014) Expert Opin Ther Targets 18:335-350).

i. Modification of the Fc portion a) Knob-in-hole Bispecific antibodies (bsAbs) contain two different antigen binding sites, allowing an alternative therapeutic approach to traditional therapeutic monoclonal antibodies (mAbs). , thereby avoiding limitations associated with mAbs such as receptor co-clustering. Small antibody fragments are easy and inexpensive to produce in high yields and can readily penetrate tissues, but are associated with limitations such as poor stability, solubility, and pharmacokinetic properties. For example, small size results in short serum half-life, reduced tissue retention, and rapid clearance from the blood through the kidneys. As a result, IgG-like bispecific (BS) Abs that do not have the same limitations are advantageous. For example, a bsAb may include an Fc region to increase serum half-life and to enable effector function if desired. However, it may be difficult to produce purified bsAbs in high yields due to the need to prevent homodimerization of the heavy chains. The "knob-in-hole" (KiH, also known as "knob-into-hole") approach provides a solution to this problem. The CH3 domain of antibody (IgG) heavy chains has been designed for heterodimerization to enable the construction of bifunctional therapeutic molecules containing non-self-associating Fc.

The knob-in-hole approach involves asymmetrically mutating the interfacial residues of the CH3 domains of two parent heavy chains in a complementary manner. A "knob" is created by replacing an amino acid with a small side chain with an amino acid with a large side chain, such as tyrosine or tryptophan, at the interface between CH3 domains, and a "hole" is created by replacing an amino acid with a large side chain with an amino acid with a large side chain, such as tyrosine or tryptophan. , created by replacing amino acids with smaller side chains, such as alanine or threonine. Knob and hole variants heterodimerize by insertion of the knob into the correspondingly designed hole of the partner's CH3 domain. Steric repulsion prevents knob-knob binding and destabilizes the hole-hole homodimer. For example, a knob mutation may be S354C, T366Y, T366W, or T394W, and a hole mutation may be Y349C, T366S, L368A, F405A, Y407T, Y407A, or Y407V (all according to EU numbering) . Knobs created toward the center of the dimer interface, such as residue T366, have been shown to disrupt homodimer formation more than those located near the edges of the dimer interface. Because residue T366 of the first CH3 domain is within hydrogen bonding distance of residue Y407 of the second or partner CH3 domain, T366Y and Y407T represent a common knob-in-hole pair; , have been shown to produce heterodimers in greater than 90% yield (see, eg, Ridgway et al. (1996) Protein Eng. 9(7):617-621).

For example, the IgG Fc region in the bispecific TNFR1 antagonist/TNFR2 agonist constructs provided herein can be modified using a knob-in-hole approach to generate heterodimerized molecules in high yield. can be generated. 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 shown in SEQ ID NO:9. Any mutation known to those skilled in the art that introduces a knob-in-hole may be used in the constructs herein.

Ligand Trap Constructs Fcs modified to have "knob-in-holes" as described above can also be used with other bispecific molecules to generate heterodimers. For example, U.S. Patent Application Publication No. 2010/0055093 and Jin et al. describes bispecific "ligand" trap constructs that target EGF receptor family ligands, including. The problem with these constructs is that they are heterogeneous and include homodimers and heterodimers, the latter being the intended therapeutic modality. RB200 and RB242 are examples of ligand traps that can be modified by replacing the Fc portion with a modified Fc region with complementary knobs and holes, and the resulting dimers are all heterodimers. RB242 targets HER1 (EGFR), HER2, and HER3 ligands, and some HER4 ligands. Because HER4 has a role in neuronal development not shared with other members of the EGFR family, it was designed not to trap HER4-specific ligands. RB242 contains HER1/ErbB1 (amino acids 1-621 of SEQ ID NO: 41) and HER3/ErbB3 (amino acids 1-621 of SEQ ID NO: 45) fused to the Fc domain (HER1-HER3/Fc) of human immunoglobulin G1 (IgG1). 621) and functions as a chimeric bispecific ligand trap. The HER3/Fc component of RB242 contains a 6x histidine tag at the COOH terminus (see, eg, Jin et al. (2009) Mol. Med. 15:11-20). RB200 binds with high affinity to HER1/ErbB1 ligands (EGF, TGF-α, HB-EGF, AR, BTC, EPR and EPG) and HER3/ErbB3 ligands (NRG1-α and NRG1-β3). RB242 inhibits EGF-stimulated and NRG1-β1-stimulated tyrosine phosphorylation of HER family proteins (HER1, HER2, and HER3) and has shown efficacy in various 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 of cell surface receptors (ErbB and human epidermal growth factor receptor (HER)) belongs to the receptor tyrosine kinase (RTK) superfamily and contains an extracellular domain (ECD) and an intracellular tyrosine kinase signaling domain. . There are four members of the EGF family: EGF receptor (EGFR)/HER1/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4, which are a large family of ligands, such as EGF, transforming Growth factor α (TGF-α), heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), β-cellulin (BTC), epiregulin (EPR), epigen (EPG) and neuregulin (NRG) activated by There are four ECDs within EGFR; domains I and III are the 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 homodimers or heterodimers between receptors. For example, TGF-α and EGF bind to EGFR/HER1/ErbB1, while NRG4 binds to HER4/ErbB4. Depending on the dimers formed, transphosphorylation of intracellular regions occurs, leading to activation of numerous downstream signaling pathways, resulting in cell proliferation, survival, and differentiation (e.g., Jin et al. (2009) Mol. Med. 15:11-20).

The epidermal growth factor receptor family is comprised of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 (Erb-B2), HER3 (ErbB-3), and HER4 (ErbB-4). ing. In many types of cancer, mutations or amplifications in one family member are associated with worse survival for cancer patients. In autoimmune diseases, TNF signaling transduces the EGFR signaling pathway by inducing the synthesis of epiregulin and heparin-binding EGF (HB-EGF) on macrophages, both growth factors that activate EGFR. Activate.

In a complementary manner, EGFR and HER2 are upregulated in synovial fibroblasts, thereby promoting their proliferation. EGFR, HER2 (ErbB2), and EGF-like growth factors, for example, are overexpressed in RA synovial fibroblasts and macrophages. Thus, the TNF and EGFR pathways cooperate in the progression of lupus and rheumatoid arthritis, and other autoimmune diseases. Among the constructs provided herein are constructs called "ligand traps." The ligand trap construct interferes with 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 TNF blocker constructs, which are TNFR1 and/or TNFR2 targeting constructs provided herein. This combination therapy, such as rheumatoid arthritis, may combine synergistically to achieve disease regression.

The EGFR family of growth factors is overexpressed in hyperproliferative/inflammatory diseases such as RA, and also in ovarian and other cancers. Elevated levels of the EGFR family and/or its cognates are a common component of multiple types of cancer. Overexpression (or even mutation) of these receptors is associated with reduced survival in many types of malignancies. Examples of targeted therapeutics that act through the EGFR family are (listed by generic name and exemplary trade name providing source) cetuximab (Erbitux®), panitumumab (Vectibix®), Trastuzumab (Herceptin®), and Pertuzumab (Perjeta®). Small molecule inhibitors also target intracellular tyrosine kinase activity of the EGFR family. Examples of small molecules include lapatinib (Tykerb®), erlotinib (Iressa®), and neratinib (Nerlynx®). These drugs target only one member of the EGFR family, with the result that other members of the family may upregulate and compensate for tumor growth. Similarly, antibodies against a single growth factor (e.g., TGF-α, EGF, HB-EGF, etc.) inhibit only that growth factor, and tumor cells compensate by upregulating other growth factors. do. The ligand trap constructs provided herein address this by blocking HER1, HER2 and HER3 together. This results in pan-inhibition of the EGFR family against cancer cells. Ovarian cancer is one of the cancers targeted for treatment.

The ligand trap constructs provided herein use modified Fc regions as described herein below for TNFR1/TNFR2 constructs to optimize heterodimer production and FcRn recycling. Improved by doing. Ligand trap constructs include TNFR1 antagonist constructs, and/or TNFR2 agonist constructs, and/or TNFR2 agonist constructs for the treatment of diseases, disorders, and conditions in which TNF plays a role, as described herein and/or known to those skilled in the art. and/or administered in a combination therapy protocol with the multispecific TNFR1 antagonist/bispecific construct and/or any other construct provided herein.

b) Modifications that enhance neonatal Fc receptor (FcRn) recycling There are numerous approaches to prolonging the short serum half-life of small polypeptide or protein therapeutics. Pegylation, which increases the serum half-life of small molecule protein therapeutics, has drawbacks. PEGylation can reduce the efficacy or activity of a protein therapeutic, can lead to heterogeneity, and can lead to immunoreactivity of the protein. Other approaches include fusion to albumin, which may improve protein circulation by increasing molecular weight and decreasing renal clearance.

Serum half-life can also be increased by fusion of IgG to the Fc portion. The long circulating half-life of approximately 2-3 weeks and slow clearance rate of IgG is due, at least in part, to neonatal Fc, which binds IgG with high affinity at acidic pH and releases them at neutral or higher pH. Due to interaction with the receptor (FcRn). FcRn binds to the Fc portion (within the CH2-CH3 domain) of pinocytosed IgG in acidic (~pH 6) endosomes in a 2:1 FcRn:IgG configuration (bivalent interaction) and carries them for lysosomal degradation. away from the cell surface and exposed to the extracellular physiological pH (approximately 7.4) where Fc-FcRn complexes dissociate, recycling them back into the circulation. As a result of insufficient binding to FcRn at acidic pH, antibodies are transported to lysosomes where they are degraded. Recycling receptors such as FcRn also provide a transport route for IgG across the epithelium and into the bloodstream (transcytosis). Interaction with FcRn can be used to improve protein transport across epithelial barriers such as the intestine and lung, allowing for non-invasive administration. Residues of the Fc CH2 and CH3 domains are involved in FcRn binding, and their mutations in mAbs have been shown to affect in vivo serum half-life. Circulation and delivery of small protein therapeutics can be improved by fusing them to the Fc domain of IgG, such that the resulting fusion protein binds to FcRn and utilizes the IgG serum stabilization pathway. Become. Fusion with an Fc domain also increases the molecular weight of the therapeutic and reduces renal clearance, which may be undesirable due to potentially reduced tissue permeability and specific activity of the fusion protein. Alternatively, studies have shown that short FcRn-binding peptides (FcRnBPs) allow interaction of small proteins with FcRn, eliminating the need for fusion to high molecular weight Fc domains. For example, fusion with FcRnBP increases molecular weight by about 3 kDa compared to fusion with Fc or albumin, which increases molecular weight by about 50-70 kDa (e.g., Datta-Mannan et al. (2019) Biotechnol. J. 14:1800007; see Sockolosky et al. (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) can be used in Fab heavy and light chains with 1 to 4 FcRnBPs per Fab. fused at the C-terminus, N-terminus, or both (FcRnBP-Fab construct). Pharmacokinetic studies in cynomolgus monkeys showed that FcRn binding of FcRnBP-Fab constructs increased as the number of peptides fused to the Fab increased. This is due to increased avidity, as constructs containing four linear FcRnBPs fused to the N- and C-termini of the heavy and light chains of the Fab exhibit maximal pharmacokinetics in cynomolgus monkeys compared to the parent Fab. shows improvement. For example, the half-life was improved from 3.7 h for the parent Fab to 15-60 h for various FcRnBP-Fab constructs (e.g., Datta-Mannan et al. (2019) Biotechnol. J. 14:1800007). (see ). These results indicate an improvement in serum half-life, which is much shorter than that of IgG, which is approximately 2-3 weeks. Also, the use of FcRnBP does not significantly increase the molecular weight of the therapeutic agent and therefore does not reduce renal clearance.

As discussed above, fusion with IgG Fc extends the half-life of small protein therapeutics by taking advantage of FcRn binding and by increasing the molecular weight of the therapeutic, resulting in, for example, kidney disease. This makes it difficult for them to be excreted from the body quickly. To improve pharmacokinetics and overall pharmacology, residues within the Fc region can be mutated to increase affinity for FcRn, typically 30-fold or more, and further increase in vivo half-life. The Fc region, which spans the interface of CH2 and CH3 domains, interacts with FcRn. Human Fc residues that have been identified to play a role in FcRn binding include, for example, L251, M252, I253, S254, L309, H310, Q311, L314, E380, N434, H435 and Y436 (according to EU numbering). , see Table 1). Mutation of residues located at the Fc-FcRn interface, including M252, S254, T256, H433, N434, and Y436 (according to EU numbering), improves the stability of the human FcRn-IgG1 complex. For example, substitutions M252Y/S254T/T256E and H433K/N434F/Y436H improved binding to human FcRn by 11-fold and 6.5-fold, respectively, at pH 6.0 and efficiency at pH 7.4 compared to wild-type IgG1. released. The combination of these substitutions increases binding affinity to FcRn by 57-fold. Additional mutations in IgG1 Fc that have shown improved binding to FcRn include, for example, M252W, M252Y, M252Y/T256Q, M252F/T256D, E380A, and N434F/Y436H (e.g., Dall'Acqua et al. (2002) J. Immunol. 169:5171-5180).

When the triple substitution M252Y/S254T/T256E was introduced into the CH2 domain of MEDI-524, a humanized anti-respiratory syncytial virus (RSV) mAb, the mAb in cynomolgus monkeys compared to unmodified MEDI-524. The serum half-life of When introduced into the Fc portion of MEDI-522, a humanized mAb optimized for affinity to the human αvβ3 integrin complex, the substitutions M252Y/S254T/T256E (YTE) inhibited its ADCC activity and human FcγRIIIA (F158 allotype). reduced its binding. The ADCC activity of MEDI-522-YTE can be reversed and increased compared to unmodified MEDI-522 by the introduction of ADCC-enhancing substitutions S239D/A330L/I332E (according to EU numbering) , thereby indicating that substituted YTE provides a reversible mechanism to modulate the ADCC function of human IgG1 (e.g., Dall'Acqua et al. (2006) J. Biol. Chem. 281(33):23514 -23524).

Residues at positions 250, 314, and 428 (according to EU numbering) of the human IgG heavy chain, which are conserved among all four human IgG subtypes, are also located near the Fc-FcRn interface. Introduction of mutations T250Q, M428L, and T250Q/M428L into the Fc of human IgG2 mAb increased binding to FcRn by approximately 3-fold, 7-fold, and 28-fold, respectively, at pH 6.0, and no binding was observed at pH 7.5. There wasn't. When the pharmacokinetics of the mutant was evaluated in rhesus monkeys, the mean clearance, i.e., the volume of serum antibody removed per unit time, was approximately 1.8-fold lower for the M428L mutant compared to the unmodified antibody. , was found to be approximately 2.8-fold lower in the T250Q/M428L mutant, but the elimination half-life was approximately 1.8-fold longer in the M428L mutant and approximately 1.9-fold longer in the T250Q/M428L mutant. . These residues are conserved among IgG subtypes, so mutations M428L and T250Q/M428L are expected to have similar effects in human IgG1, IgG3, and IgG4 antibodies (e.g., Hinton et al. (2004) J. Biol. Chem. 279(8):6213-6216). Modified T250R/M428L was shown to provide selective binding to FcRn at pH 6.0 and reduce serum IgG2 and IgG1 degradation by 2.8-fold in rhesus monkeys (e.g., Saxena et al. (2016) Front Immunol. 7:580).

Mutation N434A (according to EU numbering), when introduced into human anti-HER2 IgG1 trastuzumab, resulted in an approximately 4-fold higher affinity for human FcRn than the unmodified antibody at pH 6, but negligible at pH 7.4 It was a combination. The N434A variant had increased exposure, decreased clearance (approximately 2-fold), and increased half-life (approximately 2-fold) compared to the wild-type antibody when tested in vivo in cynomolgus monkeys. In contrast, mutant N434W showed approximately 80-fold increased binding to FcRn at pH 6 and clearance rates similar to the wild type; this mutant also showed significant binding to FcRn at pH 7.4. also show that maintaining pH-dependent binding of Fc mutants to FcRn is important for improving pharmacokinetics in vivo (Yeung et al. (2009) J. Immunol . 182:7663-7671). The N434A mutation also counteracts decreased FcRn affinity that can result from the introduction of mutations that increase binding to FcγR; N434A is typically added to mutations S298A/E333A/K333A to enhance FcγR binding. and create mutants with normal or improved FcRn binding. Fc mutations that improve FcRn binding also include N434Y, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y. E294 deletion increases sialylation of the N297 glycan on the Fc and increases antibody half-life in vivo. It has been shown that sialylation also plays a role in regulating serum half-life (see, eg, Saunders, K.O. (2019) Front. Immunol. 10:1296).

The substitution M428L/N434S (according to EU numbering) increases the affinity for FcRn by 11-fold at pH 6.0 when introduced into the humanized anti-VEGF IgG1 antibody bevacizumab (Avastin®), resulting in an 11-fold increase in the affinity for FcRn in cynomolgus monkeys in vivo. The half-life was extended from 9.7 days to 31.1 days, an improvement of 3.2 times. The M428L/N434S modification resulted in a similar increase in FcRn binding and half-life extension when introduced into the anti-EGFR antibody cetuximab, which is rapidly cleared by receptor-mediated internalization. The increased half-life of these anti-tumor antibodies correlates with enhanced tumor regression in vivo in mouse models, indicating that improved pharmacokinetics, such as clearance rates, can enhance the therapeutic efficacy of antibodies in vivo. has been done. Other mutations incorporated into the bevacizumab Fc include (by EU numbering): N434S (approximately 3-fold improved FcRn binding and approximately 2.8-fold increased serum half-life in mice); V259I/ V308F (approximately 6-fold improved FcRn binding and increased serum half-life in mice and cynomolgus monkeys approximately 3-fold and approximately 2-fold, respectively); and V259I/V308F/M428L (approximately 20-fold improvement in FcRn binding and approximately 4-5-fold and 2.5-fold increase in serum half-life in mice and cynomolgus monkeys, respectively); .6-fold increase) (Zalevsky et al. (2010) Nat Biotechnol. 28(2):157-159).

The mutations identified above, and other such mutations, can be introduced into the IgG Fc region in the constructs provided herein. These include constructs such as those of Formulas 1 and 2, where the linker comprises Fc or an Fc dimer, depending on the structure of the construct.

In some embodiments, the IgG Fc region in the constructs herein, such bispecific TNFR1 antagonist/TNFR2 agonist constructs, and the TNFR1 antagonist constructs provided herein, inhibits neonatal FcR recycling. Modified to enhance and increase in vivo half-life. This can be accomplished by mutating residues at the interface of the CH2 and CH3 domains of the IgG Fc that are involved in binding to FcRn. These include, but are not limited to, residues T250, L251, M252, I253, S254, T256, V259, T307, V308, L309, H310, L314, Q311, A378, E380, S383, M428, H433, N434 , H435 and Y436 (according to EU numbering). Exemplary Fc modifications that increase binding to FcRn include, but are not limited to, 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, T 250R/M428L, M428L/N434S, V259I/ V308F, V259I/V308F/M428L, E294del/T307P/N434Y, T256N/A378V/S383N/N434Y, and combinations thereof (according to EU numbering). Table 7 below shows the corresponding mutations according to Kabat numbering and sequential numbering with reference to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO: 9. Other modifications known in the art to confer enhanced or increased FcRn binding are also contemplated for use herein.

c) Enhancement or reduction/absence of Fc immune effector function There are four human IgG subclasses that differ in effector function, circulating half-life, and stability. IgG1 has Fc effector function, is the most abundant IgG subclass, and is the most commonly used subclass in FDA-approved therapeutic proteins. IgG2 lacks Fc effector function, but dimerizes with other IgG2 molecules and is unstable due to scrambling of disulfide bonds in the hinge region. IgG3 has Fc effector function and a very long and stiff hinge region. IgG4 lacks Fc effector function, has a shorter circulating half-life than other subclasses, and IgG4 dimers are biochemically unstable due to the presence of a single disulfide bond in the hinge region. , leading to the exchange of heavy chains between different IgG4 molecules. Fc regions derived from IgG2 and IgG4 therefore have no effector function and can be used where effector function is unnecessary or detrimental, for example, in the context of autoimmune and inflammatory diseases and disorders.

Most of the approved therapeutic mAbs belong to the human IgG1 subclass and can interact with humoral and cellular components of the immune system. For example, antibodies participate in humoral immune responses through interaction with complement protein C1q, which initiates the complement cascade, resulting in cytolysis of target cells (i.e., complement-dependent cytotoxicity (CDC)). ) and interaction with Fc gamma receptors (FcγR) leads to the formation of membrane attack complexes that are involved in cellular immune responses. FcγRs include the FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) classes, which differ in cell surface expression and Fc binding affinity. The five activating FcγRs include high affinity FcγRI, which can bind monovalent antibodies, and low affinity FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb (requiring avidity-based interactions). FcγRIIb is the only inhibitory receptor. When Fc binds to activated receptors, intracellular signaling pathways regulated through phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) trigger effector functions, e.g., antibody-dependent cell-mediated cytotoxicity ( ADCC; also called antibody-dependent cytotoxicity) and antibody-dependent cell-mediated phagocytosis (ADCP; also called antibody-dependent cellular phagocytosis), and inflammation by induction of cytokine secretion. Inhibitory FcγRIIb-mediated signaling regulated by phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) recruits phosphatases that balance activated signaling pathways (e.g., Wang et al. (2018 ) Protein Cell 9(1):63-73).

The glycosylation of the hinge and proximal CH2 amino acid sequence (lower hinge-upper CH2 domain region) and the conserved N297 residue (according to EU numbering) of the CH2 domain Asn-X-Ser/Thr glycosylation motif of the Fc region is , mediates the interaction of antibodies with FcγR and complement protein C1q. Antibody/Fc engineering has been used to alter the immune effector function of antibodies by altering binding to C1q and various Fcγ receptors. Thus, depending on the application, the CDC, ADCC, and ADCP activities of a therapeutic mAb can be increased or decreased. For example, the effectiveness of anti-cancer monoclonal antibodies depends in part on the induction of FcγR effector function. Effector functions include FcγRIIIa-mediated activation of natural killer (NK) cells and subsequent ADCC activity and release of inflammatory cytokines, induction of macrophage-mediated ADCP through interaction with multiple FcγRs, and other immune responses. Recruitment and activation of cells such as neutrophils, which are the main receptors for NK cell-mediated ADCC. There are two polymorphic variants of FcγRIIIa: one with V158, which has a high affinity for IgG1; and a variant with F158, which has a low affinity for IgG1. Cancer patients with the high affinity V158 polymorphism may have better outcomes after treatment with cetuximab, trastuzumab, and rituximab compared to patients with the lower affinity F158 polymorphism. These results highlight the therapeutic role of FcγR-mediated immune effector functions and suggest that engineering antibodies and related molecules to increase their affinity for FcγRs may increase therapeutic efficacy. (See, for example, Wang et al. (2018) Protein Cell 9(1):63-73).

Residues in the lower hinge and proximal CH2 regions of IgG have been identified as important for binding to FcγRs. Residues within 5 angstroms of the FcγR:Fc interface of FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIb include residues (according to EU numbering) P232, E233, L234, L235, G236, G237, P238, S239 (SEQ ID NO:9). (with reference to SEQ ID NO: 9, corresponding to residues P115 to S122), D265, V266, S267, H268, E269, D270 (with reference to SEQ ID NO: 9, corresponding to residues D148 to D153), Y296, N297, S298 . ~I215) (see, for example, Wang et al. (2018) Protein Cell 9(1):63-73).

Modifications of Fc that enhance or reduce ADCC activity and/or enhance affinity/binding to receptors are known to those skilled in the art. For example, Fc modifications that increase IgG1 affinity and binding to FcγRIIIa and/or enhance ADCC function include the following substitutions (by EU numbering): F243L/R292P/Y300L/V305I/P396L, L235V /F243L/R292P/Y300L/P396L, F243L/R292P/Y300L, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A, and L234Y/L235Q in one heavy chain /G236W/S239M/ H268D/D270E/S298A, and D270E/K326D/A330M/K334E in the opposite heavy chain, and L234Y/G236W/S298A in one heavy chain, and S239D/A330L/I332E in the opposite heavy chain. combination. In addition, mutations A327Q/P329A (interacts with FcγRI), D265A/S267A/H268A/D270A/K326A/S337A (interacts with FcγRIIa), G236A (interacts with FcγRIIa), and T256A/K290A/S298A/ E333A/K334A (which interacts with FcγRIIIa) results in high affinity interactions with FcγRs.

Fc modifications that increase binding to FcγRIIa and FcγRIIIa and enhance ADCC and ADCP include (by EU numbering) G236A/I332E, G236A/S239D/I332E (also increases binding to FcγRI), and G236A/S239D/A330L /I332E (for example, Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al (2016) Front. Immunol. 7:580; and Saunders, K. O. (2019) Front. Immunol 10:1296).

Glycoengineering of IgG containing a conserved N-linked glycosylation site at residue N297 of the CH2 domain may enhance Fc effector function. Glycosylation of N297 is essential to maintain the conformation of Fc and mediate its interaction with FcγR (and C1q). The glycan present at residue N297 typically has two N-acetylglucosamines (GlcNAc), three mannoses, and two or more additional GlcNAcs linked to the mannose, forming a biantennary complex glycan. Additional fucose, galactose, sialic acid, and GlcNAc may be added to the core glycan structure. Although IgG found circulating in human serum is generally fucosylated, the production of recombinant IgG can be achieved by expression of antibodies in plant cells, by knocking in or knocking out specific glycosidases, or by glycosylation. In vitro enzymatic digestion of IgG can alter the glycan composition; since both heavy chains are glycosylated, a single IgG molecule may have glycan heterogeneity. Glycans directly affect FcγR binding. For example, N297 glycans on Fc can collide with glycans on FcγRIII proteins, resulting in reduced effector cell involvement in mediating ADCC. Fc regions containing different glycans at N297 adopt different hinge region conformations, which may affect the ability of the Fc to interact with FcγRs. Expression of β(1,4)-N-acetylglucosaminyltransferase III during IgG expression produces antibodies glycosylated with a biantennary glycan at position N297; this antibody has increased binding to FcγRIIIa. and enhance ADCC activity. Fucose-deficient (defucosylated/non-fucosylated) IgG1 has been demonstrated to have up to 50-fold increased binding to FcγRIIIa and enhanced ADCC activity. Two glycoengineering (defucosylation) mAbs, obinutuzumab (anti-CD20) and mogamulizumab (anti-CCR4), have been approved for clinical use, demonstrating the potential of glycoengineering to enhance effector function and the clinically approved It shows the potential for its translation into therapeutic agents (e.g. Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al. (2016) Front. Immunol. 7:580; and Saunders, K. O. (2019) Front. Immunol. 10:1296).

Fc may be modified to bind to a broader range of Fc receptors. Certain leukocytes have Fc receptors of isotypes other than gamma (i.e., IgA, IgM, and IgE) and bind to effector cells by modifying the Fc region to bind multiple Fc receptors. Antibodies with expanded capabilities are produced. Neutrophils, the most abundant white blood cells in the body, engage the Fc of IgA antibodies through FcαRI receptors. For example, to bind FcγR and FcαRI, a single domain of IgA2 was 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 alpha1 constant region domain, generating a constant region (CH1a-CH2g-CH3g-CH3a) whose structure is closer to the alpha constant region. These four-domain, cross-isotype IgG chimeric antibodies bind to the J chain similar to native IgA2, exhibit reduced transport through polymeric Ig receptors, a 3- to 5-fold decrease in FcγRI affinity, and prolonged serum circulation of IgG1. Instead, the serum half-life of IgA2 was shortened. However, a 4-domain cross-isotype IgG chimeric antibody was capable of mediating complement-dependent lysis of sheep red blood cells and was more pH tolerant than IgG1. Another cross-isotype Fc is 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 are fused to the C-terminus of IgG1. It was done. This tandem cross-isotype IgG/IgA fusion exhibits expression levels, antigen binding, and thermostability similar to IgG1, and in vitro binds FcαRI and FcγRI, FcγRII, FcγRIIIa, and FcRn, and binds wild-type IgA and IgG1, respectively. showed similar affinity. Binding to various FcRs resulted in ADCC activity against polymorphonuclear cells and NK cells; however, C1q binding was reduced by threefold compared to IgG1. Tandem IgG/IgA had a similar in vivo half-life to IgG1 in BALB/c mice. CH3 domain and CH2α1 loop residues 245-258 of the IgG1 constant region, with structurally similar regions of the IgA constant region (corresponding to the sequence PKPKDTLMISRTPE, according to EU numbering; (residues 128-141 of SEQ ID NO: 9)) Another cross-isotype antibody was generated by substituting . This chimeric Fc could bind FcγRI, FcγRIIa, and FcαRI, and the antibody containing the chimeric Fc mediated ADCC in polymorphonuclear cells and ADCP in macrophages and activated complement, but half of the antibody lacked binding to phase-regulating FcRn; therefore, further optimization is required for effective in vivo use (see, e.g., Saunders, K.O. (2019) Front.Immunol.10:1296). thing).

Another approach to enhance FcγR binding is multimerization of IgG, which has shown promise in the treatment of autoimmune diseases. IgG multimers can be created, for example, by adding a heterologous multimerization domain such as an isoleucine zipper, or by adding another hinge region to the N-terminus of the natural hinge, or by adding another hinge region to the C-terminus of the CH3 domain. Generated by adding areas. IgG hexamers are created by adding an IgM tailpiece to the C-terminus of IgG1 Fc, creating a cysteine bond at position 309; this multimeric IgG binds strongly to FcγRI, FcγRIIa, and FcγRIIIa; It weakly bound to FcγRIIb and FcγRIIIb. Various multimeric IgGs have increased binding to FcγRI, FcγRIIb, and FcγRIII compared to monomeric IgGs and show promise in preclinical models of arthritis, neurological disorders, and autoimmune myasthenia gravis. It has been shown that This multimeric IgG design has been further optimized to fine-tune which immune receptors, including FcRn, can bind to the multimer (e.g., Saunders, K. O. (2019) Front. Immunol. 10:1296 checking).

Residues in the Fc region of IgG involved in interaction and binding with C1q (and thus CDC) include (according to EU numbering) S267, D270, K322, K326, P329, P331, and E333. Fc modifications that have been shown to enhance CDC by increasing C1q binding include, for example, K326A, E333A, K326A/E333A, K326W, K326W/E333S, K326M/E333S, C220D/D221C, H268F/S324T, S267E, H268F, S324T, S267E/H268F/S324T, and G236A/I332E/S267E/H268F/S324T (all according to EU numbering). In the upper hinge region of IgG1 Fc, substitution of Trp at positions 222, 223, and 224 (i.e., K222W, T223W, and H224W according to EU numbering) in various combinations resulted in wild C1q binding and CDC activity were increased compared to type IgG1. Specifically, mutations included K222W/T223W, K222W/T223W/H224W, and D221W/K222W. Mutants C220D/D221C and C220D/D221C/K222W/T223W also increased C1q binding and CDC activity (e.g. Wang et al. (2018) Protein Cell 9(1):63-73; Saxen et al (2016) Front. Immunol. 7:580; Saunders, K. O. (2019) Front. Immunol. 10:1296; and Dall'Acqua et al. (2006) J. Immunol. 177:1129-1138) .

IgG3 has the best in vitro binding to C1q; 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) Generate IgG3 cross-subtype antibodies and also increase C1q binding and enhance CDC activity. Other IgG1/IgG3 cross-subtype antibodies with increased C1q binding and enhanced CDC activity include CH1, IgG1 hinge and CH3, and IgG3 CH2; these modifications allow C1q to become a CH2 domain. binding allows for increased Cq1 binding and facilitates production as protein A binds to the CH3 domain. Furthermore, the modification 45R/E430G/S440Y, which resulted in the formation of an IgG hexamer with K322 positioned to favorably interact with the hexameric C1q headpiece, enhanced CDC activity. Mutant E345R alone also forms IgG hexamers, increases C1q binding, and enhances CDC activity (e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saxena et al. (2016) Front. Immunol. 7:580; see Saunders, K. O. (2019) Front. Immunol. 10:1296).

Glycoengineering can also be used to improve complement fixation; the N297 glycan within the CH2 domain of the Fc can be modified to improve CDC activity. For example, hypergalactosylation of IgG1 Fc increases C1q binding and CDC activity and also improves thermostability compared to the unmodified glycoform of IgG1. Therefore, galactosylation of Fc can be used to generate stable biologics with enhanced CDC activity (see, eg, Saunders (2019) Front. Immunol. 10:1296).

Table 8 below summarizes Fc modifications that increase binding to FcγR or C1q and thus enhance immune effector functions including ADCC, ADCP, and CDC, and identifies corresponding modifications by Kabat numbering and sequence numbering ( 9). Any one or more of these modifications, alone or in various combinations, may be introduced into the IgG1 Fc portion of the constructs provided herein. Other modifications known in the art to confer enhanced or enhanced immune effector function are also contemplated for use herein.

Therapeutic antibodies may also be engineered to reduce or eliminate immune effector function. For purposes herein, in some embodiments it is important to reduce or eliminate ADCC activity, for example. Constructs herein that include Fc are generally modified to reduce or eliminate ADCC activity.

It is of interest to reduce or eliminate immune effector function, for example: The therapeutic antibody is antagonistic to prevent receptor-ligand interaction and signal transduction; the antibody cross-links the receptor and prevents signal transduction. the antibody is a drug delivery vehicle that delivers the drug to target cells expressing the antigen; and the reduction or elimination of effector function may lead to target cell death or secretion of unwanted cytokines. prevent. Reducing effector function also prevents the antibody drug conjugate from interacting with FcγRs and reduces off-target cytotoxicity. The importance of reducing or eliminating effector function is underlined by the first approved mAb, muromonab, designed to prevent activation of T cells in patients transplanted with donor kidneys, lungs, or hearts. ) became apparent after adverse events related to administration. Patients receiving muromonab experienced a dangerous induction of pro-inflammatory cytokines (i.e., cytokine storm); this was due, in part, to the interaction of muromonab with FcγRs (e.g. , Wang et al. (2018) Protein Cell 9(1):63-73; and Saunders, K. O. (2019) Front. Immunol. 10:1296).

There are many known mutations that reduce or eliminate receptor function. For example, substitutions of L235E and F234A/L235A in human IgG4 and L235E and L234A/L235A (all according to EU numbering) in human IgG1 reduce FcγR and C1q binding and reduce effector functions such as inflammatory cytokine release. let Release of inflammatory cytokines from therapeutic antibodies can lead to adverse effects. The S228P/L235E substitution, when introduced into IgG4, also reduces binding to FcγR; the S228P mutation improves the stability of IgG4. Mutations S228P/F234A/L235A of IgG4 Fc reduce binding to FcγRI, IIa, and IIIa and reduce ADCC and CDC. The triple mutant L234E/L235F/P331S of IgG1 Fc decreases binding to FcγRI, FcγRII, FcγRIII, and C1q and reduces CDC, and the mutation L234A/L235A/P329G in IgG1 Fc decreases binding to FcγRI, FcγRII, FcγRII, and C1q. Eliminates FcγRIII, and C1q binding and reduces ADCP. Mutations L234F/L235E/P331S also reduce binding to FcγR and C1q, reducing effector function of IgG1 Fc. Mutations G237A and E318A in IgG1 Fc reduce binding to FcγRII and ADCP, respectively. Mutations D265A and E233P reduce FcγRI, FcγRII and FcγRIII and reduce ADCC and ADCP, mutations G236R/L328R reduce binding to all FcγRs and reduce ADCC. Crystal structure data show that a conformational change at residue P329, which is packed between two conserved tryptophan residues that occur in all FcγRs and form a “proline sandwich,” is detrimental to interaction with FcγRs. It has become clear that modification at residue D270 can adversely affect the interaction with C1q (e.g. Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, K. O. (2019) Front. Immunol. 10:1296; see International Application Publication No. WO2019/226750).

Induction of the complement cascade eliminates the binding of C1q to Fc, which is associated with adverse reactions at the site of antibody injection and is also an early step in CDC activation. Modification of the Fc region eliminates C1q binding and can be used to eliminate CDC in constructs containing the Fc region. As shown above, many of the mutations that eliminate FcγR binding also eliminate C1q binding. For example, mutation A330L disrupts C1q binding, reduces CDC, and also eliminates FcγRIIb binding. Mutations D270A, P329A, K322A, and P331A also result in decreased C1q binding and decreased CDC activity (see, e.g., Saunders, K. O. (2019) Front. Immunol. 10:1296).

Glycoengineering can be used to remove FcγR and C1q binding. As discussed elsewhere herein, the glycan of residue N297 is a complex biantennary glycan. Modification of this glycan to a high mannose glycan (ie, high mannose glycosylation) reduces the affinity of IgG1 Fc for C1q and reduces CDC activity. Fc mutations that reduce or eliminate binding of C1q to FcγRI may also increase galactosylation and sialylation of the N297 glycan; such mutations include, for example, F241A, V264A, and D265A. . Mutations N297A, N297Q, N297D, and N297G (according to EU numbering) eliminate effector functions such as CDC and ADCC by removing the glycosylation site of N297 and abrogating Fc interactions with C1q and FcγR, respectively. lower. The N297G/D265A combination almost completely abolishes binding to FcγR and C1q. IgG3 Fc lacking glycosylation (aglycone Fc) has reduced binding to FcγRI and C1q (e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, K. O. (2019) Front. Immunol. 10:1296).

To reduce or eliminate Fc effector function, large portions of Fc regions from different subclasses lacking opposing functions can be exchanged to generate cross-subclass Fc regions. For example, IgG2 binds C1q with weak binding to FcγR, and IgG4 lacks binding to C1q but reacts with FcγR; therefore, a combination of IgG2 and IgG4 CH domains that lack both C1q and FcγR binding can be constructed. Generally, in an IgG1/IgG4 chimera, the hinge and CH1 domains are derived from IgG2 and the CH2 and CH3 domains are derived from IgG4. Because IgG1 and IgG3 recruit complement more effectively than IgG2 and IgG4, and IgG2 and IgG4 have limited ability to induce ADCC, a cross-subclass approach may reduce effector function. For example, the anti-C5 mAb eculizumab contains IgG2 residues 118-260 (according to EU numbering; 447 (according to EU numbering; corresponds to residues 274-478 according to Kabat numbering, and residues 141-327 (with respect to SEQ ID NO: 15)) with limited or undetectable effector function. Similarly, an IgG2 variant (IgG2m4) with point mutations H268Q/V309L/A330S/P331S from IgG4 (according to EU numbering; corresponds to H281Q/V328L/A349S/P350S according to Kabat numbering and H147Q/V188L/A209S/P210S (see SEQ ID NO: 11)) lacks binding to all FcγRs and C1q, indicating reduced effector function. Mutations containing the IgG2 to IgG4 cross-subclass mutation V309L/A330S/P331S (according to EU numbering; corresponding to V328L/A349S/P350S according to Kabat numbering and corresponding to V188L/A209S/P210S (see SEQ ID NO: 11)) (referred to as IgG2σ), and the non-germline mutations V234A/G237A/P238S/H268A (according to EU numbering); ), eliminates binding to FcγR and C1q, and exhibits undetectable CDC, ADCC, and ADCP activities. The IgG1/IgG4 cross-subclass variant IgG1σ, which contains the mutations L234A/L235A/G237A/P238S/H268A/A330S/P331S, lacks binding to FcγRI and IIIa, and at high antibody concentrations binds to FcγRIIa and IIb very poorly. weakly, resulting in reduced ADCC and CDC activities (see, e.g., Wang et al. (2018) Protein Cell 9(1):63-73; Saunders, K. O. (2019) Front. Immunol. 10:1296) ).

Tables 9 and 10 below show the effects of reducing or eliminating binding to FcγR and/or C1q and thus reducing or eliminating immune effector functions, including ADCC, ADCP and CDC, that can be introduced into the Fc region in the constructs herein. Some IgG1 and IgG4 Fc modifications to eliminate are summarized. The table refers to the sequence of the IgG1 heavy chain constant domain as shown in SEQ ID NO: 9 or the IgG4 heavy chain constant domain as shown in SEQ ID NO: 15 and provides the corresponding modifications according to Kabat numbering and sequential numbering. Any one or more of these modifications, alone or in various combinations, may be introduced into the IgG1 Fc portion of the constructs provided herein. Other modifications known in the art to reduce or eliminate immune effector function are also contemplated for use herein.

ii. Other Modifications of the Fc Portion The Fc portion may also be modified to increase binding to inhibitory FcγRs resulting in suppression of the immune response. Therapeutic antibodies containing immunosuppressive Fc modifications are advantageous in the treatment of inflammatory diseases. These mutations can be incorporated into the Fc portion of the constructs herein intended for the treatment of diseases and conditions with inflammatory components or pathogenesis or involvement. For example, an immunosuppressive version of an anti-CD19 antibody (XmAb5871; of CD19+ B cells. Introducing the same mutation into a humanized anti-IgE antibody (XmAb 7195; 430-fold and is used in the treatment of allergies, including allergic asthma. Anti-CD3 antibody TRX4 (Tolerx) contains the aglycosylated Fc mutation N297A (according to EU numbering) and 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 a monomeric IgG1 Fc (mFc) containing mutations L351S/T366R/L368H/P395K (according to EU numbering), which binds to FcRn and has a similar in vivo half-life as dimeric Fc. , selectively binds FcγRI with high affinity but not FcγRIIIa, abrogating Fc-mediated cytotoxicity including ADCC and CDC. FcγRI is expressed on inflammation-related cells such as inflammatory macrophages. Targeting this receptor can be used to treat chronic inflammatory diseases such as arthritis, multiple sclerosis, and cancer. Mutant mFc kills FcγRI+ macrophage-like U937 cells when fused to Pseudomonas exotoxin A fragment (PE38). Neither the mutant mFc nor the fusion protein exhibits cytotoxicity (ADCC or CDC) in vitro (see, eg, Ying et al. (2014) mAbs 6(5):1201-1210).

Modifications that increase binding or confer selective binding to inhibitory FcγRIIb and/or FcγRI (but not FcγRIIIa) may be used to modify the IgG in the TNFR1 antagonist and TNFR1 antagonist/TNFR2 agonist constructs provided herein. It may also be operated on the Fc region. These modifications include one or more of the following EU numbering: S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F, L351S/T366R/L368H/P395K, and combinations thereof; Not limited to these. Table 11 below shows the corresponding substitutions according to Kabat numbering and according to sequential numbering with reference to the sequence of the IgG heavy chain constant domain set set out in SEQ ID NO:9.

iii. Human serum albumin (HSA)
A problem with previously provided dAbs (see, eg, International PCT Application No. 2008/149144) was that their serum half-life was insufficient for use as therapeutic agents. They were linked to anti-HSA antibodies and bound HSA; half-life was insufficient. Here, the dAb or Vhh antibody is linked to HSA. There are 33 cysteines in HSA; Cys34 is the only cysteine with a free sulfhydryl group that does not participate in a disulfide bond. HSA can be linked to a dAb via its N-terminus or C-terminus, either directly or through a linker such as a Gly-Ser linker, to extend the serum half-life of the dAb. Alternatively, they may be linked via a free cysteine. Example 6 illustrates a construct containing a dAb linked to the N-terminus of HSA via a Gly-Ser linker.

e. Multispecific TNFR1 Antagonist/TNFR2 Agonist Constructs To selectively inhibit TNFR1 signaling while enhancing the beneficial effects of TNFR2 signaling, multispecific constructs, such as bispecifics comprising a TNFR1 antagonist and a TNFR2 agonist, A construct is provided (see, eg, Equation 2 above). These multispecific constructs may optionally include linkers and activity modifiers that confer advantageous properties, as discussed above.

The TNFR1 inhibitor and TNFR2 agonist portions of the constructs provided herein can be polypeptides or small molecules or combinations thereof; they can be directly in any order or including those described herein. They may be linked indirectly via a linker such as a Gly-Ser linker and/or a hinge region, or may be linked via a chemical linker. The construct may include an activity modifier such as an Fc region or a modified Fc, and/or other activity modifiers such as a polypeptide such as HSA that extends half-life, or even a polymer such as PEG or a polymeric moiety. good.

A human TNFR1 antagonist, such as a TNFR1 antagonist set forth in any of SEQ ID NOs: 54-703, or a TNFR1 antagonist having about or at least about 95% sequence identity with a TNFR1 antagonist set forth in any of SEQ ID NOs: 54-703. The C-terminus is fused to the N-terminus of a first IgG1 Fc, such as an IgG1 Fc from trastuzumab. The order may be reversed.

The Fc region includes the CH2 and CH3 domains of the trastuzumab heavy chain (see, eg, residues 234-450 of SEQ ID NO: 26). In some embodiments, the linker between the TNFR1 antagonist and the first Fc subunit comprises all or part of the hinge sequence of an antibody such as trastuzumab (SCDKTH; corresponding to residues 222-227 of SEQ ID NO: 26). Including. To confer protease resistance and increase the plasticity of the fusion protein, the SCDKTH hinge sequence or protease cleavage site, or both, is combined with a Gly-Ser short peptide linker, such as, for example, GSGS, GGGGS, or GGGGSGGGGSGGGGS, and herein Others may be substituted as described and/or known in the art. In other embodiments, only a GS linker is included. In another embodiment, the linker comprises a PEG or branched PEG with a molecular weight of 30 kDa or greater.

In some embodiments, Fc subunits (also referred to as regions or domains) are capable of multimerization. The first Fc subunit is attached to the second Fc subunit via a disulfide bond. For bispecific constructs, the C-terminus of the second Fc subunit is a TNFR2 agonist, such as any of SEQ ID NO: 765-801, 803, and 810, or SEQ ID NO: 765-801, 803, and 810 or to a small molecule TNFR2 agonist having about or at least about 95% sequence identity to any of the TNFR2 agonists. The second Fc subunit and the TNFR2 agonist are connected via a 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 another embodiment, single chain Fv fragments (scFv), or Fab regions, or other antigen-binding fragments of TNFR2 agonist monoclonal antibodies may be used; the scFv or Fab is an N-terminal fusion with the C-terminus of the Fc. is dimerized by As provided herein, antigen binding fragments can be derived from the TNFR2 agonist mAbs MR2-1 and MAB226.

Fc subunits may be modified to alter their activity. For example, dimers can be used to prevent homodimerization and/or to support immune effector functions, e.g., antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis ( ADCP), and/or to eliminate complement-dependent cytotoxicity (CDC), and/or to enhance neonatal FcR (FcRn) recycling to improve in vivo half-life and stability of recombinant constructs. changed to.

In embodiments where the construct is for the treatment of inflammatory diseases, the Fc portion is modified to reduce or eliminate effector function. In embodiments, for example, when the construct is for the treatment of cancer, the Fc dimer is modified to enhance immune effector functions such as ADCC, ADCP and/or CDC. The specific Fc modification will vary depending on the intended disease target.

In some embodiments, the Fc subunit can include an IgG4 Fc region, such as the IgG4 Fc from nivolumab (Opdivo®), which includes the CH2 and CH3 domains of the nivolumab heavy chain (e.g., residues 224- 440 SEQ ID NO: 29). A short peptide linker comprising all or a portion of the nivolumab hinge sequence ESKYGPPCPPCP (see, e.g., residues 212-223 of SEQ ID NO: 29) sufficient to provide flexibility can be linked to the Fc region of nivolumab and the TNFR1 antagonist and and/or a TNFR2 agonist. Optionally or alternatively, a GS linker may also be included.

In an exemplary embodiment, TNFR2 may require aggregation/clustering of receptors for signal transduction, so bivalent antibody-like structures may be generated to achieve superior agonism. In this embodiment, the C-terminus of each of the first and second Fc subunits is fused to the N-terminus of the TNFR2 agonist, as described above. Fc dimers prevent homodimerization and eliminate antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), as described elsewhere herein. , has been modified to enhance neonatal FcR recycling and increase the in vivo half-life of the recombinant construct.

PEGylation to link the components of multispecific constructs, PEG-centered multispecific constructs, e.g. bispecific TNFR1 antagonist/TNFR2 agonist constructs with biocompatible and biologically inert polymers. PEGylation, which refers to the covalent attachment of certain poly(ethylene glycol) (PEG) to molecules such as proteins, peptides, drugs, and other molecules, is another modifier of construct activity. PEGylation increases the aqueous solubility of the molecule, increases the molecular weight of the molecule, prolongs in vivo circulation time, reduces peripheral clearance rate, minimizes nonspecific uptake, and enhances permeability and retention ( Tumors can be targeted through enhanced permeability and retention (EPR) effects. PEGylation of therapeutics, including protein therapeutics, masks undesirable antigenic surface markers, protects the therapeutic from the action of antibodies and antigen-processing cells, and reduces degradation by proteolytic enzymes and other inactivating processes. obtain. PEGylation also increases the molecular weight of protein therapeutics, increases their in vivo half-life, reduces peripheral clearance, and allows for less frequent administration.

Chemical attachment of therapeutic molecules to polymers such as PEG can form stable ester or amide bonds, as well as disulfide bonds. Conjugation of PEG to molecules of interest, such as the TNFR1 antagonists and TNFR2 agonists provided herein, can be accomplished using, for example, coupling agents such as dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), or as known in the art. This can be achieved by using N-hydroxysuccinimide (NHS) esters such as PEG NHS esters, etc. Other methods include PEG maleimides reacting with sulfhydryl groups of proteins or peptides; PEG pentafluorophenyl (PFP) esters reacting with primary and secondary amines; thiols reacting with thiols in the side chains of cysteine residues. PEG; as well as the use of click chemistry techniques. PEG azide, propargyl PEG, aminooxy PEG, hydroxy PEG, amino PEG, PEG acid, biotin PEG, PEG tosylate, and PEGs with other functional groups are also commercially available for conjugation to peptides and other therapeutic molecules. can be used for.

A common method to prepare PEG-protein conjugates is by coupling the protein's -NH2 group and monomethoxy PEG (mPEG) with electrophilic functional groups; this approach allows covalent attachment of globular proteins at the core. Polymer chains are formed. This property is exploited herein to provide PEG-centered constructs in which PEG or a chemically similar or suitable moiety exhibits multiple binding or interaction moieties for one or more different targets. . Multi-armed or branched PEG (or similar or branched moieties) may be used to increase drug (binding moiety) loading. Alternatively, the drug may be conjugated to small PEG dendrons (see, e.g., the PEG conjugation protocol on the BROADPHARM® website, available at Broadpharm.com/web/protocols.php; see also Banerjee et al. al. (2012) Journal of Drug Delivery, Article ID 103973). Heteromultifunctional PEG moieties, such as heterobifunctionals, having different reactive groups at each end, etc., may be used to attach multiple, such as two, different therapeutic moieties. A PEG moiety can have two, three, or more different reactive groups. Such molecules can be used to deliver two or more different ligands that target two different receptors on the same or different cells, such as TNFR1 and TNFR2, or the same receptor, as described herein. to deliver two targeting agents that bind to different sites on a receptor, or to cluster receptors, e.g. to activate or inhibit receptors, or to inhibit receptor activity, e.g. It can be used to cross-link two different receptors. Homobifunctional PEG molecules with identical reactive groups at each end can be used to cluster identical receptors on the same cell or, if the length of the PEG chain allows, on different cells. Such constructs can be used to capture circulating soluble receptors or ligands such as TNF. FIG. 3 herein provides an exemplary construct (binding reactive moiety) that uses a PEG moiety to present a drug.

To increase reactivity and plasticity, improve ligand-protein binding, and reduce steric hindrance, the constructs may include a linker molecule or molecules described herein as spacer molecules. Such spacers include, for example, amino acid spacers such as alanine, glycine, and small peptides. Any linker described herein, including GS linkers and other flexible linkers, as well as rigid linkers, can be used to bind the TNFR1 inhibitor moieties described herein and/or the TNFR2 agonists described herein, etc. The reactive moiety of can be conjugated to a multifunctional PEG molecule. These constructs may also include activity modifiers such as Fc regions.

The use of branched or multi-arm PEG moieties described herein (see, e.g., FIG. 5) in constructs containing a TNFR1 inhibitor moiety or a TNFR2 agonist, or a combination of both, with or without a linker. It may also be used, without limitation, to present other inhibitor and/or agonist moieties of any receptor of interest, and/or to generate immunotoxins and other toxic conjugates. . Methods for the synthesis of numerous PEG moieties and variations thereof are known (see, eg, US Patent Publication No. 2010/0221213; Han et al., (2014) Sci Rep 4:4387).

For example, in some embodiments that include a TNFR1 inhibitor and a TNFR2 agonist moiety, the construct includes a bifunctional PEG moiety and also includes a linker between the PEG moiety and each of the TNFR1 antagonist and TNFR2 agonist. The multispecific construct comprises a branched PEG polymer to which a linker is attached and to which one or both of the TNFR1 inhibitor moiety and the TNFR2 moiety are attached. Suitable PEG moieties may have a molecular weight of 30 kDa or more, such as 30-40 kDa or more. Exemplary branched PEG molecules include, for example, one arm with one type of reactive group (RG1; e.g., -NH2) linked to a TNFR1 inhibitor moiety, each linked to a TNFR2 agonist, and a different It can be a three-armed heterobifunctional PEG molecule, comprising two arms with a type of reactive group (RG2; eg -COOH). Such three-arm heterobifunctional branched PEG molecules are commercially available (eg from BROADPHARM®). The first PEG arm can be linked to the N- or C-terminus of the TNFR1 inhibitor moiety, the other two arms can be linked to the N- or C-terminus of the TNFR2 agonist, or to the TNFR2 agonist (if they are small molecules). (if any). In some embodiments, the construct may also include any linker described herein. Such a linker may be included between the PEG arm and the TNFR1 inhibitor moiety and/or the TNFR2 agonist. Such PEG-centered bispecific constructs provide monovalency for TNFR1 antagonist activity, prevent TNFR1 receptor clustering leading to unwanted agonism, and provide bivalency for TNFR2 receptor clustering. This enhances TNFR2 signaling. Among those shown in FIG. 3 are exemplary structures of the PEG-centered bispecific TNFR1 antagonist/TNFR2 agonist constructs described herein.

In another embodiment, the linker between the TNFR1 antagonist and TNFR2 agonist comprises a branched PEG with a molecular weight of 30 kDa or greater. The branched PEG molecule contains one branch linked to the N-terminus of the TNFR1 antagonist and two branches each linked to the TNFR2 agonist, providing bivalency for TNFR2 receptor clustering, which Enhances signal transmission.

Figures 3A-D show various configurations of multispecific 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 constructs are well known to those skilled in the art (see, eg, creativepegworks.com/pegylation_literature.php; and broadpharm.com/web/protocols.php). In FIG. 3, each n can independently be from 1 to 10, such as 1 to 7, 1 to 5, and 1 to 3, 1, or 2. In Figure 3A, each n is generally 1-3, depending on the particular ligand shown. In the rest of FIG. 3, n is generally from 1 to 5. N may be 1 in all embodiments. Those skilled in the art will recognize other routine variations in the PEG moiety that functions as the central linker; similar moieties may be used in place of PEG.

In Figure 3A:
R1 is H or lower alkyl (C1-C5, or C1 or C2) such as CH3, and n is generally 1-5, for example 1 or 2. The diagram shows a ligand or epitope that binds multiple targets (i.e., an epitope on a receptor such as target 1 (circle)), where 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 circle is the ligand for the target epitope or receptor, n is typically 1-5, usually n is 1 or 2, and typically 1. Figure 3D shows a homobifunctional construct; n is typically 1-3, generally 1 or 2, eg 1. Activity modifiers, such as Fc or others described herein or known to those skilled in the art, are optional. In all of these constructs, the PEG moiety provides activity-modifying activities such as half-life extension and/or sterically enhances the operable moiety that binds to the intended target (peptide, small molecule, aptamer, etc.). Generally "inactive" except for connection.

FIG. 3A depicts an exemplary bivalent construct in which PEG is the central moiety. One circle is, for example, a polypeptide agonist, antagonist, or binding protein, such as an antibody or antigen-binding fragment thereof, or an aptamer (nucleic acid or peptide). Other circles represent different moieties such as polysaccharides or receptor ligands. Bivalent structures provide clustering of targets for receptor activation. In some embodiments provided herein, targets include TNFR1 and TNFR2, and the circles represent TNFR1 inhibitors and TNFR2 agonists described throughout this disclosure. Figure 3B shows that monovalent single ligands such as CD3+ can prevent cytokine release syndrome, which is bivalent for receptor clustering via the PEG moiety, agonists, antagonists, or linked to binding proteins. Figure 3C shows that heterobifunctional PEG (or other such carriers). The constructs of Figures 3B and 3C may be used, for example, to cluster checkpoint control receptors for stimulation or inhibition of immune responses, or to cross-link two different receptors to determine receptor activity ( i.e., CD3 vs. CD450), or to deliver two different ligands, such as a stimulatory and co-stimulatory ligand, to two different receptors on the same cell. May be used. These constructs can also function as prodrugs that are directed to or accumulate in hypoxic regions with lower pH where the linking moiety can be chemically released by protonation. For example, in the case of tumors, this may be a toxin or a locally released TNF inactivator (ie an aptamer or peptide).

Figure 3D shows homobifunctional PEG for clustering identical receptors on the same or different cells depending on chain length, or for trapping circulating disease targets such as soluble receptors or ligands such as TNF. show. Additionally, in all of these embodiments, these constructs include an additional PEG side chain optionally linked to a serum half-life extending moiety such as HSA or another reactive or functional group such as an FcRn polypeptide. Good too.

Other structures are also possible, where X and Y refer to reactive groups, such as binding moieties, molecules that interact with the target (see Figure 4):

Other examples (see, e.g., FIG. 5) are as follows, where X and Y above can be any targeting or binding moiety or drug to interact with a target:

f. Additional Activity Modifiers - Properties of the fusion protein construct that include part or all of a polypeptide that increases serum half-life, but substantially reduce the interaction of the construct with TNFR1 and/or TNFR2 can be modified by adding unaltered or unaltered full-length polypeptides or portions thereof. This includes albuminization and other such modifications (see discussion above regarding half-life prolonging factors; reviewed in Strohl (2015) BioDrugs 29(4):215-239, and also Tan et al. (2018) Current Pharmaceutical Design 24:4932-4946).

5. Predicting and Eliminating Immunogenicity in Protein Therapies Many protein therapeutics, including those containing human germline sequences such as recombinant human cytokines and human antibodies, are immunogenic and induce host immune responses against the therapeutic. . As described herein, the constructs provided herein, including the TNFR1 antagonist molecules and TNFR2 agonists described and provided herein, as well as multispecific constructs, optionally include amino acid substitutions. or the like, to remove or eliminate epitopes that are immunogenic or interact with existing antibodies.

The constructs are subject to the prediction, identification and removal of immunogenic B cell and/or T cell epitopes, thereby reducing or eliminating potential immunogenicity and improving the safety, tolerability and efficacy of therapeutic molecules. increase. Molecules are tested in in vitro assays and in vivo animal models to determine immunogenicity before and after removal of immunogenic sequences.

As discussed in more detail below, protein therapeutics may include immunogenic B cell and/or T cell epitopes. When the immune system recognizes protein therapeutics as foreign, a coordinated and unwanted immune response to the therapeutic is induced. This reaction can lead to clinical complications such as rapid drug clearance, reduced drug functionality and efficacy, delayed infusion-like allergic reactions, anaphylaxis, and potentially life-threatening autoimmunity. Immune responses to protein therapeutics occur through two different mechanisms (classical immune response and by breaking tolerance). Immunological discrimination between self and non-self proteins determines the mechanism of immune response, with proteins recognized as foreign triggering a classical immune response, which occurs within days to weeks after administration. , often characterized by the formation of antibodies after a single injection of a protein therapeutic. This reaction is long-lasting, and once memory B cells are formed, it is difficult to reverse. Subsequent exposure to the protein induces a secondary reaction, mainly characterized by large amounts of IgG, which adversely affects therapy. Therapeutic proteins that induce classical immune responses include replacement therapies such as rhGAA (recombinant human acid alpha-glucosidase) and FVIII, as well as substitution therapies whose complementarity determining regions (CDRs) are highly immunogenic; monoclonal antibody (mAb) therapy that results in the production of anti-idiotypic alloantibodies due to lack of central tolerance to. Therapeutic proteins that are homologous to endogenous proteins usually do not elicit an immune response because immune tolerance is established, but after repeated administration, as in the case of IFN-γ, INF-β, and erythropoietin (EPO). Can be immunogenic by breaking B cell tolerance (e.g. Baker et al. (2010) Self/Nonself 1(4):314-322; Choi et al. (2017) Methods Mol.Biol. 1529 :375-398; see Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654).

Factors that influence the immunogenicity of protein therapeutics 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 long-term Treatments administered frequently over a long period of time are highly immunogenic. Patient-related factors such as the patient's immune status and polymorphisms in MHC (or HLA in humans) molecules also influence the immunogenicity of a protein. For example, the MHC molecule is highly polymorphic and several different alleles of MHC II exist, including various subunits such as DP, DM, DOA, DOB, DQ, DR; these receptors Because subtypes differ in their binding affinity for epitopes, differences in MHC subtypes between patients can influence the immune response to protein therapy. The patient's immune status may also influence immunogenicity. This is because autoimmune patients respond more strongly to protein therapy than immunocompromised patients. Other factors that influence immunogenicity include the properties of the protein product, such as the presence of immunogenic epitopes recognized by MHC II, the formation of aggregates in the final product, oxidation of the protein, and aggregation in the formulation. aggregation, and post-translational modifications such as glycosylation. Recombinant proteins can be, for example, E. It can be produced in several different cell types, including bacterial cells such as E. coli, and mammalian cells such as CHO cells. Proteins expressed in bacteria do not undergo post-translational modifications such as glycosylation, whereas proteins produced in mammalian cells can lead to different immunogenicity. For example, interferon β-1b, an interferon sold under the trademark Betaseron®, is an interferon sold under the trademark Betaseron®. A product (interferon beta-1a) produced in E. coli cells and unglycosylated, but later developed and sold under the trademark Avonex®, is produced in CHO cells using recombinant DNA technology. Ru. Betaseron's immunogenicity is much higher than Avonex® interferon, 35% vs. 5%, respectively. This difference may be partially due to differences in glycosylation patterns that can lead to aggregation (see, e.g., Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654). thing).

The effect after administration of protein therapeutics is the generation of high affinity anti-therapeutic antibodies, also known as anti-drug antibodies or ADA. The production of ADA involves stimulation of adaptive and non-adaptive immune responses. These are primarily polyclonal and can have neutralizing and non-neutralizing effects on protein therapeutics. ADAs may contain multiple isotypes (e.g., IgM, IgE, and IgG) and subclasses (e.g., IgG1-4) of heavy chain constant regions, and contain variable regions that bind protein therapeutics with high affinity. , undergoing somatic hypermutation of variable region genes. Immune responses are induced by recognition of immunogenic peptide fragments such as B cell and T cell epitopes in protein therapeutics. Therefore, many protein therapeutics require deimmunization while maintaining the desired therapeutic activity before they can be applied clinically (e.g., Baker et al. (2010) Self/Nonself 1(4):314 -322; see Choi et al. (2017) Methods Mol. Biol. 1529:375-398).

The formation of anti-drug antibodies (ADA) 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 sequence of protein therapeutics can trigger the generation of humoral responses to protein therapeutics. For example, DCs stimulated via pattern recognition receptors (PRRs) stimulate T cells and induce the generation of T cell-dependent high affinity ADA responses. In the first step of a T cell-dependent antibody response, APCs phagocytose protein therapeutics, process the antigen into peptide epitopes, and transfer the epitopes to the major histocompatibility complex on the APC cell surface for naive T cells. (MHC) by binding to class II molecules. Full activation of T cells, which is necessary to activate B cells, requires the T cell receptor (TCR) to interact with the MHCII-epitope complex, which is provided by the APC. additional signals from co-stimulatory molecules such as CD80 and CD86 are required. Naive B cells are activated by interactions 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 into plasma cells, leading to engagement of CD40 and CD40 ligands, leading to clonal expansion of B cells and antibody-secreting plasma cells and memory B cells. Provides additional signals that lead to antibody production by cell differentiation. While memory B cells remain quiescent until subsequently exposed to a therapeutic protein, plasma cells secrete antibodies that recognize specific epitopes on the therapeutic protein presented by APC MHC receptors. Many protein therapeutics, including recombinant human proteins, contain potent T cell epitopes. For example, the immunogenicity of IFNβ1b was improved after mapping and removal of a single immunodominant (but not subdominant) T cell epitope by amino acid mutation.

Immunogenicity can also occur in T cell-independent processes where the antigen directly engages B cells. High molecular weight aggregates of protein therapeutics can induce T-cell dependent and independent anti-drug antibody responses by stimulating DCs or by cross-linking B cell receptors. For example, if a protein therapeutic forms a multimeric structure that can cross-link B cell receptors (BCRs) effectively enough to circumvent the need for co-stimulation from T cells, T-independent stimulation can occur. There is a correlation between enhanced immunogenicity and aggregated or multimeric proteins. For example, aggregated, rather than monomeric, recombinant human interferon (IFN) α produces IFNα-specific antibodies in human IFNα transgenic mice. Aggregate formation depends on drug solubility and production process (e.g. Baker et al. (2010) Self/Nonself 1(4):314-322; Dingman et al. (2019) J. Pharm. Sci 108(5):1637-1654; see De Groot, A. S. and Moise (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340).

ADA responses to protein therapeutics may take the form of neutralizing antibodies or binding antibodies. Neutralizing antibodies recognize regions within a protein therapeutic that are necessary for biological activity and directly eliminate that activity. Humoral responses are directed against B cell epitopes within protein therapeutics, resulting in the ability to neutralize the therapeutic protein. For example, human anti-mouse (HAMA) or human anti-human (HAHA) responses to idiotypes of antibody therapeutics can be neutralized and generated against humanized and fully human antibodies. For example, 30% of hemophilia A patients develop neutralizing ADA to administered recombinant FVIII, rendering its hemostatic effect ineffective, and 89-100% of Pompe disease patients receiving rhGAA. In this case, anti-rhGAA neutralizing antibodies destroy the therapeutic effect. Binding antibodies alter the pharmacokinetic properties of a protein therapeutic, indirectly affecting its efficacy by reducing systemic exposure, for example, by promoting rapid clearance of the protein. For example, long-term use of adalimumab leads to the development of ADA in approximately 28% of patients, resulting in decreased adalimumab concentrations and worse clinical outcomes (Baker et al. (2010) Self/Nonself 1(4): 314-322; see Dingman et al. (2019) J. 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 involving labeling of drugs and detection of ADA forming a bridge between two labeled drug molecules. Bridging assays can be used for all antibody classes and any type of sample. Ligand binding assays (LBA) used to detect binding to targets include surface plasmon resonance (SPR), electrochemiluminescence, and biolayer interferometry, and can also be used to detect ADA. Protein-specific assays may be used, such as the Bethesda Assay, which has been used to measure the concentration of neutralizing anti-FVIII antibodies. Anti-PEG antibodies may also be measured in an assay in which biotin-PEG is conjugated to magnetic beads, and a sensor that detects changes in the size of the complex is used to determine the amount of anti-PEG antibody bound to the beads. Measure. Drug resistance assays overcome the limitations caused by the presence of drugs in the sample and improve the quantification of ADA. These include, for example, pH shift idiotype antigen binding assays, acid dissociation assays, temperature shift assays, and electrochemiluminescence assays. Enzyme-linked immunosorbent assay (ELISA) can be used to detect ADA; a protein therapeutic is coated onto a plate and incubated with the sample to measure bound ADA. ELISA for the detection of ADA may be limited by the lack of standards for ADA. Other methods include Immune-PCR, an extension of the bridging assay that labels the complex with biotin, which is detected using an anti-biotin antibody conjugated to DNA. The DNA is then amplified using PCR and quantified to assess ADA levels. Immuno-LC/MS can be used for the detection of ADA in plasma samples; the sample is prepared by tagging the drug with biotin or by spiking excess drug into the sample to saturate ADA binding. (see, e.g., Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654).

Prediction and removal of immunogenic epitopes from protein therapeutics (i.e., deimmunization) increases the efficacy and safety of therapeutics and prevents side effects that can lead to drug failure in clinical trials. obtain. For example, depletion of T cell epitopes from protein therapeutics by deimmunization has been successful in advancing protein therapeutics, particularly antibodies, into clinical trials. These results demonstrate the importance of T cell epitopes in generating ADA responses and that deimmunization provides safer and less immunogenic treatments (e.g., Baker et al. (2010) Self/Nonself 1(4):314-322). These methods may be used to detect or identify or predict the immunogenicity of constructs provided herein, including identifying amino acid mutations that eliminate or reduce immunogenicity or immune response in a subject. can be used to Constructs are provided that are modified to reduce or eliminate immunogenicity. Deimmunization 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 evaluation of immunogenic regions within protein therapeutic sequences includes in silico tools focused on epitope mapping, in vitro methods, e.g. epitope mapping, MHC/HLA affinity assays, and T cell proliferation assays, as well as the use of in vivo tests in animal models. Computational epitope prediction tools are used to increase the efficiency of protein therapeutic deimmunization. In silico tools include databases and algorithms for rapidly predicting immunogenic sequences of peptide libraries. The results may then be confirmed and the specific immunogenic effect of the epitope on B cells or T cells may be further evaluated using in vitro assays. Effects on immune responses to protein therapeutics can be assessed 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. Removal methods include random or site-directed mutagenesis to remove immunogenic sequences (ie, deimmunize the epitope). After mutagenesis, the immunogenic sequences are re-evaluated to confirm that they are no longer immunogenic. There are in silico tools that streamline this process; for example, sequentially substituting each amino acid of an immunogenic sequence with one of the other 19 naturally occurring amino acids (particularly alanine) and then determining the immunogenicity of the sequence. Re-evaluation programs are available. In this way, non-immunogenic sequences may be efficiently narrowed down to the most promising candidates prior to peptide synthesis and in vitro and/or in vivo re-evaluation of immunogenicity. However, prediction of immunogenic epitopes and mutagenic deletion are not always sufficient to deimmunize proteins. This is because proteins must remain folded and in a stable active conformation in order to maintain their therapeutic efficacy. Therefore, it is necessary to select epitope deletion mutations that are compatible with the structure and function of the protein.

The methods and approaches discussed below are used to predict, identify, and exclude epitopes from the constructs provided herein.

a. B Cell and T Cell Epitopes Interactions between antigens and antibodies are important in the induction of humoral immune responses against invading pathogens. Specific antibodies recognize particular antigens at distinct regions known as antigenic determinants or B-cell epitopes. B-cell epitopes include membrane-bound immunoglobulins that are solvent-exposed and surface-accessible and induce cellular or humoral immune responses, or by secreted antibodies or by B-cell receptors (BCRs). contains clusters of amino acids that are recognized and bound by

Identification of B cell epitopes is part of the development of antibodies and other protein-based therapeutics. B-cell epitopes are classified based on their spatial structure as continuous (also known as linear) epitopes, which contain consecutive residues, and discontinuous (also known as conformational) epitopes, which are nonlinear and three-dimensional. Discontinuous B-cell epitopes contain groups of solvent-exposed amino acid residues that are not completely contiguous but come together in close proximity when the protein/antigen is folded into a three-dimensional structure. join to. Approximately 90% of B cell epitopes are conformational. Linear B cell epitopes are recognized by antibodies after denaturation of the antigen, whereas conformational epitopes are no longer recognized once the antigen is denatured. The minimum amino acid sequence, or contact residue span, required for proper folding of a discontinuous B cell epitope ranges from about 20 to 400 residues in native proteins. The majority of linear B-cell epitopes identified are thought to be components of conformational B-cell epitopes, with more than 70% of discontinuous B-cell epitopes consisting of 1 to 5 linear segments (each (1 to 6 amino acids in length) (e.g., Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830; Sanchez-Trincado et al. (2017) Journal of Immunology Research, Article ID 2680160).

T cell epitopes are linear and bind to major histocompatibility complex (MHC) molecules through interactions between amino acid side chains and the binding pocket of the MHC epitope binding groove. The presence or absence of specific side chains determines whether and to what extent an epitope binds to MHC (e.g., De Groot, A. S. and Moise, L. (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340). T cells have T cell receptors (TCRs) that are displayed on the surface of antigen presenting cells (APCs) and recognize antigens bound to MHC molecules. T cell epitopes are presented by MHC class I (MHCI) and II (MHCII) molecules and are recognized by CD8+ and CD4+ T cells, respectively; therefore, there are CD8+ and CD4+ T cell epitopes. CD8+ T cells form cytotoxic T lymphocytes (CTLs) after CD8+ T cell epitope recognition, whereas primed CD4+ T cells form helper T (Th) cells, or regulatory T (Tregs), which amplify the immune response. cells that are immunosuppressive (see, eg, Sanchez-Trincado et al. (2017) Journal of Immunology Research, Article ID 2680160).

b. In Silico Epitope Prediction Methods Experimental studies and in silico analyzes have shown that the majority of epitopes span a region of 15-25 residues and 600-1000 Å2, are organized in loops, and that the epitope sequence has exposed side chains. and show that certain amino acid pairs are enriched in tyrosine, tryptophan, charged amino acids, and polar amino acids. However, the differences between epitope and non-epitope residues are not significant, demonstrating that amino acid composition alone is insufficient to distinguish between epitopes and non-epitopes. The combination of epitope mapping techniques and bioinformatics has enabled the use of computational methods in immunology to identify the structure of antibodies, B cells, T cells, and allergens, predict MHC binding, model epitopes, and analyze immune networks. including, led to the development of immunoinformatics (see, e.g., Potocnakova et al. (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 may be replaced/deimmunized, eg, for therapeutic protein production. Databases of known B cell epitopes have been developed, including pleiotropic databases such as the Immune Epitope Database (IEDB), 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 (rostlab.or g/services/epitome /), and the Structural Database of Allergenic Proteins (SDAP; available at fermi.utmb.edu/); and monopathogenic organism-specific databases, such as the HIV Molecular Immunology Database (hiv.lanl. gov/content/immunology/index.html), FLAVIdB (available at cvc.dfci.harvard.edu/flavi/), and Influenza Sequence and Epitope Database (ISED, available at influenza.cdc.go.kr). available). Other B-cell epitope databases include the Conformational Epitope Database (CED; available at immunonet.cn/ced/), the Protein Data Bank (PDB; available at rcsb.org), and the Structural Epitope Database (CED; available at immunonet.cn/ced/). e Database (SEDB; sedb.bicpu .edu.in) (see, for example, Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830).

Several algorithms are available for predicting B cell epitopes from their sequence or structure. Algorithms were developed that initially relied on linear epitope identification with propensity measures, but machine learning techniques such as hidden Markov models (HMMs), recurrent neural networks (RNNs), and support vector machines (SVMs) improved through the development of methods based on In silico B cell epitope prediction tools include tools that predict continuous/linear B cell epitopes and tools that predict discontinuous/conformational B cell epitopes. Prediction of discrete 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). ), BCPRED (BCPred and FBCPred (available at ailab.ist.psu.edu/bcpred/), BepiPred (available at cbs.dtu.dk/services/BepiPred/), LBtope (crdd.osdd.net/ra ghava /lbtope/), BcePred (available at crdd.osdd.net/raghava/bcepred/), EPMLR (available at bioinfo.tsinghua.edu.cn/epitope/EPMLR/), BEST (support vector B cell epitope prediction using machine tools; 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 (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 (epitopia.ta available at u.ac.il/), CBTOPE (available at crdd.osdd.net/raghava /cbtope/), EPCES (available at sysbio.unl.edu/EPCES/), EPSVR (Antigenic Epitopes Prediction with Support Vector Regressio n server); sysbio.unl.edu/ EPMeta (available at sysbio.unl.edu/EPMeta/), PEASE (Predicting Epitopes using Antibody Sequence); ofranlab.org /PEASE), EpiPred (available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/EpiPred.php), 3DEX (3D-Epitope-Explorer, not available online), PEPOP (pepop.sys2diag.cn rs.fr/ ), PEPOP 2.0 (available at sys2diag.cnrs.fr/index.php?page=pepop), and EpiSearch (available at curie.utmb.edu/episearch.html) (e.g., Potocnakova et al. See (2016) Journal of Immunology Research, Article ID 6760830; Sanchez-Trincado et al. (2017) Journal of Immunology Research, Article ID 2680160; Sun et al. (2013) Comput. Math Method M., Article ID 943636. ).

Sequence-based binding site prediction methods can also be used to predict B cell epitopes. Sequence-based prediction tools rely on the primary sequence of the antigen and use propensity measures to measure the probability that each residue is part of an epitope. Sequence-based prediction tools include BEST, which predicts conformational B cell epitopes. Binding site prediction tools aimed at identifying binding sites for conformational B cell epitopes on antibodies include, for example, ProMate, ConSurf, PINUP, and PIER (for example, Sun et al. (2013) Comput (See Math Method M., Article ID 943636).

Conformational B cell epitopes of proteins or antigens with 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 antibodies raised against natural antigens. Mimotope-based methods require input of a peptide (ie, mimotope) selected by antibody affinity and the 3D structure of the selected antigen. Epitope prediction methods based on mimotopes derived from phage display experiments are available, using statistical features of mimotopes to map mimotopes to overlapping positional patches on the antigen surface, or using mimotope mapping through alignment. may be converted back into the antigen sequence to indicate the location of the B cell epitope. To identify affinity selection peptides or mimotopes, random peptides are displayed on the surface of filamentous phage, and peptides that bind to the monoclonal antibody with some affinity are screened, eluted, and amplified. This selection process is repeated a total of 3-5 times to narrow down the peptides to those with the highest affinity. Mimotopes and epitopes have similar functions since they can bind the same paratope of a monoclonal antibody and elicit an immune response. The selected mimotopes share high sequence similarity, indicating the existence of certain important binding motifs and physicochemical preferences during interaction with antibodies. Therefore, mapping mimotopes back to the original antigen can help find true epitopes more precisely. Mimotopes have similar physicochemical properties and spatial organization, but rarely show sequence similarity to native antigens. Databases providing information on mimotopes include, for example, ASPD (available at mgs.bionet.nsc.ru/mgs/gnw/aspd), RELIC Peptides (available on request), PepBank (pepbank.mgh.harvard). .edu), and MimoDB (available at immunot.cn/mimodb). In silico mimotope-based prediction tools are essential for mapping mimotopes back to the surface of the original antigen, locating optimal alignment sequences, and predicting possible epitope regions. In silico B cell epitope prediction tools based on mimotope analysis include, for example, MIMOX (available at immunot.cn/mimox/), MimoPro (available at informatics.nenu.edu.cn/MimoPro), Pep-3D-Search (available at kyc.nenu.edu.cn/Pep3DSearch), MIMOP/MimCons (available upon request), MIMOP/MimAlign (available upon request), LocaPep (atenea.monstes.upm.es/# software), EpiSearch (available at curie.utmb.edu/episearch.html), Pepitope/PepSurf (available at pepitope.tau.ac.il/sources.html), PepMapper (infor matics.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.il), and SiteLight (not available online) (e.g., Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830; Sanchez-Trincado et al. (2017) ) Journal of Immunology Research, Article ID 2680160; see Sun et al. (2013) Comput. Math Method M., Article ID 943636).

ii. In silico prediction of T-cell epitopes Linear T-cell epitopes bind to MHC through the interaction of their amino acid side chains with the binding pocket of the MHC epitope-binding groove, and the presence or absence of a particular side chain determines whether a T-cell epitope is It determines whether it binds to MHC and the degree of binding. For in silico prediction, there are databases that provide libraries of existing epitopes, such as IEDB, Epitome, and SEDB, which provide information on two-dimensional T cell epitopes. Other in silico T cell epitope databases include CED, AntiJen, Bcipep, and the HLA epitope registry. There are several web pages and programs available that can be used to analyze sequences and predict immunogenic epitopes for protein therapeutic candidates. For example, a number of MHC binding motif-based tools are available that scan protein sequences for potential T cell epitopes. 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 (e.g., De Groot, A.S. and Moise, L. (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340). For example, the MHCPred algorithm provides information on the MHC binding capacity of amino acid sequences to various alleles, and EpiMatrix/JanusMatrix predicts allele-specific binding of protein therapeutics to MHC class II receptors and Bonding at the body interface can be evaluated. Other algorithms and programs for epitope prediction include, for example, ProPred, MMBPred, and Protean 3D. Epitope prediction needs to be combined with in vitro methods and activity evaluation to ensure that any modifications to remove immunogenic sequences retain the activity of the therapeutic protein (e.g., Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654).

iii. Peptide-MHC Class II Binding Prediction Structure-based methods for identifying T-cell epitopes rely, for example, on modeling peptide-MHC structures and evaluating interactions using molecular dynamics simulations. 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 prediction are based on peptide sequences known to bind to MHC molecules; peptide sequences are available in the IEDB, EPIMHC, AntiJen, and as described herein and in the art. are available in epitope databases such as others known in the US. Peptide-MHC binding predictions can be based on sequence motifs (SMs) that contain frequently occurring amino acids at specific positions (anchor residues) known to bind to MHC molecules. Motif matrices (MM) evaluate the contribution of every residue, including non-anchor residues, to the binding of MHC molecules, but do not take binding affinity into account. Quantitative affinity matrices (QAM) predict peptide-MHC binding and binding affinity. Quantitative structure-activity relationship (QSAR) additive models predict the binding affinity of a peptide to MHC as the sum of the amino acid contributions at each position plus the contribution of adjacent side chain interactions. Machine learning (ML), the most common and robust approach, uses algorithms trained on datasets consisting of peptides that either bind or do not bind to MHC molecules, and is an example of a ML-based discriminative model. include models based on artificial neural networks (ANNs), support vector machines (SVMs), decision trees (DTs), and hidden Markov models (HMMs) (e.g., Sanchez-Trincado et al. (2017) Journal of Immunology Research, Article ID 2680160).

Models for predicting the immunogenicity of protein therapeutics include in silico peptide MHC class II algorithms that predict with reasonable accuracy the ability of peptide sequences to bind to MHC class II. Such algorithms allow rapid screening of libraries of sequences. However, in silico and in vitro MHC class II binding assays generate high levels of false positives and identified immunogenic peptides fail to stimulate T cell responses in vitro and in vivo. Such analyzes do not take into account other factors that influence epitope formation, such as protein processing, recognition by the T cell receptor (TCR), and T cell resistance to the peptide. To address this, in vitro T cell assays are used. The combination of in silico analysis and in vitro assays is very useful for identifying epitopes and designing peptide variants containing epitope-depleted protein sequences with reduced MHC binding capacity (e.g., Baker et al. (2010) Self/ Nonself 1(4):314-322).

Prediction of T cell epitopes by peptide-MHC binding models is also complicated by MHC polymorphisms; in humans, MHC molecules, known as human leukocyte antigens (HLA), bind different peptides and There are hundreds of allelic variants of class I and class II HLA molecules that require specific models to predict (e.g., Sanchez-Trincado et al. (2017) Journal of Immunology Research, Article ID 2680160) (see ). Examples of T cell epitope prediction tools based on the peptide-MHC binding model include EpiDOCK, MotifScan, Rankpep, SYFPEITHI, MAPPP, PREDIVAC, PEPVAC, EPISOPT, Vaxign, MHCPred, EpiTOP, BIMAS, and TEPITO. PE, Prored, Prored-1, EpiJen , IEDB-MHCI, IEDB-MHCII, IL4pred, MULTIPRED2, MHC2PRED, NetMHC, NetMHCII, NetMHCpan, NetMCHIIpan, nHLApred, SVMHC, SVRMHC, NetCTL, and WAP P (e.g., Sanchez-Trincado et al. (2017) Journal of Immunology Research, (See Article ID 2680160).

EpiSweep is a suite of protein design algorithms that integrates computational prediction of immunogenic T-cell epitopes with sequence- or structure-based evaluation of the effects of epitope deletion mutations on protein stability, structure, and function. , allowing the selection of mutation combinations that optimize protein therapeutics for low immunogenicity and high activity and stability (e.g., for a step-by-step guide to the application of the EpiSweep suite of deimmunization algorithms, see (See Choi et al. (2017) Methods Mol. Biol. 1529:375-398).

c. In vitro epitope prediction methods In vitro methods can be used to determine cellular mechanisms of immune responses, identify immunogenic epitopes, and assess MHC affinity, T cell proliferation, and immunogenic effects of whole protein treatments. . 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 indicative of an inflammatory immune response. Epitope mapping of complete proteins is labor intensive, and in silico programs are used in conjunction with mapping to identify potentially immunogenic regions 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, as well as antigen fragmentation/antigen binding assays, competitive binding assays, modification testing/mutagenesis, phage Display technologies such as display and yeast display, and functional epitope mapping methods such as mimotope analysis.

i. In Vitro B Cell Epitope Prediction Methods Experimental methods for identifying B cell epitopes include, for example, elucidating the 3D structure of antigen-antibody complexes, screening peptide libraries for antibody binding, or when the antigen is mutated. Includes performing functional assays in which effects on antigen-antibody interactions are analyzed. Antibody-producing B cells recognize structural epitopes, approximately 16 to 22 residues in size, and amino acids that contact the antibody, approximately 3 to 5 residues in size, which influence the affinity between proteins and antibodies. containing functional epitopes. The most accurate method to identify structural epitopes is by X-ray crystallography of antigen-antibody complexes, which identifies the sequences that bind to the antibody and pinpoints the exact location of the epitope within the protein structure. can be used to identify both continuities and discontinuities and provide information about the strength of bonds. Structural epitope mapping identifies residues that make direct contact with the antibody, but does not always provide information about residues that contribute to binding strength. The freely available FTProd program can be used as an alternative calculation for 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 less than 25 kDa in size. NMR provides data on the structure, dynamics, and binding energies of antigen-antibody complexes and is performed in solution, so there is no need to generate crystals. Two additional methods for epitope mapping at moderate resolution include saturation transfer difference NMR and antibody inhibition of hydrogen-deuterium exchange on the antigen. Electron microscopy can also be used for epitope mapping, but this is a low-resolution structural method typically used for larger antigens such as whole virus particles or viral capsids. Electron microscopy cannot detect contact residues but can be used to confirm surface accessibility of epitopes. Cryo-electron microscopy is an alternative method for viewing rapidly frozen antigen-antibody complexes in physiological buffers, eliminating the need for stains and fixatives (e.g., Dingman et al. (2019) J. Pharm Sci. 108(5):1637-1654; see Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830).

Functional epitopes can be identified in a variety of ways, including antigen fragmentation, competitive binding, and modification studies. For example, functional B cell epitope mapping methods generally involve screening for antibody binding of proteolytic fragments or peptides derived from antigens, and testing of antigen-antibody reactivity of mutant proteins that have undergone site-directed or random mutagenesis. including. Therefore, functional epitope mapping tools are used to identify and characterize residues within epitopes that are important for antibody binding. The majority of functional methods use antigen fragments, synthetic peptides, or recombinant antigens, e.g., mutant variants, antigens sequenced by in situ cell-free translation, and/or selectable systems such as phage display. The binding of the antibody to the antigen expressed is detected. For example, antigen fragmentation and binding assays include immobilizing the peptide to a solid support and using Western blots, dot blots, and/or ELISA to determine whether the epitope fragment binds to the antibody. ; binding indicates that the peptide fragment may be immunogenic. Competitive binding assays that provide low-resolution mapping can be used to determine whether multiple antibodies can bind to an epitope on a protein simultaneously, or whether they compete with each other for binding to the same epitope. (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 that replaces individual residues (called hotspots) of a functional epitope and assesses the effect of the modification on antibody binding to the immunogenic sequence. Hotspots, most frequently including Tyr, Arg, and Trp residues, are energetically important residues that include part of the complete protein-protein interface region. Mutation of individual residues can identify deleterious residues that can be replaced, provided the protein retains its structure and activity. In epitope mapping by mutagenesis, peptide libraries are generated by random or site-directed mutagenesis. The combination of mutagenesis and display techniques allows the screening of large numbers of mutant proteins. Saturation mutagenesis is another method of epitope mapping in which amino acid residues at specific positions within an epitope are replaced with all 20 naturally occurring amino acids and loss of antibody binding is monitored. A disadvantage of this method is that loss of immunoreactivity may be due to disruption of antigenic structure, making interpretation of the results difficult. Most of the contacts made between epitopes and antibodies are made through amino acid side chains, and alanine scanning mutagenesis can be used to define the contribution of each residue side chain to antibody binding. This is done by sequentially replacing each non-alanine residue with alanine, one at a time, cleaving the side chain down to the β carbon without adding plasticity to the protein backbone. This method identifies key residues whose side chains make the highest energetic contribution to paratope-epitope interactions. Computational alanine scanning can also be used to rapidly determine the effect of alanine mutations on the binding free energy of protein-protein complexes by using a simple free energy function. Combinatorial mutagenesis is based on combinatorial randomization of individual antigenic regions and grouping of mutated residues (primary sequence proximity), allowing for the identification of combined effects mediated by neighboring residues. This allows the identification of residues that are not important for binding but contribute to the formation of the epitope, or that form multiple interactions with the paratope that are individually 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., an alanine substitution) to generate a mAb of a natively folded protein. This involves testing reactivity directly in cells. Shotgun mutagenesis can identify both linear and conformational epitopes with a mapping rate of >20 epitopes per month (e.g., Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830).

Other techniques for epitope mapping include display techniques and mimotope analysis, which are inexpensive, flexible and fast. Display technologies such as phage display and yeast display evaluate the binding ability of various peptides displayed on a display platform to mAbs of interest through biopanning affinity selection methods (e.g., binding of peptides to ribosome-mRNA complexes). or to the surface of phages, bacteria, mammals, insects or yeast cells (see e.g. Potocnakova et al. (2016) Journal of Immunology Research, Article ID 6760830) ).

ii. 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 assess T cell responses to protein therapeutic antigens. 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, AS and Moise, L. (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340).

MHC/HLA Binding Assay T cell epitope prediction identifies the shortest peptide sequences within an antigen that stimulate CD8+ or CD4+ T cells and are therefore immunogenic. Immunogenicity of T cell epitopes depends on antigen processing, peptide binding to MHC molecules, and recognition by cognate TCRs; MHC peptide binding is the most selective process and is the most selective process for predicting T cell epitopes. It is the main foundation. MHC binding assays can be used to detect high affinity peptides and are often applied in combination with epitope mapping to identify regions of a protein that are likely to be immunogenic. In vitro MHC class II binding assays include cell-based binding assays and soluble HLA binding assays. High-throughput MHC binding assays involve incubating various doses of the peptide of interest with a control peptide and soluble MHC protein to assess binding affinity; high-affinity peptides bind more tightly to MHC and are more likely to be recognized by T cells. For example, MHCII:epitope binding can be assessed by measuring the ability of an exogenously added peptide 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 potential immunogenic epitopes are commercially available, for example from ProImmune (e.g., Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654; See De Groot, AS and Moise, L. (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340; Sanchez-Trincado et al. (2017) Journal of Immunology Research, Article ID 2680160. thing).

iii. In Vitro T Cell Assays The presence of T cell epitopes in protein therapeutics can be detected by assessing T cell responses in vitro in T cell assays. T cells proliferate and release cytokines upon stimulation by immunogenic proteins. T-cell epitopes induce the secretion of cytokines such as IL-2, IL-4, IL-5, and IFNγ by effector T cells, and the secretion of cytokines TGFβ and TNFα and chemokines such as MIP1α/1β by regulatory T cells. Induced by cells (Tregs). Proliferation of T cells in response to immunogenic peptides/epitopes can be measured by radiolabeling with thymidine or by labeling with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). ELISA or ELISpot methods and flow cytometry can be used to measure the levels of cytokines secreted by T cells, such as IL-2 and IFN-γ, to determine immunogenicity. The ELISpot method is very 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. For example, ELISpot assays for measuring IL-2 and IL-4 are commercially available. Flow cytometry may also be used to measure T cell responses, whereby T cells responsive to particular epitopes 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 that respond to antigens 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 and/or can be determined by identifying intracellular cytokine expression. Identification of T cell epitopes can therefore be combined with phenotypic studies to assess whether the immune response is inflammatory or suppressive. Peripheral blood mononuclear cell (PBMC) assays using PBMC preparations include several types of immune cells (such as CD4+ and CD8+ T cells) that better mimic the in vivo immune system and It can be used to assess a protein's immunogenicity and potential immune response without testing. PBMC are stimulated with whole therapeutic proteins, or peptides derived from therapeutic proteins, in in vitro culture. Innate immune screens using PBMC preparations lacking CD8+ reactive T cells or innate cell lines such as innate lymphocytes (ILCs) are used to distinguish between innate and adaptive immune responses to immunogenic proteins. obtain. To be useful, in vitro T cell assays require testing peptides on PBMC from large cohorts of donors with a wide range of MHC class II allotypes. In vitro T cell assays can provide information on the number and potency of T cell epitopes, which can determine the risk of immunogenicity during preclinical development and guide removal of such epitopes by targeted amino acid substitutions. (e.g., Baker et al. (2010) Self/Nonself 1(4):314-322; Dingman et al. (2019) J. Pharm. Groot, ASand Moise, L. (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340).

d. In Vivo Epitope Prediction Methods In vivo evaluation of the immunogenicity of protein therapeutics in humans uses animal models such as mice. Generally, any human or humanized protein therapeutic can be immunogenic when administered to a non-human animal. However, animal models are useful for predicting immunogenicity, comparing relative immunogenicity between products, formulations or routes of administration, determining immunogenicity of aggregates, and elucidating 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. Because animal MHC receptors do not directly mimic human HLA receptors, and because HLA and MHC genes are highly polymorphic and HLA/MHC expression varies widely between subjects, human protein therapy Assessing the immunogenicity of drugs in animals can be difficult. To overcome these limitations, HLA transgenic mice have been generated that mimic human subjects and can be tolerized to specific proteins; the mice tolerate and develop the protein therapeutics being evaluated. Immunogenicity is due to the breakdown of self-tolerance and is not due to the classical immune response to foreign antigens. In vivo methods for determining the immunogenicity of protein therapeutics include exposing HLA transgenic mice to whole proteins or epitope peptides. Several transgenic mouse lines expressing common HLA gene products, such as HLA-A, HLA-B, and HLA-DR molecules, have been generated and are useful for protein therapy by T cell response, ELISA, and neutralizing antibody assays. can be used to measure antibodies induced by exposure to B cell epitopes in protein therapy can also be identified by immunizing HLA transgenic mice with the protein (e.g., Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654; De See Groot, AS and Moise, L. (2007) Curr. Opin. Drug Discov. Devel. 10(3):332-340).

NOD scid γ (NSG) mice are highly immunodeficient, lacking most immune cells and complement and cytokine signaling, and can be transfected to investigate the human immune system in an in vivo model. For example, a CD34+ humanized NSG mouse model is transplanted with cord blood-derived hematopoietic stem cells to develop a functional immune system with normal T cell and inflammatory functions. Animal models also include non-human primates such as rhesus macaques and chimpanzees, as their proteins show a high degree of homology with human proteins, and their immune mechanisms are similar to those of humans. (See, eg, Dingman et al. (2019) J. Pharm. Sci. 108(5):1637-1654).

e. Removal of predicted B cell and T cell epitopes (deimmunization)
As described herein, the prediction and removal of immunogenic epitopes from protein therapeutics (i.e., deimmunization) increases the efficacy and safety of the constructs provided herein; The potential for adverse effects may be reduced or prevented. For example, removal of an identified epitope, such as a B-cell epitope, may prevent the formation of ADAs, which may be inhibited by neutralizing the therapeutic agent and/or inducing rapid clearance from the body. thereby reducing the effectiveness of the administered protein therapeutic.

Deimmunization 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 mentioned above, prediction and evaluation of immunogenic regions within protein therapeutic sequences includes the use of various in silico, in vitro, and in vivo methods. Once an immunogenic epitope is identified, the amino acid sequence of the epitope is modified by random or site-directed mutagenesis to remove the immunogenic sequence and deimmunize the epitope. For example, most of the contacts made between an epitope and an antibody are made through amino acid side chains, and alanine scanning mutagenesis can be used to define the contribution that each residue side chain makes to antibody binding. This is performed one at a time by successive alanine substitutions of each non-alanine residue to identify key residues whose side chains make the highest energetic contribution to the paratope-epitope interaction. However, prediction of immunogenic epitopes and mutagenic deletion are not sufficient to deimmunize the protein, as the protein retains a folded and stable active conformation to maintain its therapeutic efficacy. This is because it is necessary to select epitope deletion mutations that are compatible with the structure and function of the protein.

There are in silico tools that increase the efficiency of this process. For example, programs are available that sequentially replace each amino acid of an immunogenic sequence with one of the other 19 naturally occurring amino acids and then reevaluate the immunogenicity of the new sequence. For example, OptiMatrix is a tool that repeatedly substitutes all 20 amino acids at any position in a peptide sequence and then reanalyzes the predicted immunogenicity of the modified sequence (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 prediction of immunogenic T-cell epitopes with sequence- or structure-based evaluation of the effects of epitope deletion mutations on protein stability, structure, and function. , allowing the selection of mutation combinations that optimize protein therapeutics for low immunogenicity and high activity and stability (e.g., for a step-by-step guide to the application of the EpiSweep suite of deimmunization algorithms) , see Choi et al. (2017) Methods Mol. Biol. 1529:375-398). Computational alanine scanning can also be used to quickly determine the effect of alanine mutations on the binding free energy of protein-protein complexes by using simple free energy functions (e.g., Potocnakova et al. 2016) Journal of Immunology Research, Article ID 6760830).

G. Pan-growth factor trap construct 1. receptor tyrosine kinase (RTK)
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, such as cell division, proliferation, differentiation, migration, and metabolism. RTKs can be activated by ligands that specifically bind to their cognate receptors. Such activation, in turn, triggers autocrine or paracrine cell signaling pathways, activating events in the signaling pathway, e.g., by activating a second messenger that produces a specific biological effect. become Approximately 20 different classes of RTKs have been identified, including, for example, the epidermal growth factor receptor (EGFR) family (class I, also known as the ErbB family); the insulin receptor family (class II); Platelet-derived growth factor receptor (PDGFR) family (class III); vascular endothelial growth factor receptor (VEGFR) family (class IV); fibroblast growth factor receptor (FGFR) family (class V); hepatocyte growth factor receptors (HGFR) family (class VIII); and the Eph receptor family (Ephs, after erythropoietin-producing human hepatocyte receptors; class IX).

RTKs have been implicated in the regulation of pathways involved in physiological and angiogenesis, including tumor angiogenesis, and are involved in the regulation of cell proliferation, migration, and survival. RTKs are involved in many diseases including autoimmune diseases and cancers, such as breast and colorectal cancers, gastric cancers, gliomas, and tumors of mesodermal origin. Dysregulation of RTKs is associated with several cancers. For example, breast cancer is associated with amplified expression of p185-HER2. RTKs have also been associated with eye diseases such as diabetic retinopathy and macular degeneration. Additionally, members of the epidermal growth factor receptor (EGFR) family, and EGF-like growth factors (ligands), have been shown to be overexpressed in synovial fibroblasts and macrophages of rheumatoid arthritis (RA) patients.

a. Human Epidermal Growth Factor Receptor (HER) Family Some of the disease-associated RTKs include class I human There is a family of receptors in the EGFR (HER; also called ErbB) family. HER1, HER3, and HER4 are involved in epidermal growth factor (EGF), transforming growth factor (TGF)-α, heparin-binding (HB)-EGF, amphiregulin, β-cellulin (BTC), epiregulin, epigen, and neurules. Collectively binds over 11 canonical ligands, including Grin (NRG) 1-4. HER2 does not bind any of these ligands, but functions as a signal amplifier through heterodimerization with other HER family members such as HER3 and HER4 (Jin et al. (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 through the phosphatidylinositol 3-kinase pathway. do.

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, L1, CR1, L2, and CR2, where "L" refers to a leucine-rich repeat domain and "CR" refers to a cysteine-rich repeat domain. (also known as the furin-like repeat domain); the four subdomains are also referred to as domains I-IV, respectively. Domains I and III are the 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 necessary for dimerization known as dimerization arms, and domain IV contains sequences that enable domain II/IV tethering, but the tether height HER2, which does not have the following structure, is an exception. In the absence of ligand, EGFR, HER3, and HER4 subdomains II and IV form an intramolecular autoinhibitory tether. Upon binding of ligand, the subdomains undergo conformational changes that allow subdomains I and III to form a high-affinity ligand-binding pocket. Mutagenic disruption of the tether formed by subdomains II and IV, or C-terminal deletion of subdomain IV, has been shown to increase ligand binding affinity by up to 15-fold (e.g., Jin et al. (2009) Mol. Med. 15(1-2):11-20).

Members of the HER family are expressed in a variety of tissues of epithelial, mesenchymal, and neuronal origin. Under normal physiological conditions, activation of 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 activation of the intrinsic kinase domain, autophosphorylation of specific tyrosine residues in the cytoplasmic tail, and in some cells triggers internal protein recruitment and phosphorylation and binding to multiple downstream signaling cascades. Activated signaling pathways include the Ras-Raf-mitogen-activated protein kinase mitogen pathway, the phosphatidylinositol 3-kinase-AKT cell survival pathway, and the stress-activated protein kinase C and Jak/Stat pathways. The induced signaling pathways lead to a variety of cellular responses including, for example, cell migration, invasion, proliferation, survival, and differentiation (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 HER family members and their ligands, through overexpression or mutation, has been shown to play a role in cancer and other diseases. ing. For example, HER1 and HER2 are involved in the development and pathology of many human cancers, and alterations in these receptors are associated with more aggressive disease and worse clinical outcomes. Overexpression of TGF-α is associated with cancers of the prostate, pancreas, lung, ovary, and colon, and overexpression of NRG1 is associated with breast cancer. HER1 overexpression is associated with glioma, as well as head and neck cancer, breast cancer, bladder cancer, prostate cancer, kidney cancer, and non-small cell lung cancer, and mutations in HER1 are associated with glioma, as well as lung cancer, Associated with breast cancer and ovarian cancer. HER2 overexpression is associated with breast, lung, pancreatic, colon, esophageal, endometrial, and cervical cancers; HER3 is associated with breast, colon, gastric, prostate, and oral squamous cancers. Associated with epithelial cancer; HER4 is associated with breast and prostate cancer, and childhood medulloblastoma (e.g., Yarden et al. (2001) Nat. Rev. Mol. Cell Biol. 2:127-137 checking).

The EGF family of ligands and receptors has been shown to be involved in the development of inflammatory arthritis. For example, expression of HER2 and the presence of the EGFR ligands EGF, amphiregulin, and TGF-α have been detected in RA synovium. Adenoviral delivery of the human EGFR family inhibitor herstatin, an alternative splice variant of HER2, has been shown to abolish all clinical signs of collagen-induced arthritis (CIA) in mice. Herstatin prevents dimerization and functions as a natural inhibitor of natural HER1, HER2, and HER3. A long-standing RA patient previously treated with rituximab and adalimumab experienced a significant reduction in joint pain after treatment with the anti-EGFR/HER1 antibody cetuximab for head and neck cancer. These results indicate that treatments targeting HER may be therapeutically useful in the treatment of autoimmune and inflammatory conditions such as rheumatoid arthritis (RA) (e.g., Gompels et al. 2011) Arthritis Research & Therapy 13:R161).

Macrophages are the source of TNF in chronically inflamed RA joint tissue. Phenotypic analysis of macrophages from synovial tissue of RA patients revealed that the inflammatory genes NR43A (nuclear receptor subfamily 4 group A member 3), PLAUR (plasminogen activator, urokinase receptor), and CXCL2; It was revealed that HBEGF+ (hairpin-binding EGF-like growth factor+) inflammatory macrophages were enriched, overexpressing the growth factors HB-EGF and epiregulin (EGFR family ligand). HBEGF+ inflammatory macrophages also produced the proinflammatory cytokine IL-1 and promoted synovial fibroblast invasiveness in an epidermal growth factor receptor-dependent manner. The majority of drugs used in the treatment of RA have been shown to target HBEGF+ macrophages in ex vivo synovial tissue assays, and EGFR inhibitors have been shown to target HBEGF+ macrophages in ex vivo assays. were shown to effectively block EGFR responses, indicating that blocking the EGFR response may provide a non-immunosuppressive therapeutic approach for RA (e.g., Kuo et al. (2019) Sci. Transl. Med. 11(491)). Such an approach is advantageous over the use of conventional anti-TNF therapies, which are immunosuppressive and often associated with the development of serious infections such as tuberculosis.

HER family signaling has also been implicated in 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 is dependent on transactivation by HER1/EGFR in response to HB-EGF. EGFR expression is also associated with psoriasis; in normal skin, EGFR expression is limited to the basal layers, but in psoriatic patients, EGFR and its ligand amphiregulin are highly expressed throughout the epidermal layers. (eg, Yarden et al. (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, some diseases and conditions are associated with exposure to one or more neuregulin (NRG) ligands, such as NRG1, NRG2, NRG3, and/or NRG4, including types I, II, and III, e.g. , may be caused by or aggravated by them. Examples of NRG-related diseases include neurological or neuromuscular diseases, including schizophrenia and Alzheimer's disease (see, eg, US Patent Application Publication No. 2010/0055093).

Due to its role in cancer and other proliferative diseases, rheumatoid arthritis, neurodegenerative diseases, and autoimmune diseases, HER is a target for therapeutic intervention. Anti-HER therapeutics include antibodies that target the extracellular domain (or ectodomain), referred to herein as the ECD, and small molecule tyrosine kinase inhibitors. Therapeutic agents approved for the treatment of cancers caused by proteins of the HER family include monoclonal antibodies such as trastuzumab (directed against HER2), pertuzumab (directed against HER2), panitumumab (directed against HER1/EGFR) ), and cetuximab (targeting 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 to treat node-positive or node-negative breast cancer that overexpresses HER2; cetuximab is used to treat metastatic colorectal cancer, as well as head and neck cancer; panitumumab is used to treat metastatic colorectal cancer, as well as head and neck cancer; Used to treat colorectal cancer; lapatinib is used as first-line treatment for triple-positive breast cancer and as adjunctive therapy for patients who have progressed on trastuzumab; erlotinib is used to treat non-small cell lung cancer and pancreatic cancer used for.

Anti-HER therapeutics have limited efficacy and limited duration of response. Trastuzamab (eg, sold as Herceptin®) is a humanized version of a murine monoclonal antibody that targets the extracellular domain of HER2. However, the efficacy of trastuzumab requires high expression of HER2 (at least 3-5-fold overexpression), so that less than 25% of breast cancer patients are eligible for treatment. Within this population, the majority do not respond to treatment. Furthermore, small molecule tyrosine kinase inhibitors often lack specificity. Except for patients who highly express HER2 and are treated with trastuzumab in combination with chemotherapy, efficacy observed with single-targeted anti-HER antibodies or small molecule tyrosine kinase inhibitors ranges from 10-15%. . Treatments, especially those targeting only one HER family member, also suffer from intrinsic or acquired resistance associated with co-expression and ligand activation of other RTKs, especially other HER family members. For example, drug resistance is often associated with upregulation or compensation of other HER family members such as HER3 and HER4, or increased expression of HER1 or HER3 ligands by tumor cells. Homo- and heterodimerization between members of the HER family of receptors also has implications for therapy directed against single HER family receptors. Due to the limited efficacy of available treatments, alternative anti-HER treatments are needed. Provided herein are more effective alternative therapies that target the HER family of RTKs and their ligands.

2. Pan-growth factor inhibition As described herein, resistance to single-target anti-HER therapies such as trastuzumab, cetuximab, gefitinib and erlotinib is often due to co-expression and/or up-regulation of other HER family members. , and/or overexpression of their ligands. One strategy to alleviate or overcome this resistance and improve the efficacy of HER-targeted therapy is to inhibit multiple ligand-induced HER family members simultaneously. This can be achieved, for example, by chimeric HER ligand binding molecules that behave like receptor decoys and sequester multiple HER family ligands, preventing ligand-dependent receptor activation and downregulating aberrant HER family activity. do.

a. RB242 Ligand Trap An antagonist called RB242, a chimeric bispecific ligand trap that is an Fc-mediated heterodimer of 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 IgG1. In RB200, the extracellular domains of EGFR (corresponding to residues 1 to 621 of the mature EGFR protein shown in SEQ ID NO: 41) and HER3 (corresponding to residues 1 to 621 of the mature HER3 protein shown in SEQ ID NO: 45) ( Gly-Arg-Met-Asp added to the N-terminus of the Fc fragment, respectively, to the N-terminus of the Fc fragment of human IgG1 (corresponding to residues P100-K330 of SEQ ID NO: 9). (GRMD) linker. The HER3/Fc component of RB200 contains a 6xHis tag at the COOH terminus for purification.

RB200 inhibits EGFR and HER3 ligands (including EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, epiregulin, and epigen, and NRG1-α, NRG1-β1, and NRG1-β3, respectively). , binds with high affinity and inhibits ligand-induced tyrosine phosphorylation of HER family members (HER1, HER2 and HER3), inhibits the growth of a diverse range of tumor cells in vitro and tumor xenografts in nude mouse models. It has been shown to inhibit the growth of cancer (epidermoid carcinoma and non-small cell lung cancer). RB200 also showed synergy with tyrosine kinase inhibitors for EGFR/HER1 and HER2 kinases, such as AG-825, erlotinib, gefitinib, or lapatinib, in inhibiting tumor cell growth in vitro. Inhibition of ligand-stimulated phosphorylation of HER1, HER2, and HER3 was more effective by RB200 compared to monoclonal antibodies targeting HER1 (C225) or HER2 (trastuzumab and 2C4) (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 proteins, vectors encoding HER1/Fc and HER3/Fc constructs were cotransfected into HEK293T cells at a ratio of 1:3 (HER1/Fc:HER3/Fc). It was effected. As a result, in addition to the desired HER1/Fc:HER3/Fc heterodimer, HER1/Fc and HER3/Fc homodimers are expressed. The expressed protein was purified using a combination of Protein A, Ni-Sepharose and EGFR-Affibody column chromatography methods. Analytical reverse-phase high-performance liquid chromatography (HPLC) revealed that the RB242 heterodimer contains approximately 10% contamination with two homodimers (e.g., Sarup et al. (2008) (See Mol. Cancer Ther. 7 (10):3223-3236). Therefore, improved methods are needed to improve the yield and purity of heterodimers.

b. RB200 and RB242 for the treatment of autoimmune diseases
As discussed elsewhere herein, a significant proportion of RA patients do not respond to treatment with anti-TNF therapies, such as anti-TNF antibodies, which are associated with an increased risk of serious infections, including tuberculosis. or stop responding. Therefore, alternative treatments are needed. Increased expression of EGF ligands and receptors (HERs) in the synovium and synovial fluid of rheumatoid arthritis (RA) patients has been reported, and EGFR-targeted therapies can be used to treat RA and other autoimmune diseases. It has been shown that inflammatory diseases and disorders can be treated.

Bispecific EGFR ligand trap RB200 (and its derivative RB242) dose-dependently reduces disease severity of collagen-induced arthritis (CIA). Mice with CIA were treated 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. The patient was treated intraperitoneally. Treatment with 1 mg/kg or 10 mg/kg RB200 inhibited the increase in clinical scores and paw swelling in a dose-dependent manner. EGF has been shown to promote angiogenesis, and mice treated with RB200 showed decreased CD31 immunopositive staining, reflecting a reduction in synovial blood vessels, and inhibition of synovial angiogenesis. Joint sections from mice treated with PBS control showed numerous infiltrating cells in the inflamed synovium, as well as infiltration and erosion of the bone by the synovium, which was also associated with significant CD31 expression. Joints of mice treated with RB200 at 1 mg/kg or 10 mg/kg were protected and had normal appearance, well-preserved joint architecture, and few CD31-positive blood vessels. These results indicate that inhibition of EGFR-mediated responses may have therapeutic use in the treatment of RA (see, eg, Gompels et al. (2011) Arthritis Research & Therapy 13:R161).

The combination of TNF inhibition and inhibitors of EGFR-mediated signaling enhances the therapeutic efficacy of anti-TNF therapy and may be useful in the treatment of RA. Coadministration of a low dose of RB200 (0.5 mg/kg) and a suboptimal dose of etanercept (1 mg/kg) inhibited the increase in clinical score and paw swelling, completely abolished CIA, and suboptimal dose of etanercept (5 mg/kg) alone was shown to be of similar efficacy to that observed. In contrast, administration of low-dose RB200 alone or low-dose etanercept alone had no effect. Fluorescently labeled monoclonal antibodies against E-selectin may be used to localize endothelial activation in inflamed tissues in vivo and are sensitive, specific and quantifiable for the assessment of CIA. It is a molecular imaging technology. The combination of low-dose RB200 and low-dose etanercept reduced the amount of E-selectin detected in the paw to levels seen in healthy animals, whereas E-selectin was significantly reduced by low-dose RB200 alone or low-dose etanercept. was detected in the paws of CIA mice fed alone. RB200 alone and etanercept alone had dose-dependent effects on joint structure, with progressively fewer joints with severe destruction and more joints with mild or moderate destruction, but the most pronounced effect was observed with combined treatment. and 64% of the joints were normal compared to 0% in mice treated with either low-dose RB200 alone or low-dose etanercept alone. Combination treatment was also more effective than high-dose etanercept alone, demonstrating the efficacy of combined pan-EGFR and TNF-targeted therapies in promoting joint protection (e.g., Gompels et al. (2011) Arthritis Research & (See Therapy 13:R161).

c. RB242 Ligand Trap The ligand trap called RB242, derived from RB200, contains 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 the HER3 ECD subdomains I and IV. An affinity-optimized Fc-mediated triple mutant EGFR:HER3 heterodimer containing Y246A in domain II (see sequence of mature HER3 protein (SEQ ID NO: 45)). Compared to the parent molecules RB200, RB242, the affinity for various ligands such as EGF, TGF-α, HB-EGF, and NRG1-β was improved by an average of 22 times, and the affinity for cultured monolayer BxPC3 pancreatic cancer cells was improved by an average of 22 times. and improved antiproliferative activity in a mouse model of human non-small cell lung cancer. RB242 also showed a 10- to 60-fold improvement in inhibiting ligand-induced HER phosphorylation compared to RB200 (e.g., Jin et al. (2009) Mol. Med. 15(1-2):11 -20).

3. Optimized multispecific growth factor trap constructs, including bispecific growth factor trap constructs herein specifically target more than one cell surface receptor, as long as the activity of more than one cell surface receptor is modulated. is a pan-cell surface receptor therapeutic, e.g., by binding to a ligand for one or more receptors and/or by interacting with one or more cell surface receptors. Multispecific, including bispecific, growth factor trap constructs designed to do so are provided. The constructs include constructs that target two or more HER family members, as well as constructs that target one or more HER and additional receptors, e.g., HERs that contribute to or are involved in the development of resistance to anti-HER therapy. can be mentioned. The growth factor trap constructs provided herein target the multimerization domain of one receptor, particularly EGFR/HER1, HER2, HER3 or HER4, such as the Fc of human immunoglobulin (Ig), such as the Fc of human IgG. a plurality, in particular two chimeric fusion polypeptides, each comprising all or part of the extracellular domain (ECD) of a member of the HER family, such as a HER family member, such as a HER family member, such as a HER family member, such as a HER family member such as a HER family member. The ECD or portion thereof in the chimeric fusion polypeptide may be linked to the Fc directly 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 a multimerization domain, such as an IgG Fc.

The growth factor trap constructs herein are described, for example, in Sarup et al. (2008) Mol. Cancer Ther.7(10):3223-3236; Gompels et al. (2011) Arthritis Research & Therapy 13:R161; al. (2009) Mol. Med. 15(1-2):11-20; and expressed and constructed as described in US Patent Publication No. 2010/0055093. The following sections describe each portion of the multispecific growth factor trap constructs provided herein.

a. Extracellular domain (ECD) polypeptides As used herein, multispecific, such as bispecific, growth factors comprising the extracellular domain (ECD) of two or more cell surface receptors (CSR) or portions thereof A trap construct is provided. In certain embodiments, the construct is a bispecific heterodimeric construct comprising two different cell surface receptors. The construct includes a first ECD polypeptide and a second ECD polypeptide, each linked directly or indirectly to a multimerization domain via a linker. 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 HER2 ECD (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, in particular 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 comprises at least a sufficient portion of subdomains I, II and III for ligand binding and receptor dimerization. For example, the ECD may include sufficient portions of subdomains I and III for ligand binding, and/or the ECD may include a sufficient portion of subdomain II to dimerize with a cell surface receptor. may include a sufficient portion of. In some embodiments, the ECD includes subdomains I, II, and III, and at least module 1 of domain IV.

In some examples, multispecific, such as bispecific, growth factor trap constructs include a first ECD polypeptide comprising all or a portion of the ECD of HER1/EGFR, HER2, HER3 or HER4, particularly EGFR/HER1. peptide, and a second chimeric polypeptide comprising an ECD derived from a different CSR, e.g. HER2, HER3, HER4, insulin growth factor-1 receptor (IGF1-R), vascular endothelial growth factor receptor (VEGFR, e.g. , VEGFR1), fibroblast growth factor receptor (FGFR, e.g. FGFR2 or FGFR4), TNFR, platelet-derived growth factor receptor (PDGFR), hepatocyte growth factor receptor (HGFR), immunoglobulin-like and EGF-like domain 1 a tyrosine kinase (TIE, eg, TIE-1 or TEK (TIE-2)), a receptor for advanced glycation end products (RAGE), an Eph receptor, or a T cell receptor.

In certain embodiments, the first ECD polypeptide is the full-length ECD of HER1/EGFR (corresponding to residues 1-621 of SEQ ID NO: 41), or a portion thereof (e.g., residues 1-621 of SEQ ID NO: 41). ~501, which corresponds to subdomains I-III of domain IV and module 1 of domain IV), and the second ECD polypeptide comprises the full-length ECD of HER3 (residue 1 of SEQ ID NO:45). 621), or a portion thereof (e.g., residues 1-500 of SEQ ID NO: 45, corresponding to subdomains I-III and module 1 of domain IV), where the ECD portion comprising a sufficient portion of the ECD to dimerize with a cell surface receptor, including at least a sufficient portion of domains I and III to bind a ligand of the HER receptor, and a sufficient portion of subdomain II. . The first and second ECD polypeptides form a multimer, eg, a dimer, through interaction of their multimerization domains. The resulting multimeric construct provided herein binds additional ligands compared to the first or second chimeric polypeptide alone or a homodimer thereof, and/or The chimeric polypeptide dimerizes with more cell surface receptors than the chimeric polypeptide alone or its homodimer. For example, the first and second ECD polypeptides form a heterodimer that binds HER1 and HER3 ligands.

b. Modifications to the Extracellular Domain In some embodiments, at least one of the ECD domains, or a portion thereof, alters ligand binding, specificity, or other activity or property as compared to an unmodified ECD polypeptide. including modifications. In such multimeric constructs, the second ECD portion may be the same ECD domain, wild type or mutant form, or may be an ECD from any other cell surface receptor. Each monomeric ECD or portion thereof is linked directly or via a linker to a multimerization domain, or directly or via a linker to a second ECD or portion thereof. For example, the modification alters the ligand binding, specificity, or another activity or property of an ECD or full-length receptor containing such an ECD as compared to an unmodified ECD or full-length receptor, thereby rendering the heterogeneous Multimers exhibit altered activities or properties, such as altered ligand binding or specificity. Such modifications include any modification that removes or adds or enhances activity, such as binding to additional ligands. An example of such a multimeric construct is a construct that includes at least one HER1 ECD that includes mutations in subdomain III that increase its affinity for ligands other than EGF. Such increase in affinity is at least 2-fold to 10-fold, typically 100-fold, 1000-fold, 104-fold, 105-fold, 106-fold or more.

In certain embodiments, the growth factor trap construct is a heterodimer comprising a HER1 (EGFR) chimeric fusion polypeptide and a HER3 chimeric fusion polypeptide, where each chimeric fusion polypeptide optionally carries a peptide linker. It contains the ECD of the receptor linked to the Fc of human IgG1 via the Fc of human IgG1. 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 a multimerization domain, such as an IgG1 Fc.

In some examples, the HER1 portion has enhanced ligand binding and/or biological activity. In other examples, the HER3 portion has enhanced ligand binding and/or biological activity. In yet another example, both the HER1 and HER3 portions are enhanced for ligand binding and/or biological activity.

Exemplary modifications include, for example, S418F of HER1 (see the sequence of the mature protein shown in SEQ ID NO: 41), which allows the HER3 ligand NRG2-β to stimulate HER1. The resulting ECD binds or interacts with at least two ligands, one for HER1 such as EGF and one for HER3 such as NRG2-β. Other modifications include, for example, mutations T15S and G564S in the EGFR/HER1 ECD subdomains I and IV, respectively (with respect to the sequence of the mature EGFR protein (SEQ ID NO: 41)), and Y246A in the HER3 ECD subdomain II (with respect to the mature HER3 (with respect to the protein sequence (SEQ ID NO: 45)), which when combined provide an average 22-fold improvement in affinity for various ligands including EGF, TGF-α, HB-EGF and NRG1-β. Additional mutations in the HER1 ECD include E330D/G588S, S193N/E330D/G588S, and T43K/S193N/E330D/G588S, with respect to the sequence of precursor HER1 (including the signal peptide) shown in SEQ ID NO: 40; E306D/G564S, S169N/E306D/G564S, and T19K/S169N/E306D/G564S (referring to the sequence of the mature HER1 polypeptide shown in SEQ ID NO: 41). These mutations increase HER1 binding affinity for the ligands EGF, HB-EGF, and TGF-α (see, eg, US Patent Publication No. 2010/0055093).

c. Multimerization Domain In certain embodiments, the multimerization domain is an Fc domain or variant thereof that effects multimerization. The Fc domain can be derived from any immunoglobulin (Ig) molecule, including from IgG, IgM, or IgE. For example, the Fc domain can be derived from IgG1, IgG2, IgG3 or IgG4 and includes CH2 and CH3 domains, and optionally includes all or part of the hinge region. In particular examples, the Fc portion is human IgG1 Fc, optionally comprising all or part of the hinge region, eg, residues 99-330, 100-330, 104-330, 109-330 of SEQ ID NO:9; 111-330, 113-330, or 114-330. Also included are Fc domains that have been modified as described in the above section to have knob-in-holes and have altered properties.

Each ECD polypeptide in the multispecific growth factor trap construct is linked directly to an Fc or indirectly through a linker, such as a chemical or polypeptide linker, to form a chimeric fusion polypeptide (i.e., an ECD/Fc fusion polypeptide). ) to form. The multimerization domains, such as the Fc domains, of each chimeric fusion polypeptide interact (via disulfide bonds in the case of the Fc domain) to form heteromultimers, such as heterodimers.

The linker between the ECD and Fc portions of each chimeric fusion polypeptide may be a flexible peptide linker, such as the hinge region of IgG, or from small amino acids such as glycine, serine, threonine, and/or alanine. Other polypeptide linkers configured in various lengths and combinations are also possible. For example, the linker can be (Gly)n, (GGGGS)n, (SSSSG)n, or (AlaAlaProAla)n, where n is 1-6, or GKSSGSGSESKS, GGSTSGSGKSSEGKG, GSTSGSGKSSSEGSGSTKG, GSTSGSGKPGSGEGSTKG, EGKSSGSGSESKEF, Gly-Arg-Met-Asp (GRMD), Ser-Cys-Asp-Lys-Thr (SCDKT), or Glu-Lys-Thr-Ile-Ser (EKTIS) (SEQ ID NOS: 816-834) of reference or any other linker described herein or known in the art that is suitable for such purposes.

d. Modifications to the Fc Domain The Fc domain in the growth factor trap constructs provided herein can be modified to increase, for example, increase in vivo half-life and/or alter immune effector function and predominate, as described below. to improve or enhance protein expression and purification, and to improve pharmacokinetic and pharmacological properties, including by resulting in the production of heterodimers as primary or sole products; will be modified.

i. Knob-in-Hole Introduction The Fc domain in the growth factor trap constructs provided herein is such that steric interactions promote stable interactions and homodimerization from mixtures of chimeric ECD polypeptide monomers. can be engineered to promote the formation of heterodimers rather than dimers. As discussed elsewhere herein, "knob-in-hole"(KiH; also known as "knob-into-hole") into the CH3 domain of an antibody (e.g., IgG) heavy chain. Introduction optimizes heterodimer production. In the knob-in-hole approach, the interfacial residues of the CH3 domains of two Fc monomers are complementary and asymmetrically mutated. Generally, a "knob" or protrusion is created by replacing an amino acid with a small side chain with an amino acid with a large side chain, such as tyrosine or tryptophan, at the interface between CH3 domains, making it identical or similar in size to the knob. Compensatory "holes" or cavities are created by replacing amino acids with large side chains and amino acids with small side chains such as alanine and threonine. Knob and hole variants of Fc monomers heterodimerize by insertion of the knob into the correspondingly designed hole of the partner CH3 domain. Steric repulsion prevents knob-knob binding and destabilizes the hole-hole homodimer.

In some embodiments, the Fc portion of the heterodimeric growth factor trap constructs provided herein is engineered to include a knob-in-hole. The knob mutation corresponds to, for example, S354C, T366Y, T366W, or It may be T394W (according to EU numbering). The hole mutations can be Y349C, T366S, L368A, F405A, Y407T, Y407A, or Y407V according to EU numbering, which are respectively Y232C, T249S, L251A, F288A, Y290T, Y290A, or Y290V (SEQ ID NO: 9 (see the sequence of the human IgG1 heavy chain constant domain shown in ). The introduction of the knob-in-hole increases the yield of the desired heterodimer and reduces the amount of homodimer impurities, e.g., when compared to RB200 and RB242, the dual Specificity of heterodimeric growth factor trap constructs facilitates the protein purification process.

ii. Modifications that enhance neonatal Fc receptor (FcRn) recycling As described elsewhere herein, fusion with IgG Fc can be Increasing the molecular weight of the therapeutic agent also increases the half-life of the small protein therapeutic agent, resulting in slower removal of the therapeutic agent from the body, for example, by the kidneys. To improve pharmacokinetics and overall pharmacology, residues within the Fc region of the growth factor trap constructs provided herein are mutated to generally increase affinity for FcRn by 30-fold or more; In vivo half-life may be further increased.

In some embodiments, the Fc portion of the growth factor trap constructs herein are modified to enhance neonatal FcRn recycling and increase in vivo half-life. This may be done by mutating residues at the interface of the CH2 and CH3 domains of the IgG Fc that are involved in binding to FcRn. Exemplary Fc modifications that increase binding to FcRn and may be introduced into the Fc portion of the growth factor trap constructs herein include, but are not limited to, 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/T 256E, H433K/N434F/Y436H, N434F/ One or more of Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y , as well as combinations of these ( (according to EU numbering). With reference to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO: 9, the corresponding mutations according to Kabat numbering and sequential numbering are shown in Table 7 (IgG1 Fc modifications to enhance FcRn binding) in the section describing Fc modifications. is shown. Other modifications known in the art to confer enhanced or increased FcRn binding are also contemplated for use herein.

Modifications of the Fc portion of the growth factor trap constructs provided herein to enhance FcRn binding and recycling extend the in vivo half-life of therapeutic agents, necessitating the administration of lower doses. and/or less frequent administration, resulting in improved therapeutic efficacy compared to RB200 and RB242.

iii. Effector Functions As described herein, immune effector functions mediated by IgG Fc include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis), and antibody-dependent cell-mediated phagocytosis (ADCP; also called antibody-dependent cell phagocytosis). The Fc region of the growth factor trap constructs herein eliminates, reduces, or eliminates immune effector functions, including, for example, any one or more of CDC, ADCC, and ADCP, as discussed below and elsewhere herein. or may be mutated or modified to enhance it.

Because the growth factors targeted by the growth factor trap constructs exist as membrane proteins and free (i.e., soluble) ligands, in certain embodiments, the immune effector functions of the Fc portion of the ECD/Fc fusion polypeptide, particularly ADCC is retained. In another embodiment, in addition to human IgG1 Fc, other Fc regions may also be included in the ECD/Fc chimeric fusion polypeptides provided herein. For example, when effector function mediated by Fc/FcγR interactions is minimized, complement or effector cell recruitment is insufficient and IgG isotypes that do not exhibit effector function, e.g., IgG2 or IgG4, are combined with Fc. Fusion is considered. This approach may be used when effector function is not required or would be detrimental, such as in the context of autoimmune and inflammatory diseases and disorders.

In certain instances, the Fc portion may be modified to enhance or increase immune effector function. This can be achieved, for example, by modifications that increase binding to C1q (for CDC) and/or specific activating FcγRs (eg, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb). Fc regions modified to have increased binding to Fc receptors may be more effective in promoting the destruction of cancer cells in a patient, even when linked to an ECD polypeptide. Antibodies may be used for antiproliferative purposes, such as antiproliferation by blocking growth pathways, intracellular signaling leading to apoptosis, enhancing receptor downregulation and/or turnover, promoting ADCC, ADCP, CDC, and adaptive immune responses. Destroys tumor cells through many mechanisms. Thus, in embodiments where the growth factor trap constructs herein are used to treat cancer, the Fc portion of the construct may be modified to enhance or increase immune effector function. Section F. 4. d. i. Table 8 (IgG1 Fc modifications that enhance immune effector function) (in c) (enhancing or reducing/eliminating Fc immune effector function) increase binding to FcγR or C1q and thus improve immunity, including ADCC, ADCP and CDC. A collection of Fc modifications that enhance effector function and provide corresponding modifications according to Kabat numbering and sequential numbering with reference to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO:9. Any one or more of these modifications, alone or in various combinations, may be introduced into the IgG1 Fc portion of the growth factor trap constructs provided herein. Other modifications known in the art to confer enhanced or enhanced immune effector function are also contemplated for use herein. These lists in the sections above describe IgG1 Fc modifications that enhance immune effector function.

In another embodiment, the Fc portion of the growth factor trap constructs provided herein is modified to reduce or eliminate immune effector function. This can be achieved, for example, by modifications that reduce or abolish binding to C1q (in the case of CDC) and/or specific activating FcγRs (eg, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb). This may be used, for example, when it is desirable to antagonize, but not kill, cells bearing the target antigen, or when undesirable or deleterious immune effector functions, e.g. release of undesired pro-inflammatory cytokines and off-target cytotoxicity, are desired. desirable when reduction of Accordingly, in embodiments where the growth factor trap constructs provided herein are used to treat chronic inflammatory and autoimmune diseases and disorders, such as RA, the Fc portion of the construct is modified to Functionality may be reduced or eliminated.

Section F. 4. d. i. c (IgG1 Fc modifications that reduce or eliminate immune effector function) Table 9 (Enhance or reduce/eliminate Fc immune effector function) reduce or eliminate binding to activated FcγR and/or C1q, thereby reducing ADCC , ADCP, and CDC, and summarizes exemplary IgG1 Fc modifications that can be introduced into the Fc region of the growth factor trap constructs herein. This table refers to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO: 9 and provides the corresponding modifications by Kabat numbering and sequential numbering. Any one or more of these modifications, alone or in various combinations, may be introduced into the IgG1 Fc portion of the growth factor trap constructs provided herein. Other modifications known in the art to reduce or eliminate immune effector function are also contemplated for use herein.

The Fc portion of the growth factor trap constructs provided herein can also be modified to increase binding to inhibitory FcγRs resulting in suppression of the immune response. Therapeutic antibodies containing immunosuppressive Fc modifications are advantageous in the treatment of inflammatory diseases. These mutations affect the Fc portion of the growth factor trap constructs herein for the treatment of diseases and conditions with an inflammatory component or pathogenesis or involvement, such as RA, and other inflammatory and autoimmune diseases. may be incorporated into.

Modifications that increase binding or confer selective binding to inhibitory FcγRIIb and/or FcγRI (but not FcγRIIIa) are engineered into the IgG1 Fc region in the growth factor trap constructs provided herein. obtain. These modifications include, but are not limited to, S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F, L351S/T366R/L368H/P395K, and combinations thereof (according to EU numbering). There are more than one. Section F. 4. d. i. Table 11 (IgG1 Fc modifications that increase binding to inhibitory FcγRIIb) in i refers to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO: 9, with corresponding substitutions (by Kabat numbering and by sequential numbering). ). These modifications are summarized in the section above describing IgG1 Fc modifications that increase binding to inhibitory FcγRIIb.

4. Compositions, Therapeutic Uses and Methods of Treatment Nucleic acid molecules encoding chimeric fusion polypeptides (ie, ECD/Fc) and growth factor trap constructs, vectors comprising the nucleic acid molecules are provided. Also provided are cells comprising the vectors described herein, and pharmaceutical compositions comprising any of the growth factor trap constructs encoding the nucleic acid molecules, vectors or cells described herein. The growth factor trap constructs herein are, for example, Sarup et al. (2008) Mol. Cancer Ther. 7(10):3223-3236; Gompels et al. (2011) Arthritis Research & Therapy 13:R161; Jin et al. al. (2009) Mol. Med. 15(1-2):11-20; and as previously described in US Patent Publication No. 2010/0055093.

Multispecific (including bispecific) growth factor trap constructs herein are defined by directly or indirectly linking two or more, particularly two, same or different ECD polypeptides to a multimerization domain. Two or more chimeric proteins, especially two chimeric proteins, are produced. In some instances, 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 protein is transformed with a recombinant expression vector into a host cell, particularly a mammalian cell (e.g., HEK293T or CHO cells, or as described herein or known in the art). (any other suitable mammalian cell) and assemble into multimers, such as dimers, where the multimerization domains interact to form a multivalent polypeptide. The resulting chimeric polypeptide, and multimers formed therefrom, may be purified by any suitable method known in the art, such as, for example, affinity chromatography on a Protein A or Protein G column. Additionally or alternatively, for example, gel electrophoresis, dialysis, ion exchange chromatography, ethanol precipitation, HPLC such as reversed-phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatofocusing, SDS-PAGE, Other techniques for protein purification may be used, including and ammonium sulfate precipitation. When two nucleic acid molecules encoding different ECD chimeric polypeptides are transformed into a cell (eg, HER1/Fc and HER3/Fc), homodimer and heterodimer formation occurs. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation. For example, the ratio of nucleic acid molecules encoding different ECD chimeric polypeptides can be adjusted such that an excess of one nucleic acid molecule reduces the formation of homodimers. Furthermore, as mentioned above, the introduction of knobs-in-holes into the Fc monomer favors the formation of heterodimers over homodimers.

ECD chimeric polypeptides containing the Fc region may also contain tags with metal chelates or other epitopes, such as 6xHis tag, c-myc tag, FLAG tag, maltose binding protein (MBP), glutathione-S-transferase (GST). , or may be engineered to include thioredoxin (TRX) and the like. Tagged domains can be used for rapid purification by metal chelate chromatography and/or antibodies, allowing detection in Western blots, immunoprecipitation, or activity depletion/blocking in bioassays.

a. Pharmaceutical Compositions Provided herein are pharmaceutical compositions comprising a multispecific, such as bispecific, growth factor trap construct provided herein or encoding a nucleic acid molecule. . Also provided are pharmaceutical compositions comprising isolated cells comprising the nucleic acid molecules or vectors provided herein. Such compositions include a therapeutically effective amount of a growth factor trap construct. Pharmaceutical compositions are formulated in any conventional manner by mixing a selected amount of a growth factor trap construct or nucleic acid molecule with one or more physiologically acceptable carriers or excipients. obtain. Pharmaceutical compositions may be used for therapeutic, prophylactic, and/or diagnostic uses. The concentration of active compound in the composition depends on absorption, inactivation, and excretion rates of the active compound, dosing schedule, and amount administered, as well as other factors known to those skilled in the art.

Pharmaceutical carriers or vehicles suitable for administering the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. The choice of carrier or excipient is within the skill of the administering professional and may depend on a number of parameters. These include, for example, the mode of administration (ie, systemic, oral, nasal, pulmonary, topical, topical, or any other mode) and the disorder being treated. Pharmaceutical compositions containing multispecific, such as bispecific, growth factor trap constructs, or therapeutically effective amounts of the nucleic acid molecules described herein, can also be prepared as a lyophilized powder, reconstituted with sterile water, etc. It may also be provided immediately prior to administration.

The pharmaceutical compositions provided herein may be in various forms, such as solid, semi-solid, liquid, powder, aqueous, or lyophilized. The pharmaceutical compositions provided herein may be formulated for single-dose (direct) administration or for dilution or other modification. The concentration of the compound in the formulation, upon administration, is effective to deliver the intended therapeutically effective amount. Typically, compositions are formulated for single administration. The compound may be suspended in micronized or other suitable form, or may be derivatized to produce a more soluble active product. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. Effective concentrations are sufficient to ameliorate the targeted condition and can be determined empirically. To formulate a composition, weight fractions of the compounds are dissolved, suspended, dispersed, or otherwise mixed in the selected vehicle at an effective concentration such that the target condition is alleviated or ameliorated. Ru.

Methods of producing nucleic acids encoding growth factor trap constructs provided herein include those 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 methods of administration described in Section I can be adapted to manufacture compositions and formulations containing the growth factor trap constructs and encoding nucleic acid molecules described herein. Dosages and methods of administration may be determined by the administering expert and are known in the art and described elsewhere herein.

b. Therapeutic Uses and Methods of Treatment The multispecific, including bispecific, growth factor trap constructs provided herein can be used for any purpose known to those skilled in the art for the 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 purposes. In particular, the multispecific growth factor trap constructs provided herein are of use in the treatment of various diseases and conditions involving the CSR, including RTKs, and in particular the HER family of proteins, including those described herein. be able to. HER signaling is involved in the pathogenesis of a variety of diseases and disorders, which are contemplated for treatment with the growth factor trap constructs provided herein.

The growth factor trap constructs and encoding nucleic acid molecules and pharmaceutical compositions provided herein are useful for anti-HER therapies (e.g., trastuzumab, cetuximab, gefitinib, erlotinib, and lapatinib, as well as the cancer and other proliferative diseases and disorders, angiogenesis-related diseases and disorders, rheumatoid arthritis and other chronic inflammatory and autoimmune diseases. It can be used to treat any condition, including, but not limited to, diseases and disorders of the central nervous system (CNS), and neurodegenerative diseases and disorders of the central nervous system (CNS). For example, treatments using the growth factor trap constructs provided herein include angiogenesis-related diseases and conditions, inflammatory diseases and conditions, autoimmune diseases and conditions, neurodegenerative diseases, and cell proliferation-related diseases. including, but not limited to, treatment of conditions such as: Such diseases and conditions include, for example, eye disease, atherosclerosis, vascular damage, Alzheimer's disease, cancer, smooth muscle cell-related conditions, rheumatoid arthritis (RA), and various autoimmune diseases. .

Dosage levels and regimens can be determined based on known doses and regimens and, if necessary, extrapolated based on changes in the properties of the polypeptides and constructs provided herein. , and/or can be determined empirically based on various factors. Such factors include, for example, the individual's body weight, general health, age, sex, and diet, as well as the activity of the particular compound used, time of administration, rate of excretion, drug combinations, severity of the disease, and These include the course of the disease, as well as the patient's predisposition to the disease, as well as the judgment of the attending physician. The active ingredient is typically combined with a pharmaceutically effective carrier. The amount of active ingredient that may be combined with the carrier materials to produce a single or multiple dosage form may vary depending on the host treated and the particular mode of administration.

The dosage depends on the particular disorder, disease or condition being treated, as well as the particular subject. Typical doses are similar to those of known anti-HER therapies, such as antibodies such as trastuzumab, cetuximab, pertuzumab, and panitumumab, and small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib, and lapatinib. Exemplary doses for subjects, including humans and other animals, range from about 0.1 to 100 mg/kg, such as from 1 mg/kg to about 30 mg/kg, such as from 5 mg/kg to 25 mg/kg. Doses can be determined based on the assumption that the average human mass is approximately 75 kg. Doses can be adjusted for children, infants, and smaller adults.

Once the patient's condition improves, maintenance doses of the compound or composition can be administered as needed; the dosage, dosage form, or frequency of administration, or combinations thereof, can be modified. In some cases, the subject may require intermittent treatment on a long-term basis if symptoms of the disease recur or based on scheduled dosage.

Treatment of diseases and conditions with the multispecific growth factor trap constructs provided herein can be carried out by, by way of example and without limitation, by any suitable route of administration using suitable formulations as described herein. , by injection, subcutaneous injection, and inhalation, or by intramuscular, intradermal, oral, topical, and transdermal administration.

Provided herein are methods of treating a HER-mediated or HER-related disease or condition, wherein a subject with the disease is tested to identify which HER receptors are expressed or overexpressed. and, based on the results, selecting a multispecific growth factor trap construct that targets at least one, typically two, of the HER receptors. In one embodiment, the disease is cancer. Examples of cancers for treatment herein include glioma, as well as pancreatic cancer, gastric cancer, head and neck cancer, cervical cancer, lung cancer, colorectal cancer, endometrial cancer, prostate cancer, esophageal cancer, ovarian cancer. cancer, uterine cancer, bladder cancer or breast cancer. Cancers treatable with the growth factor (HER ligand) trap constructs herein are generally those that express at least one HER receptor, typically two or more HER receptors. Such cancers can be identified by any means known in the art for detecting HER expression. For example, HER2 expression can be assessed using commercially available diagnostic/prognostic assays such as HercepTest™ (Dako). Paraffin-embedded tissue sections from tumor biopsies are subjected to immunohistochemistry (IHC) assays and HER2 protein staining intensity criteria are applied. Tumors below the threshold score are characterized as not overexpressing HER2, while tumors above the threshold score are characterized as overexpressing HER2. In one example of treatment, HER2 overexpressing tumors are evaluated as candidates for treatment with multispecific growth factor trap constructs such as any provided herein.

In another embodiment, the HER-mediated or HER-related disease or condition is an inflammatory or autoimmune disease, particularly rheumatoid arthritis. Animal models of arthritis, such as the collagen-induced arthritis (CIA) mouse model, can be used to test the growth factor trap constructs provided herein. A reduction in symptoms of arthritis, including paw swelling, erythema and ankylosis, can be observed in mice treated with the growth factor trap constructs herein, such as by local injection of the protein. A reduction in synovial neovascularization and synovial inflammation can also be observed.

Multispecific growth factor trap constructs, including bispecific encoding nucleic acid molecules and pharmaceutical compositions provided herein, can be used to describe how HER receptors and/or ligands play a role in some of their pathogenesis, pathology, or pathogenesis. Any disease, condition or disorder implicated in the embodiments can be used to treat HER (ErbB) related diseases or HER receptor mediated diseases. HER-related diseases for treatment include cancers such as glioma or pancreatic cancer, gastric cancer, head and neck cancer, cervical cancer, lung cancer, colorectal cancer, endometrial cancer, prostate cancer, esophageal cancer, ovarian cancer , uterine cancer, bladder cancer, kidney cancer, or breast cancer. Other diseases that can be treated include, for example, non-cancerous proliferative diseases such as smooth muscle cell proliferation and/or migration, inflammatory or autoimmune diseases, skin diseases, and eye injuries. Diseases and conditions for treatment include, for example, rheumatoid arthritis, diabetic retinopathy, anterior segment disease, psoriasis, restenosis, stenosis, atherosclerosis, hypertension due to thickening of blood vessels, bladder, heart or other muscle thickening, bladder disease, endometriosis, obstructive airway disease, and one or more neuregulin (NRG) ligands, such as NRG1 (including types I, II, and III), NRG2, NRG3, and/or diseases or conditions associated with (eg, caused or exacerbated by) exposure to NRG4, or other HER family ligands. Examples of NRG-related diseases, as well as diseases associated with other HER family ligands, include neurological or neuromuscular diseases, including schizophrenia, Parkinson's and Alzheimer's diseases, cardiomyopathy, pre-eclampsia, neurological diseases, and heart failure. is included.

Examples of cancers that can be treated include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies, such as squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, Bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, renal cell cancer, esophageal cancer, glioma, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer , and head and neck cancer.

The multispecific growth factor trap constructs provided herein, when administered, target a single targeting anti-HER antibody, such as trastuzumab, cetuximab, and other antibodies described herein or known in the art. therapy and small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and lapatinib can generally result in increased therapeutic efficacy and decreased drug resistance. As described herein, resistance to single-targeted anti-HER therapy is associated with co-expression and/or upregulation of other HER family members and overexpression of their ligands. The HER ligand-binding constructs provided herein behave as receptor decoys, sequester multiple HER family ligands, prevent ligand-dependent receptor activation, and downregulate aberrant HER family activity; Simultaneously inhibits multiple ligand-induced HER family members. This increases therapeutic efficacy and reduces the likelihood of drug resistance developing.

5. Combination Therapy Combination therapy can be used. Combination therapy involves combining the multispecific (including bispecific) growth factor trap constructs, nucleic acid molecules, and pharmaceutical compositions provided herein with another agent or treatment, including radiation and surgery. This includes administering. Additional agents or therapeutic agents can be administered simultaneously with, before, after, or intermittently with the treatments provided herein. They may be separate compositions or combinations.

Multispecific, such as bispecific, heteromultimeric growth factor trap constructs, nucleic acid molecules, and pharmaceutical compositions provided herein include, but are not limited to, TNF antagonists/blockers, chemotherapeutic agents, single Targeted anti-HER therapies (including antibodies and tyrosine kinase inhibitors), anti-angiogenic agents, antibodies, cytotoxic agents, anti-inflammatory agents, cytokines, growth inhibitors, anti-hormones, cardioprotective agents, steroids, immunostimulants one or more other therapeutic agents, including immunosuppressants, biological or non-biological disease-modifying antirheumatic drugs (DMARDs), agents for treating infectious diseases (including antibodies), or other therapeutic agents. It can be administered before, after, intermittently, and simultaneously with a therapeutic regimen or drug. In particular, growth factor trap constructs are administered in conjunction with the TNFR1/TNFR2 axis constructs provided herein. They can also be administered with other anti-TNF therapies, including those described in the sections above or known to those skilled in the art.

The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific constructs, nucleic acids, and other constructs provided herein can be administered in combination with other anti-TNF therapies. Examples of anti-TNF therapies that can be used in the combination therapy herein include, for example, conventional synthetic DMARDs such as methotrexate (MTX), hydroxychloroquine (HCQ; Plaquenil®), sulfasalazine (Azulfidine), etc. ), and leflunomide (Arava®); biological DMARDs such as abatacept (Orencia®), anakinra (Kineret®), rituximab (Rituxan®, Truxima ® , MabThera ® ), tocilizumab (atlizumab, Actemra ® , RoActemra ® ), corticosteroids (e.g., dexamethasone, methylprednisolone, prednisolone, prednisone, or triamcinolone), tofacitinib ( Xeljanz®), as well as TNF inhibitors/anti-TNF agents such as certolizumab pegol (Cimzia®), infliximab (Remicade®), adalimumab (Humira®), golimumab (Simponi®), and etanercept (Enbrel®). Combination therapy can also include, for example, immunotherapeutic agents such as cyclosporine, methotrexate, adriamycin or cisplatin, and immunotoxins.

In certain examples, the growth factor trap constructs provided herein can be used to treat any of the chronic inflammatory, autoimmune, and/or neurodegenerative/demyelinating diseases and conditions described herein; administered with any of the TNFR1 antagonist constructs, TNFR2 agonist constructs, or multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs provided herein, particularly for the treatment of rheumatoid arthritis (RA). .

In some examples, growth factor trap constructs provided herein are administered with one or more anti-angiogenic agents. For example, anti-angiogenic factors can be small molecules or proteins (eg, antibodies, Fc fusions, or cytokines) that bind to growth factors or growth factor receptors involved in promoting angiogenesis. Examples of anti-angiogenic agents include antibodies that bind vascular endothelial growth factor (VEGF) or antibodies that bind VEGF-R, RNA-based therapeutics that reduce VEGF or VEGF-R expression levels, VEGF-toxin fusions. , Regeneron's VEGF-Trap, Angiostatin (plasminogen fragment), Antithrombin III, Angiozyme, ABT-627, Bay12-9566, Benefin, Bevacizumab, Bisphosphonates, BMS-275291, Cartilage-derived inhibitor (CDI), CAI , CD59 complement fragment, CEP-7055, Col3, combretastatin A-4, endostatin (collagen XVIII fragment), farnesyltransferase inhibitor, fibronectin fragment, GRO-beta, halofuginone, heparinase, heparin hexasaccharide fragment, HMV833, Human chorionic gonadotropin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon-inducible protein 10 (IP-10), interleukin 12, kringle 5 (plasminogen fragment), marimastat, metalloproteinase inhibitors (e.g. TIMPs), 2-methoxyestradiol, MMI270 (CGS27023A), plasminogen activator inhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16kDa fragment, proliferin-related protein ( PRP), PTK787/ZK222594, Retinoid, Solimastat, Squalamine, SS3304, SU5416, SU6668, SU11248, Tetrahydrocortisol-S, Tetrathiomolybdate, Thalidomide, Thrombospondin-1 (TSP-1), TNP470, Transforming Growth Factor These include, but are not limited to, beta (TGF-β), vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.

In some examples, a growth factor trap construct provided herein is administered in conjunction with one or more tyrosine kinase inhibitors and, optionally, a TNFR1/TNFR2 axis construct provided herein. Examples of tyrosine kinase inhibitors include quinazolines such as PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP59326, CGP60261 and CGP62706; )-7H-pyrrolo(2,3-d)pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostin containing a nitrothiophene moiety; PD-0183805 (Warner- Lambert); antisense molecules (e.g., those that bind to a nucleic acid encoding ErbB); quinoxaline (see, e.g., U.S. Pat. No. 5,804,396); tyrphostin (e.g., U.S. Pat. No. 5,804,396); Pan-ErbB inhibitors such as ZD6474 (Astra Zeneca); PTK-787 ((Novartis/Schering AG); C1-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lill); y); Imatinib mesylate (STI571, Gleevec®; Novartis); PKI166 (Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD 6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1 C11 (ImClone); gefitinib (Iressa®, ZD1839, AstraZeneca); and OSI-774 (OSI Pharmaceuticals/Genentech, sold under the trademark Tarceva®), or The following patent publications: U.S. Patent No. 5,804,396, and International Application Publication Nos. 33979, and WO96/33980, but are not limited thereto.

Other compounds useful in combination therapy include steroids such as antiangiogenic 4,9(11)-steroids and C21-oxygenating steroids, angiostatin, endostatin, vasculostatin, canstatin and maspin, angiopoietin, Bacterial polysaccharide CM101 and antibody LM609 (e.g., U.S. Patent No. 5,753,230), thrombospondin (TSP-1), platelet factor 4 (PF4), interferon, metalloproteinase inhibitor, AGM-1470/TNP -470, thalidomide, and drugs containing carboxamide triazole (CAI), cortisone, e.g. in the presence of heparin or heparin fragments, anti-invasive factors, retinoic acid and paclitaxel, shark cartilage extract, anionic polyamides or polyureas. Examples include oligomers, oxindole derivatives, estradiol derivatives and thiazolopyrimidine derivatives.

Examples of anti-cancer antibodies that can be co-administered with the growth factor trap constructs provided herein include anti-17-IA cell surface antigen antibodies, such as edrecolomab (sold under the trademark Panorex®) anti-4-1BB antibodies; anti-4Dc antibodies; anti-A33 antibodies such as A33 and CDP-833; anti-α1 integrin antibodies such as natalizumab; anti-α4β7 integrin antibodies such as LDP-02; F-200, M-200, and SJ anti-αVβ1 integrin antibodies such as -749; anti-αVβ3 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, and Medi-523 (sold under the trade name Vitaxin); anti-complement factor 5 (C5) such as 5G1.1; Antibodies; anti-CA125 antibodies such as oregovomab (sold under the trademark OvaRex®); anti-CD3 antibodies such as vsilizumab (Nuvion®) and Rexomab; such as IDEC-151, MDX-CD4, and OKT4A anti-CD4 antibodies; anti-CD6 antibodies such as Oncolysin B and Oncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodies such as B43, MT-103, and Oncolysin B; 2H7, 2H7. v16, 2H7. v114, 2H7. anti-CD20 antibodies such as v115, tositumomab (Bexxar®), rituximab (Rituxan®), and ibritumomab tiuxetan (Zevalin®); CD22 antibodies; anti-CD23 antibodies such as IDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax® (daclizumab); anti-CD30 antibodies such as AC10, MDX-060, SGN-30; gemtuzumab ozogamicin Anti-CD33 antibodies such as (Mylotarg®), Oncolysin M, and Smart Ml 95; anti-CD38 antibodies; anti-CD40 antibodies such as SGN-40 and tralizumab; CD40L antibody; anti-CD44 antibody such as bivatuzumab; anti-CD46 antibody; anti-CD52 antibody such as Campath (registered trademark) (alemtuzumab); anti-CD55 antibody such as SC-1; anti-CD56 antibody such as huN901-DM1; MDX-33, etc. anti-CD64 antibodies such as XR-303; anti-CD74 antibodies such as IMMU-110; anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89 antibodies such as MDX-214; anti-CD123 antibodies; B-B4 - anti-CD138 antibodies such as DM1; anti-CD146 antibodies such as AA-98; anti-CEA antibodies such as cT84.66, labetuzumab, and Pentacea®; anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4 antibodies; anti-EGFR antibodies such as ABX-EGF, Erbitux® (cetuximab), panitumumab, IMC-C225, and Merck Mab425; anti-EGFR antibodies such as Crucell's anti-EpCAM, ING-1, and IS-IL-2; EpCAM antibodies; anti-ephrinB2/EphB4 antibodies; anti-HER2 antibodies such as Herceptin® (trastuzumab), pertuzumab, and MDX-210; anti-FAP (fibroblast activation protein) antibodies such as sibrotuzumab; NXT-211, etc. anti-ferritin antibodies; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies; anti-fibrin antibodies; anti-G250 antibodies such as WX-G250 and gilentuximab (Rencarex®) ; anti-GD2 ganglioside antibodies such as EMD-273063 and TriGem; anti-GD3 ganglioside antibodies such as BEC2, KW-2871, and mitumomab; anti-gpIIb/IIIa antibodies such as ReoPro; anti-heparinase antibodies; anti-HLA antibodies such as Oncolym and Smart 1D10 ; anti-HM1.24 antibodies; anti-ICAM antibodies such as ICM3; anti-IgA receptor antibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164; anti-IGF-1R antibodies such as IMC-A12; CNTO-328 and anti-IL-6 antibodies such as ercilimomab; anti-IL-15 antibodies such as HuMax®-IL15 antibody; anti-KDR antibodies; anti-laminin 5 antibodies; anti-Lewis Y antigen antibodies such as Hu3S193 and IGN-311; anti-MCAM antibodies ; anti-Muc1 antibodies such as BravaRex and TriAb; anti-NCAM antibodies such as ERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn and Therex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptk antibodies; anti-PTN antibodies; anti-RANKL antibodies such as AMG-162; anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such as Monopharm C; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA and MDX-220; anti-TGF-β such as CAT-152. Antibodies; anti-TNF-α antibodies such as CDP571, CDP870, D2E7, adalimumab (Humira®), and infliximab (Remicade®); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin 2 antibodies; and anti-VLA-4 antibodies, such as the Antegren® antibody. Anti-idiotypic antibodies can be used including, but not limited to, the GD3 epitope antibody BEC2 and the gp72 epitope antibody 105AD7. Bispecific antibodies can also be used, including but not limited to the anti-CD3/CD20 antibody Bi20.

Examples of antibodies that can be co-administered with the growth factor trap constructs provided herein that can treat autoimmune or inflammatory diseases, graft rejection, and/or GVHD include LDP anti-α4β7 integrin antibodies such as -02; anti-beta2 integrin antibodies such as LDP-01; anti-complement (C5) antibodies such as 5G1.1; anti-CD2 antibodies such as BTI-322 and MEDI-507; OKT3, SMART anti- anti-CD3 antibodies such as CD3; anti-CD4 antibodies such as IDEC-151, MDX-CD4, and OKT4A; anti-CD11a antibodies; anti-CD14 antibodies such as IC14; anti-CD18 antibodies; anti-CD23 antibodies such as IDEC152; anti-CD25 such as Zenapax. Antibodies; anti-CD40L antibodies such as 5c8, Antova, and IDEC-131; anti-CD64 antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114, anti-CD147 antibodies such as ABX-CBL, anti-E-selectin such as CDP850 Antibodies, anti-gpIIb/IIIa antibodies such as ReoPro (registered trademark)/Abcixima, anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740, anti-FcγR1 antibodies such as MDX-33, and anti-fcγR1 antibodies such as rhuMab-E25. IgE antibodies, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodies such as SB-240563 and SCH55700, anti-IL-8 antibodies such as ABX-IL8, anti-interferon gamma antibodies; CDP571, CDP870, D2E7, These include, but are not limited to, anti-TNFα antibodies such as adalimumab, infliximab, and MAK-195F, and anti-VLA-4 antibodies such as Antegren. Examples of other Fc-containing molecules that can be co-administered to treat autoimmune or inflammatory diseases, graft rejection, and GVHD include the TNFRII receptor/Fc fusion Enbrel® ( etanercept) and Regeneron's IL-1 Trap.

Examples of antibodies that can be co-administered to treat infections include anti-anthrax antibodies such as ABthrax; anti-CMV antibodies such as CytoGam and sebilumab; anti-Cryptosporidium antibodies such as CryptoGAM, Sporidin-G; Pyloran, etc. anti-Helicobacter antibodies; anti-hepatitis B antibodies such as HepeX-B, and Nabi-HB; anti-HIV antibodies such as HRG-214; anti-RSV antibodies such as Felbizumab, HNK-20, Palivizumab, and RespiGam; and Aurexis, Aurograb , BSYX-A110, and SE-Mab.

In some instances, the growth factor trap constructs described herein are administered with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; calsterone, dromostanolone propionate, epithiostanol, Androgens such as mepithiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; aclacinomycin, actinomycin, anthramycin, azaserine , bleomycin, cactinomycin, calicheamicin, carbicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin Antibiotics such as , marcellomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, pepromycin, porphyromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin antiestrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifen, keoxifene, LY117018, onapristone, and (Fareston); methotrexate and 5-fluorouracil (5-FU ); antimetabolites such as denopterin, methotrexate, pteropterin, and trimetrexate; aziridines such as benzodepa, carbocone, metredepa, and uredepa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethio Ethyleneimine and methylmelamine, including phosphoramide and trimethylolmelamine; folic acid supplements such as folic acid; chlorambucil, chlornafadine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan , novembichin, fenesterine, prednimustine, trophosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine, 6-mercaptopurine, thiamipurine, and thioguanine; ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine, and pyrimidine analogs such as 5-FU; paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.C.); J. ) and taxanes such as docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); topoisomerase inhibitors such as RFS2000; thymidylate synthase inhibitors such as Tomudex; additional chemotherapeutic agents including aceglatone; Famidoglycoside; aminolevulinic acid; amsacrine; bestrabcil; bisanthrene; edatrexate; defosfamide; demecolcin; diazicon; difluoromethylornithine (DMFO); eflornithine; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; Santron; mopidamole; nitraculine; pentostatin; phenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2" trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobroman; gacytocin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; and topoisomerase inhibitors, such as, but not limited to. Pharmaceutically acceptable salts, acids or derivatives of any of the above may also be used.

Chemotherapeutic agents can be administered as prodrugs. Examples of prodrugs that can be administered with the growth factor trap constructs described herein include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D- Amino acid modified prodrugs, glycosylated prodrugs, beta-lactam containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, and 5-fluorocytosine and other 5-fluorouridine prodrugs. drugs, which can be converted into more active cytotoxic free drugs.

In some instances, the multispecific growth factor trap constructs described herein are administered with one or more immunomodulatory agents. Such agents may increase or decrease the production of one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or increase or decrease the production of one or more types of immune cells. Proliferation, differentiation, migration, or activation can be promoted. Examples of immunomodulators include non-steroidal anti-inflammatory drugs such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketorolac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, naproxen, ketoprofen, and nabumetone. (NSAIDs); steroids such as glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone and triamcinolone; eicosanoids such as prostaglandins, thromboxanes, and leukotrienes; such as anthralin, calcipotriene, clobetasol, and tazarotene topical steroids; cytokines such as TGFβ, IFNα, IFNβ, IFNγ, IL-2, IL-4, IL-10; or etanercept (Enbrel®), adalimumab (Humira®), infliximab (Remicade BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152, complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ, IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9, IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrin, Cytokines, chemokines or receptors, including antibodies, soluble receptors and receptor-Fc fusions against LFA-1, LFA-3, MHC, selectins, TGFβ, TNFα, TNFβ, TNFR1, TNFR2, and T-cell receptors. Antagonists; xenoantilymphocyte globulins; and other immunomodulatory molecules, such as 2-amino-6-aryl-5-substituted pyrimidines, anti-idiotypic antibodies against MHC-binding peptides and MHC fragments, azathioprine, brequinar, bromocriptine, cyclophosphamide D, cyclosporine A, D-penicillamine, deoxyspergualine, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamide (e.g., leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and sulfasalazine , but are not limited to these.

In some instances, the multispecific growth factor trap constructs described herein are administered with one or more cytokines. Examples of cytokines include, but are not limited to, lymphokines, monokines, and traditional polypeptide hormones. Cytokines include interferons such as interferon-alpha, interferon-beta, and interferon-gamma; macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF). Colony stimulating factors (CSF) such as ); IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 , interleukins (IL) such as IL-10, IL-11, IL-12 and IL-15; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptides including LIF and kit ligand (KL). Contains peptide factors.

In some instances, the multispecific growth factor trap constructs described herein are administered with one or more cytokines or other agents that stimulate cells of the immune system and enhance desired effector functions. . For example, agents that stimulate natural killer (NK) cells, including but not limited to IL-2, can be administered with the multispecific growth factor trap constructs described herein. In another embodiment, C5a and a formyl peptide such as N-formyl-methionyl-leucyl-phenylalanine (see, e.g., Beigier-Bompadre et al. (2003) Scand. J. Immunol. 57:221-228) Agents that stimulate macrophages, including but not limited to, can be administered with the multispecific growth factor trap constructs described herein. Agents that stimulate neutrophils, including but not limited to G-CSF and GM-CSF, can also be administered with the multispecific growth factor trap constructs described herein. Agents that promote migration of such immunostimulatory cytokines can be administered with the multispecific growth factor trap constructs described herein. Additional agents can promote one or more effector functions, including but not limited to interferon gamma, IL-3 and IL-7. In some instances, the multispecific growth factor trap constructs described herein are administered with one or more cytokines or other agents that inhibit effector cell function.

In some examples, the multispecific growth factor trap constructs described herein can be used to treat aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycin, butyrosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin). , ribostamycin, sisomicin, spectinomycin), aminocyclitol (e.g., spectinomycin), amphenicol antibiotics (e.g., azidamphenicol, chloramphenicol, florfenicol, and thiamphenicol) ansamycin antibiotics (e.g., rifamide and rifampin), carbapenems (e.g., imipenem, meropenem, and panipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatridine, cefazedone, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefuroxime, cefixime, cephalexin, and cefrazine), cephamycins (e.g., cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan), lincosamides (e.g., clindamycin, and lincomycin), macrolides (e.g. , azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, and tobramycin), monobactams (e.g., aztreonam, carmonam, and tigemonam), mupirocin, oxacephems (e.g., flomoxef, latamoxef, moxalactam), penicillins (e.g., amdinocillin) , amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamecillin, penetamate hydroiodide, penicillin o-benetamine, penicillin O, penicillin V, penicillin V benzoate, penicillin V-hydrabamine, penimepicycline, and pheneticillin potassium), polypeptides (e.g., bacitracin, colistin, polymyxin B, teicoplanin, vancomycin), quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, enrofloxacin) sacin, fleroxacin, flumequin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, and trovafloxacin), rifampin, streptogramins (e.g., quinupristin, and dalfopristin), sulfonamides (e.g., sulfanilamide, and sulfamethoxazole), and tetracyclines (e.g., chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, duramycin, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin).

In some examples, the multispecific growth factor trap constructs provided herein include amphotericin B, ciclopirox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, terbinafine, terconazole , and one or more antifungal agents including, but not limited to, tioconazole.

In some examples, the multispecific growth factor trap constructs described herein contain protease inhibitors, reverse transcriptase inhibitors, and type I interferons, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir, amantadine , including, but not limited to, amprenavir, clevudine, enfuvirtide, entecavir, foscarnet, ganciclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine1 Administered with one or more antiviral agents.

The multispecific growth factor trap constructs provided herein can be combined with other therapeutic regimens. For example, in one embodiment, a patient treated with a multispecific growth factor trap construct provided herein can undergo radiation therapy. Radiation therapy can be administered according to protocols commonly used in the art and known to those skilled in the art. Such therapies include, but are not limited to, cesium, iridium, iodine, or cobalt radiation. Radiation therapy may be whole-body radiation or may be directed locally to specific sites or tissues in or on the body, such as the lungs, bladder, or prostate. Radiation therapy can also include treatment with isotopically labeled molecules such as antibodies. Examples of radioimmunotherapeutics include the trademarks Zevalin® (Y-90 labeled anti-CD20), LymphoCide® (Y-90 labeled anti-CD22), and Bexxar® (I-131 labeled anti-CD20). CD20) is available.

Typically, radiation therapy is administered in pulses over a period of about 1 to 2 weeks. However, radiation therapy can be administered over a longer period of time. For example, radiation therapy can be administered to head and neck cancer patients for about 6 weeks to about 7 weeks. Radiation therapy may be administered as a single dose or as a series of multiple doses. A skilled practitioner can empirically determine an appropriate dose or doses of radiation therapy useful herein. In some instances, multispecific growth factor trap constructs, and optionally one or more other anti-cancer therapies, are used to treat cancer cells ex vivo. It is contemplated that such ex vivo treatment may be useful for bone marrow transplants, particularly autologous bone marrow transplants. For example, prior to transplantation of cells or tissues containing cancer cells into a recipient patient using a multispecific growth factor trap construct as described herein and treatment with one or more anti-cancer therapies. cancer cells can be depleted or substantially depleted.

Additionally, it is contemplated that the multispecific growth factor trap constructs provided herein can be administered to a patient or subject in combination with other therapeutic techniques such as surgery or phototherapy.

For example, provided herein are methods for treating cancer by administering any of the multispecific growth factor trap constructs, nucleic acid molecules, or pharmaceutical compositions provided herein in combination with another anti-cancer agent. This is a method of treating. Anti-cancer agents can include radiation and/or chemotherapeutic agents. For example, the anti-cancer agent can be a tyrosine kinase inhibitor or an antibody. Exemplary anti-cancer agents include quinazoline kinase inhibitors, antisense or siRNA or other double-stranded RNA molecules, antibodies that interact with HER family receptors, and antibodies or cytotoxic agents conjugated to radionuclides. . Other exemplary anti-cancer agents include gefitinib, lapatinib, elortinib, panitumumab, cetuximab, trastuzumab, imatinib, platinum complexes, or nucleoside analogs. Examples of cytotoxic or chemotherapeutic agents include, for example, taxanes (such as paclitaxel and docetaxel) and anthracycline antibiotics, doxorubicin/adriamycin, carminomycin, daunorubicin, aminoopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, Porphyromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin or podophyllotosin derivatives, such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurocidin ( leurosidine), vindesine, leurosidne, maytansinol, epothilone A or B, taxotere, taxol, estramustine, cisplatin, combretastatin and analogs, and cyclophosphamide. Any of the other anti-cancer antibodies and chemotherapeutic agents described elsewhere herein or known in the art may be combined with the multispecific growth factor trap constructs, nucleic acid molecules provided herein. It is contemplated for use in the treatment of cancer, or in combination with a pharmaceutical composition.

In another example, provided herein can be used to combine any of the multispecific growth factor trap constructs, nucleic acid molecules, or pharmaceutical compositions provided herein with another anti-rheumatic drug, e.g. A method of treating rheumatoid arthritis (RA) by administration in combination with TNF therapy. Examples of anti-TNF therapies that can be used in combination with the multispecific growth factor trap constructs, nucleic acid molecules, or pharmaceutical compositions provided herein include conventional synthetic DMARDs, such as methotrexate (MTX), hydroxychloroquine ( HCQ; such as Plaquenil®), sulfasalazine (Azulfidine®), and leflunomide (Arava®); biological DMARDs, such as abatacept (Orencia®), anakinra (Kineret®); ), rituximab (Rituxan®, Truxima®, MabThera®), tocilizumab (atlizumab, Actemra®, RoActemra®), corticosteroids (e.g. dexamethasone, methylprednisolone, prednisolone, prednisone, or triamcinolone), tofacitinib (Xeljanz®), and TNF inhibitors/anti-TNF agents, such as certolizumab pegol (Cimzia®), infliximab (Remicade®) )), adalimumab (Humira®), golimumab (Simponi®), and etanercept (Enbrel®). Combination therapy can also include, for example, immunotherapeutic agents such as cyclosporine, methotrexate, adriamycin or cisplatin, and immunotoxins.

By administering any of the multispecific growth factor trap constructs described herein together with any of the TNFR1 antagonist, TNFR2 agonist, or bispecific TNFR1 antagonist/TNFR2 agonist constructs provided herein. Also provided herein are methods of treating chronic inflammatory diseases, autoimmune diseases, neurodegenerative diseases, and/or demyelinating diseases, particularly RA, as described elsewhere herein. Any other therapeutic agent useful in the treatment of chronic inflammatory, autoimmune, neurodegenerative, and/or demyelinating diseases, such as immunosuppressive agents, anti-angiogenic agents, cardioprotective agents, antibodies, cells Injurious agents, anti-inflammatory agents, cytokines, growth inhibitory agents, chemotherapeutic agents, biological or non-biological disease-modifying antirheumatic drugs (DMARDs), agents for the treatment of infectious diseases (including antibodies), or herein as well as any other suitable therapeutic agent described in or known in the art, such as methotrexate, or described above or elsewhere herein, or known in the art. Additional anti-TNF therapies may also be administered, such as those of.

Angiogenesis plays an important role in pannus formation and maintenance in RA. The multispecific growth factor trap constructs provided herein can be used in combination with other therapeutic agents to modulate angiogenesis. For example, angiogenesis inhibitors can be used in combination with the multispecific growth factor trap constructs provided herein to treat RA. Exemplary angiogenesis inhibitors include angiostatin, angiogenesis inhibitor antithrombin III, canstatin, cartilage-derived inhibitor, fibronectin fragment, IL-12, vasculostatin, and those known in the art and described herein. including, but not limited to, others listed elsewhere in the book.

In some embodiments, the growth factor trap constructs provided herein are used in combination with TNF blockers and/or other DMARDs, such as methotrexate, to compare standard of care RA therapy. For example, the growth factor trap constructs provided herein can be combined with etanercept and/or methotrexate, such as suboptimal doses of etanercept and/or methotrexate. To assess efficacy, combinations can be treatment with etanercept and/or methotrexate alone, including optimal and suboptimal doses of etanercept and/or methotrexate. Growth factor trap constructs allow for reduced doses of other treatment agents, thereby reducing harmful or undesirable side effects. In other embodiments, the growth factor trap constructs provided herein are combined with other anti-TNF therapies, such as adalimumab or infliximab (including suboptimal doses thereof), and methotrexate (including suboptimal doses of methotrexate). ), with or without methotrexate, and the efficacy of treatment is compared to treatment with anti-TNF therapy alone, with or without methotrexate. In yet another embodiment, a growth factor trap construct provided herein is a TNFR1 antagonist, a TNFR2 agonist, or a multispecific, such as a bispecific, TNFR1 antagonist/TNFR2 agonist construct provided herein. Compare standard-of-care RA therapies such as etanercept, adalimumab, or infliximab, with or without methotrexate.

H. Evaluation of the Activity and Efficacy of TNFR1 Antagonist and TNFR1 Antagonist/TNFR2 Agonist Constructs Where appropriate, the constructs provided herein are evaluated based on the properties of the construct and/or suitability for treating a particular disease, disorder, or condition. Any in vivo and/or in vitro assay known to those skilled in the art can be used to assess activity and efficacy. These assays can also be used to monitor treatment and/or predict response or select subjects for treatment. Exemplary assays are described in the sections below.

Generally, the antagonist constructs herein are non-competitive; they are constructs that generally lock the receptor into an inactive conformation, which, as discussed above, selects for high affinity. This means that selecting for antagonist activity is less important.

1. Disease activity score (DAS28)
The 28-joint disease activity score (DAS28; or 28-joint disease activity score) is a measure of disease activity in rheumatoid arthritis (RA), simplifying the original DAS score, which required counting 44 joints. This is what I did. The 28 joints counted include proximal interphalangeal joints (10 joints), metacarpophalangeal joints (10 joints), wrists (2), elbows (2), shoulders (2), and knees (2). It will be done. DAS28 is used in clinical trials to evaluate therapeutics for RA because it indicates RA disease activity and response to treatment. DAS28 is based on the number of 28 swollen and tender joints, and scores range from 0 to 10, with higher values indicating more disease activity. In addition to counting the number of swollen and tender joints (out of 28), DAS28 includes measurements of erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP), which , an acute phase reactant/blood marker of inflammation, is a general health (GH) assessment, represents a patient's self-assessment of disease activity, and is scored on a 100 mm visual analogue scale (VAS), 0. A value of 100 means "no activity" and a value of 100 means "highest possible activity." DAS28 typically assesses physical function using the Health Assessment Questionnaire (HAQ) in combination with other measures of disease severity such as pain and grip strength.

To calculate DAS28 values using ESR or CRP levels, use the following formulas, respectively:
DAS28 (ESR) = 0.56x√ (TJC28) + 0.28x√ (SJC28) + 0.014xGH + 0.70xln (ESR);
DAS28(CRP)=0.56x√(TJC28)+0.28x√(SJC28)+0.014xGH+0.36xln(CRP+1)+0.96;
where TJC = tender joint number and SJC = swollen joint number.

A value <2.6 indicates remission, a value ≦3.2 (>2.6 but ≦3.2) indicates low disease activity, a value >3.2 A value of ≦5.1 indicates moderate disease activity, and a value greater than 5.1 indicates high disease activity (ie, active disease). Improvement (i.e., decrease in DAS28 score/value) >1.2 indicates good response/improvement; improvement >0.6 to ≦1.2 indicates moderate response; A decrease in DAS28 of ≦0.6 indicates no improvement (e.g., Prevoo et al. (1995) Arthritis & Rheumatism 38(1):44-48; Wells et al. (2009) Ann. Rheum Dis. 68:954-960).

The therapeutic efficacy of the bispecific constructs containing selective TNFR1 antagonists, TNFR2 agonists, and/or combinations thereof provided herein can be demonstrated before, during, and after treatment with DAS28 (ESR) or DAS28. It can be evaluated by calculating (CRP).

2. SOMAscan® Proteomics Analysis and Other Proteomics Tools for Quantifying Analytes The SOMAscan® Proteomics Assay (SomaLogic, Inc.; Boulder, CO.) is a small volume serum, plasma or It is an aptamer-based, multiplexed, highly sensitive, quantitative and reproducible proteomics tool that can simultaneously measure the abundance of over 5,000 protein analytes in samples such as cerebrospinal fluid. Other biological matrices can also be used, such as cell culture supernatants, cell and tissue lysates, synovial fluid, and bronchoalveolar and nasal washes. The SOMAscan® assay is optimized for protein biomarker discovery as it can simultaneously quantify a wide range of protein targets, including non-small cell lung cancer, Alzheimer's disease, cardiovascular disease, and inflammatory bowel disease. have been used to identify biomarker signatures associated with several diseases. The SOMAscan® assay detects protein signatures in, e.g., RA patients by analyzing samples taken before and after initiation of treatment with TNFR1 antagonists, TNFR2 agonists, and bispecific constructs provided herein. can be used to determine. In this way, patient response can be monitored at the beginning of treatment and throughout treatment.

The SOMAscan® assay employs a protein capture reagent known as a Slow Off-rates engineered adapter (sold as SOMAmer® aptamer) reagent, which contains amino acid side chains. Short, single-stranded DNA-based protein affinity reagents constructed with chemically modified nucleotides that mimic the It is. This assay measures proteins in their native folded conformation (ie, tertiary structure) and does not detect unfolded, denatured (ie, inactive) proteins. In the SOMAscan® assay, the SOMAmer®-protein binding step is followed by a series of partitioning and washing steps, which separates the proteins in the biological sample to correspond to each individual protein concentration. quantification by converting to SOMAmer® reagent concentration (SOMAmer®-based DNA signal), which is then quantified by standard DNA detection techniques such as microarray or qPCR. This assay takes advantage of the dual nature of SOMAmer reagents as protein affinity binding reagents with defined three-dimensional structures and containing unique nucleotide sequences that are recognizable by specific DNA hybridization probes. There is.

The SOMAmer® reagent was prepared with three tags and contains a fluorophore attached to biotin via a photocleavable linker. Briefly, for the assay, the biological sample of interest is diluted, which is then incubated with the respective SOMAmer® reagent mixture pre-immobilized on streptavidin (SA) coated beads. do. SOMAmer® reagents are bound to proteins in the biological sample and the beads are washed to remove unbound proteins. Any non-specific complexes formed have a rapid dissociation rate. Proteins that remain bound to the cognate SOMAmer® reagent are tagged using NHS-biotin reagent and a polyanion competition solution is added to resolve non-specific complexes. The protein-SOMAmer® complex and unbound (free) SOMAmer® reagent are released from the streptavidin beads by cleaving the photocleavable linker using ultraviolet light. The photocleavage eluate containing all the SOMAmer® reagents (both bound and free from the biotinylated protein) is then combined with the biotinylated protein and the biotinylated protein-SOMAmer® complex. and unbound material is removed by a subsequent washing step. In the final elution step, the protein-bound SOMAmer® reagent is released from the cognate protein using denaturing conditions, and the SOMAmer® reagent is used for hybridization to custom DNA microarrays and measurement of fluorophore tags. , quantified by standard DNA quantification techniques. Data are reported in relative fluorescence units (RFU) after normalization and calibration to correlate the measured SOMAmer® reagent signal with protein levels found in biological samples (e.g., Gold et al. (2010) PLoS ONE 5(12):e15004; Candia et al. (2017) Sci. Reports 7:14248; Tanaka et al. (2018) Aging Cell. 17:e12799).

3. Transcriptome analysis to predict response to therapy and select subjects likely to benefit from treatment Conventional anti-TNF therapies, i.e. TNF blockers such as etanercept, infliximab, are associated with non-responsiveness in RA patients The gender is about 30%. However, there are clinical markers that can predict the effectiveness of these anti-TNF therapies. Analysis of differentially expressed genes after anti-TNF therapy with etanercept was performed using global transcriptome analysis to determine RNA expression signatures in peripheral blood mononuclear cells (PBMCs). Similar transcriptomic analyzes can be performed to assess the efficacy of and/or patient responsiveness to the TNFR1 antagonist and TNFR1 antagonist/TNFR2 agonist constructs herein.

In an exemplary protocol, blood samples are obtained from patients before and after treatment, PBMCs are separated using a Ficoll density gradient, and then flow cytometry is used to determine populations of CD3+, CD14+, CD19+, and CD56+ cells. evaluate. Total RNA is then extracted using, for example, a Qiagen RNeasy® kit, and microarray analysis (e.g., using Affymetric® microarray technology) is used to extract tens of thousands of known proteins within PBMCs. to identify responder/non-responder profiles. Gene expression profiles can be determined early in treatment to quickly identify non-responders. For example, by using this method to identify reliable biomarkers for predicting the therapeutic effect of etanercept in RA patients, we identified gene pairs with a predictive accuracy of >89%, and gene pairs with a predictive accuracy of >95%. A gene triplet was identified. These include, for example, genes involved in TNF signaling through the NF-κB pathway, genes involved in NF-κB-independent signaling, and genes involved in the regulation of cellular and oxidative stress responses. For example, the identified gene triplet includes TNFAIP3, which encodes TNFα-inducible protein 3, a zinc finger protein that has been shown to inhibit NF-κB activation; involved in NF-κB-independent signal transduction; and RAPGEF1, which encodes Rap guanine nucleotide exchange factor 1, an activator of RAS signaling. Expression of all three of these genes was downregulated 3 days after etanercept administration in responders compared to non-responders. Other genes evaluated include CCL4, CXCR4, CCL3, PIGO, FSD1, RUNX1, LGALS13, PTPRD, IL1B, ADAM12, and HCG4P6 (e.g., Koczan et al. (2008) Arthritis Research & Therapy 10: (See R50).

Expression levels of a subset of genes of interest can be measured by quantitative real-time PCR (RT-PCR) using pre-designed primers and probes to validate the results obtained with microarray analysis. In order to calculate the change in gene expression of a selected gene, the ΔΔCT method can be used, whereby the threshold cycle (CT) value of specific mRNA expression in a sample can be calculated from the CT of GAPDH mRNA in a sample. Gene expression change (ΔΔCT) is defined by the difference between pre- and post-treatment CT values (see, eg, Koczan et al. (2008) Arthritis Research & Therapy 10:R50).

4. L929 Cytotoxicity Assay TNFR1-mediated processes and cellular responses can be determined, for example, by assessing TNF-induced cell death in the L929 cytotoxicity assay, where TNFR1 antagonists inhibit TNF-induced cytotoxicity. Briefly, L929 mouse fibroblasts are plated in microtiter plates and incubated overnight with the TNFR1 antagonists, 100 pg/ml TNF and 1 mg/ml actinomycin D. Cell viability was determined after incubation with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Measure by reading absorbance at 490 nm. TNFR1 antagonists reduce TNF-mediated cytotoxicity and result in increased absorbance compared to TNF-only controls.

5. HeLa IL-8 Assay The activity of TNFR1 antagonists can be determined using the HeLa IL-8 assay, which assesses the ability of antagonists to neutralize TNF-induced IL-8 secretion in HeLa cells. Briefly, HeLa cells are plated overnight in microtiter plates in the presence of various concentrations of TNFR1 antagonist and 300 pg/ml TNF. The supernatant is then aspirated and the concentration of IL-8 is measured using a sandwich ELISA. TNFR1 antagonist activity reduces IL-8 secretion into the supernatant compared to TNF-only controls.

6. HUVEC Assay A human umbilical vein endothelial cell (HUVEC) assay can be used to determine the activity of the TNFR1 antagonists herein. Treatment of HUVEC with TNF results in upregulation of VCAM-1 expression on the cells, which can be determined, for example, by ELISA. Because TNFR1 antagonists inhibit the action of TNF, VCAM-1 expression is decreased in HUVECs in the presence of antagonists. The level of inhibition of TNF-induced VCAM-1 expression by a TNFR1 antagonist is then determined by plotting the concentration of antagonist against the percent inhibition of VCAM-1 expression.

According to the protocol for this assay, HUVECs are cultured overnight, then incubated with TNFR1 antagonist for 1 hour, followed by stimulation with TNF (1 ng/mL) for 23 hours. Cells incubated with medium only are used as a negative control and cells incubated with TNF only are used as a positive control. The cell culture supernatant was then aspirated and cells were washed three times with ice-cold PBS and incubated with ice-cold Tris-glycerol lysis buffer (40mM Tris, 274mM NaCl, 2% Triton-X-100, 20% glycerol, 50mM Add and dissolve NaF, 1mM Na3VO4, 1x protease inhibitor tablets per 10mL, then incubate on ice for 15 minutes. Cell lysates are then used in a VCAM-1 sandwich ELISA. To calculate inhibition of TNF-induced VCAM-1 expression, % inhibition of maximal VCAM-1 expression (i.e., measured in positive control) = {100 - [(OD value at antagonist concentration) / (OD of positive control value)]}x100.

EC50 values are then determined by plotting antagonist concentration against percentage inhibition using available software such as, for example, GraphPad Prism software.

7. Quantification and Evaluation of Treg Cell Activity To determine the effects of TNFR1 antagonist and TNFR1 antagonist/TNFR2 agonist constructs on Tregs, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using e.g. Ficoll-Paque The number of Tregs is determined by isolating CD4+CD25+ Tregs and CD4+CD25− non-regulatory T cells using monoclonal antibodies (mAbs) and paramagnetic beads, or other similar methods known in the art. can be quantified before and after and during treatment. Each cell was analyzed using flow cytometry and immunostaining with mAbs against CD4 and CD25 (for Tregs) or CD4 and CTLA-4 (for non-regulatory T cells). The number of types can be quantified (see, eg, Vigna-Perez et al. (2005) Clin. Exp. Immunol. 141(2):372-380).

CD4+CD25+ Tregs suppress the proliferation of CD4+CD25-T cells. To test the activity of Tregs in treated patients, a cell proliferation assay can be used in which Tregs and T cells are cultured for 48 hours with phytohemagglutinin (PHA, which stimulates T cells). 3H-TdR (tritiated thymidine) is added during the last 12 hours of culture, after which cells are harvested and proliferation determined using a liquid scintillation counter. CD4+CD25- T cells cultured alone were used as a control and results are expressed as the stimulation index (SI) of cell proliferation, calculated using the following formula:
SI = (cpm of cells containing PHA) ÷ (cpm of cells cultured in medium only), where cpm is counts per minute, determined by the counted radioactivity (e.g. Vigna-Perez et al. (2005) Clin. Exp. Immunol. 141(2):372-380).

To test immunoreactivity against M. tuberculosis, PBMCs are cultured in complete medium for 72 hours in the presence of bacterial total protein extract. 3H-TdR (tritiated thymidine) is added during the last 12 hours of culture, after which cells are harvested and proliferation determined using a liquid scintillation counter. As above, results are expressed as irritation index. For in vivo reactivity against Mycobacterium tuberculosis, a standard PPD (purified protein derivative) skin test can be used (e.g., Vigna-Perez et al. (2005) Clin. Exp. Immunol. 141(2):372 -380).

8. Evaluation of the binding properties of TNFR1 antagonist/TNFR2 agonist constructs Specificity of antibodies or antibody fragments or multispecific constructs (such as the constructs provided herein) to TNFR1 and/or TNFR2 (e.g., human TNFR1 and/or TNFR2) Binding can be assessed by any of a variety of known methods. Affinity is the concentration (EC50) of the TNFR1 antagonist, TNFR2 agonist, or multispecific construct required to achieve half-maximal potentiation of TNFR1 and/or TNFR2 signaling in vitro; It can be expressed quantitatively by a variety of metrics, including the equilibrium constant (KD) of antagonist-TNFR1 and/or agonist-TNFR2 complex dissociation. The equilibrium constant KD representing the interaction of TNFR1 or TNFR2 with a binder, such as a construct (binder), provided herein is a It is a chemical equilibrium constant of the dissociation reaction between solvent-separated TNFR1 or TNFR2 and a binder molecule.

TNFR1 antagonists, TNFR2 agonists, and multispecific constructs can also be characterized by various in vitro binding assays. Examples of experiments that can be used to determine KD or EC50 include, for example, surface plasmon resonance (SPR, e.g., BIAcore™ analysis), isothermal titration calorimetry, fluorescence anisotropy, and ELISA, among others. -based assays included. ELISA is a particularly useful method for analyzing antibody activity because such assays usually require minimal concentrations of antibody. A common signal analyzed in a typical ELISA assay is luminescence, which is usually the result of the activity of a peroxidase conjugated to a second antibody that specifically binds to the first antibody (e.g., as described herein). TNFR1 antagonist or TNFR1 agonist TNFR2 agonist bispecific construct provided in

The kinetics of association and dissociation of a TNFR1 antagonist and TNFR1 or a TNFR2 agonist and TNFR2 can be characterized quantitatively, for example, by monitoring the rate of antibody-antigen complex formation according to established procedures. For example, surface plasmon resonance (SPR) can be used to determine the rate constants of formation (kon) and dissociation (koff) of antagonist-TNFR1 or agonist-TNFR2 complexes. Since the equilibrium constant of this unimolecular dissociation can be expressed as the ratio of koff to the kon value, the equilibrium constant (KD) can be determined from these data. SPR is advantageous for determining the kinetic and thermodynamic parameters of receptor-antibody (or other binder) interactions because the experiment does not require modifying one component by attachment of a chemical label. It's technology. Rather, the receptor is typically immobilized on a solid metal surface that is pulsed with solutions of increasing concentrations of antibody or binder (i.e., TNFR1 antagonist or TNFR2 agonist, or bispecific constructs thereof). be converted into Antibody-receptor binding induces a distortion of the angle of reflection of incident light at the metal surface, and this change in refractive index over time when the antibody is introduced into the system is coupled to the binding and dissociation of antibody-receptor interactions. To calculate rate constants, established regression models known in the art can be fitted.

9. Antibody Dependent Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) Assays Antibody Dependent Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) assays are used to determine Fc monomers. The immune effector function/cytotoxicity of the TNFR1 antagonists, TNFR2 agonists, and multispecific constructs provided herein containing antibodies or dimers can be assessed. Generally, the Fc portion(s) are modified to eliminate or substantially reduce ADCC or ADCC and CDC effector function (eliminating or reducing adverse side effects to acceptable levels). Such assays are well known in the art (see, eg, Ying et al. (2014) mAbs 6(5):1201-1210). For example, in an exemplary ADCC assay, mesothelin-negative A431 or mesothelin-positive H9 cells are incubated for 30 minutes with a TNFR1 antagonist, TNFR2 agonist, or multispecific construct provided herein, followed by a 50:1 Target cells are added to wells containing effector cells (eg, PBMC) at an effector to target cell ratio. After 24 hours of incubation, target cell lysis is measured using the CytoTox-ONE Homogenous Membrane Integrity Assay (Promega) according to the manufacturer's protocol.

In an exemplary CDC assay, A431 and H9 cells are washed with serum-free RPMI and the density is adjusted to 1 million/mL with serum-free RPMI. 50 μL of cell suspension is incubated with 50 μL of TNFR1 antagonist, TNFR2 agonist, or multispecific construct diluted in RPMI. Negative controls included 50 μL of cell suspension with 50 μL of RPMI, and positive controls included target cells lysed with 1% Triton X-100 in a final volume of 150 μL. Untreated human plasma was diluted in PBS (1:4), clarified by centrifugation, and 50 μL of diluted plasma was added to each cell/construct mixture and incubated in a 96-well plate at 37°C to obtain complement. Mediated cell lysis can be performed. After 3 hours of incubation, 100 μL of supernatant is transferred to a white plate and 100 μL of substrate from the CytoTox-ONE Homogenous Membrane Integrity Assay kit (Promega) is added. The plate is then incubated at room temperature for 10 minutes and the fluorescence signal is read using a fluorometer with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. CDC of target cells is expressed as a percentage of the experimental sample relative to the positive control.

10. Disease Models The selective TNFR1 antagonists, TNFR2 agonists, and multispecific constructs provided herein are mediated by TNF in pathogenesis, as evaluated in any clinically relevant disease model known to those of skill in the art. or their effects on autoimmune and inflammatory and other diseases or disorders in which TNF is implicated can be determined. Exemplary disease models include collagen-induced arthritis (CIA), rheumatoid arthritis synovial mononuclear cell cultures, Tg197 mouse model of arthritis, ΔARE mouse model of arthritis/IBD, mouse dextran sodium sulfate (DSS) induction of IBD. models, and experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. Other models are known to those skilled in the art. For example, Malaviya et al. (2017) Pharmacol Ther. 180:90-98; anti-TNF therapy for COVID-19, which provides numerous models for testing constructs to treat inflammatory lung diseases; and models, targeting the use of treatments Feldmann et al. (2020) Lancet 395:1407-1409; use of anti-TNF therapy to treat H1N1, and models of viral infection, Shi et al. (2013) ) Crit. Care 17(6):R301; and describes the advantage of activating TNFR2 to treat Alzheimer's disease; TNF blockers that inhibit TNFR1 and TNFR2 are problematic. See Orti-Casan et al. (2019) Front. Neurosci.13:49, which demonstrates the approach herein. Below is a non-exhaustive discussion of diseases, disorders, and conditions that can be treated with the constructs provided herein, as well as exemplary models for each. These are examples; one skilled in the art can select the appropriate model for the particular construct and target disease, disorder, or condition. Since the anti-TNFR1 and TNFR2 antagonist/agonist constructs provided herein are intended for use in targeting human TNFR1/TNFR2, they may be derived from non-human, particularly non-primate species. It is not expected to function similarly in reaction/interaction with TNFR1/TNFR2. For testing, non-human models such as rodent models, such as mouse models, are transgenic for human TNFR1 and human TNFR2. They can be used against a murine TNFR1/2 knockout mouse background in in vivo models of inflammation and autoimmune diseases. Alternatively, severely immunodeficient mice (such as NOD/NSG mice) transplanted with human CD34+ stem cells can be used for this purpose. Alternatively, human rheumatoid arthritis synovial cells can be transplanted into immunodeficient mice to induce RA-like inflammation (for a description of such models, see e.g. Schinnerling et al. (2019) Front Immunol. 10: 203).

a. Collagen-induced arthritis (CIA)
Type II collagen-induced arthritis (CIA) is a mouse model of an autoimmune inflammatory joint disease that histologically resembles RA and is characterized by inflammatory synovitis, pannus formation, and cartilage and bone erosion. It can be induced by To induce CIA, bovine type II collagen (B-CII) is injected intradermally at the base of the tail in the presence of complete Freund's adjuvant. After 21 days, mice can be re-immunized using the same protocol. To examine the effects of selective TNFR1 antagonists, TNFR2 agonists, and multispecific constructs provided herein, selective TNFR1 Antagonists, TNFR2 agonists, or multispecific constructs or controls can be administered intraperitoneally twice a week for three weeks. Mice can be sacrificed 7 weeks after the first immunization for histological analysis.

To evaluate the therapeutic effect of the construct on established disease, it can be administered daily for a total of 10 days after the onset of clinical arthritis in one or more limbs. The degree of swelling in the initially affected joint can be monitored by measuring the thickness of the foot using calipers. Inflammatory cytokines and chemokines, such as granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-10 (IL-10), IL-1β, IL-6, IL-8, RANTES (CCL5), and monocyte chemotaxis. Serum can be collected from mice for measurements such as catalytic protein 1 (MCP-1; also known as CCL2).

In another example, primate models are available for RA treatment. To evaluate therapeutic efficacy and treatment, tender and swollen joint responses (e.g., clinical arthritis scores ) can be monitored.

b. Rheumatoid Arthritis Synovial Mononuclear Cell Culture Human rheumatoid arthritis (RA) synovial mononuclear cells (MNCs) expressing TNFR1 and TNFR2 can also be used to test the therapeutic efficacy of the constructs provided herein. can. RA synovial MNCs can be obtained from RA patients undergoing joint replacement surgery and cultured ex vivo to assess cytokine production and regulation of RA synovial cells. RA synovial MNC cultures spontaneously produce inflammatory cytokines and chemokines in the absence of exogenous stimuli; antibody-mediated neutralization of TNF in these cultures, and constructs such as provided herein. Selective blockade of TNFR1 by inducing production of pro-inflammatory cytokines and chemokines such as GM-CSF, IL-10, IL-1β, IL-6, IL-8, RANTES (CCL5), and MCP-1 (CCL2) is inhibited.

In an exemplary assay, to prepare RA synovial MNCs, RA synovial tissue is cut into small pieces and incubated with 5 mg/ml collagenase A and 0.15 mg/ml DNase in RPMI 1640 for 1 hour at 37°C; The digested tissue is then passed through a 170 μm filter and washed three times with RPMI 1640 containing 100 units/ml streptomycin, 100 μg penicillin, and 10% FCS. Heterogeneous RA synovial MNCs are then used without passaging. For ex vivo cell culture, single cell suspensions of RA synovial MNCs were cultured in 96-well flat bottom plates (2 x 10 cells/well) in RPMI 1640 medium containing 5% FCS with TNFR1 antagonist, TNFR2 agonist, or bispecific constructs, or in the presence or absence of controls, for 2-5 days at 37°C and 5% CO2. The supernatant is then collected and used immediately or stored at −20°C for analysis by cytokine and chemokine ELISA (e.g., Schmidt et al. (2013) Arthritis & Rheumatism 65(9):2262- 2273). Alternatively, cytokines in the culture supernatant can be quantified by cytokine bead array analysis.

c. Tg197 Mouse Model of Arthritis The Tg197 transgenic strain of mice, a mouse model of erosive arthritis, is an established animal model of RA. Tg197 mice are human TNF transgenic C57BL/6 mice that overexpress human TNF and develop symmetric polyarthritis with pannus formation, bone destruction, and cartilage damage that are characteristic of human RA. In addition to exhibiting features of chronic destructive joint disease, this model exhibits symptoms such as enthesitis or bilateral sacroiliitis characteristic of other inflammatory diseases such as spondyloarthritis (Bluml et al. (2010) Arthritis & Rheumatism 62(6):1608-1619). Tg197 mice develop arthritis with 100% penetrance and provide a rapid in vivo model for evaluating human therapeutics targeting RA. For example, infliximab (originally sold as Remicade®) was the first anti-TNF therapy successfully applied in the clinic and is recommended by the FDA for screening potential anti-RA therapies. The Tg197 mouse model was used to evaluate the therapeutic effects of .

Tg197 mice carry five copies of the human TNF gene construct in which the 3' region containing the 3' untranslated and 3' flanking sequences is replaced with the 3' region of the human β-globin gene. Because a set of highly conserved UA-rich sequences in the 3' untranslated region of TNF mRNA is important for controlling mRNA stability and translation efficiency, this genetic construct was microinjected into mouse fertilized eggs and regulated. An in vivo model of deregulated TNF gene expression is created (see, eg, Keffer et al. (1991) EMBO J. 10(13):4025-4031).

d. ΔARE Mouse Model of Arthritis/IBD Mice lacking the 3'AU-rich element (ARE) of TNF mRNA (TnfΔARE) overproduce TNF between 4 and 8 weeks of age and histopathologically exhibit Crohn's disease. Develop a similar inflammatory bowel disease. Mice also develop clinical signs of RA. The efficacy of TNFR1 antagonists, TNFR2 agonists, and bispecific constructs provided herein can be evaluated by evaluating their inhibitory effects on Crohn's disease-like pathology and arthritis in TNFΔARE mice after i.p. injection. (see, eg, US Pat. No. 9,028,822).

e. Humanized TNF/TNFR2 Mouse Many human anti-TNF therapies do not interact with mouse TNF or TNFR2, as human TNF can bind and engage mouse TNFR1 but not TNFNR2. , a limitation in the development of therapeutics targeting the TNF/TNFR2 signaling pathway is the lack of preclinical animal models. Humanized TNF/TNFR2 mice harboring a functional human TNF-TNFR2 (hTNF-hTNFR2) signaling module are used to evaluate therapeutic agents such as agonist and antagonist antibody constructs human TNF or human TNFR2 in various models of autoimmunity. can be used for. Such TNF/TNFR2 dual humanized mice can be used, for example, to evaluate the TNFR2 agonist constructs provided herein.

Humanized TNF/TNFR2 mice can be generated as described in Atretkhany et al. (2018) Proc. Natl. Acad. Sci. U.S.A. 115(51):13051-13056. Briefly, human TNFR2 knock-in (hTNFR2KI) and human TNF knock-in (hTNFKI) mice are generated using standard genetic engineering techniques. Next, hTNFKI mice in which the mouse TNF gene has been replaced by the human TNF gene are crossed with hTNFR2KI mice containing humanized TNFR2 ligand binding moieties, and then reciprocally crossed to double humanized double homozygous hTNFKI× Generate hTNFR2KI mice. To assess the role of TNFR2 signaling in specific cells, such as Tregs, we inserted two LoxP sites within the hTNFR2 locus, which allow conditional Cre-mediated excision of the extracellular portion of TNFR2. Become. For Treg-specific deletion of TNFR2, these mice are crossed with FoxP3-Cre transgenic mice (e.g., Atretkhany et al. (2018) Proc. Natl. Acad. Sci. U.S.A. 115(51):13051- 13056).

I. Methods of Producing Nucleic Acids Encoding TNFR1 Antagonist and TNFR1 Antagonist/TNFR2 Agonist Constructs TNFR1 antagonist polypeptides, TNFR2 agonist polypeptides, and TNFR1 antagonist/TNFR2 agonist polypeptide constructs provided herein that are polypeptides can be obtained by methods well known in the art for protein purification and recombinant protein expression, and for the preparation of recombinant antibodies. Constructs provided herein that include moieties such as linkers that are not polypeptides can optionally be prepared by chemical conjugation. Polypeptide moieties are produced by standard recombinant techniques such as expression in a suitable host (bacteria if glycosylation is not desired, or eukaryotic cells such as HEK293 and CHO cells if glycosylated forms are desired). can be done. Active antibodies and antibody fragments have been produced in E. coli but often suffer from aggregation and lysis problems due to improper folding, which can be ameliorated by further mutagenesis of the coding sequence. (See, e.g., Kunz et al. (2018) Sci Rep. 8(1):7934).

Polypeptides can also be chemically synthesized. Fusion polypeptides can be synthesized by standard methods of recombinant production. The above components of the various constructs can be synthesized separately and combined using standard methods to produce the constructs.

Nucleic acids encoding polypeptide constructs or polypeptide portions thereof, including modifications or variants, including truncated forms, can be prepared from the nucleic acids. Modified or variant polypeptides can be engineered from nucleic acids encoding wild-type polypeptides using standard recombinant DNA methods. For example, a modified TNF polypeptide, such as a TNF mutein, that selectively binds to TNFR1 or TNFR2, and/or selectively antagonizes TNFR1 or selectively agonizes TNFR2, can be modified by site-specific binding of the encoding DNA. It can be engineered from wild-type TNF, such as by mutagenesis. Any method known to those skilled in the art can be used. The following discussion and description of the Examples are exemplary.

1. Isolation or Preparation of Nucleic Acids Encoding TNFR1 Antagonist and TNRF2 Agonist Polypeptides Nucleic acids encoding TNFR1 antagonist polypeptides, TNFR2 agonist polypeptides, and TNFR1 antagonist/TNFR2 agonist polypeptide constructs are used for cloning and isolating nucleic acid molecules. can be cloned or isolated using any available method known in the art. Such methods include screening of libraries, including polymerase chain reaction (PCR) amplification of nucleic acids, nucleic acid hybridization screening, antibody-based screening, and activity-based screening. For example, if the polypeptide is produced by recombinant means, any method known to those skilled in the art for identifying nucleic acids encoding the desired polypeptide can be used.

Nucleic acid molecules encoding the polypeptides herein are optionally prepared using conventional procedures (e.g., heavy chains of antibody fragments such as single domain antibodies (dAbs), scFv fragments, and Fab antibody fragments). and/or by using oligonucleotide probes that can specifically bind to the gene encoding the light chain), or can be produced synthetically or easily isolated and sequenced. . For example, any cell source known to produce or express TNFR1 antagonist or TNFR2 agonist antibodies or fragments thereof can serve as such a source of DNA. In another example, once the DNA encoding a TNFR1 antagonist or TNFR2 agonist antibody or fragment thereof has been sequenced, gene synthesis techniques can be used to construct the nucleic acid sequence.

For example, nucleic acid amplification methods, including polymerase chain reaction (PCR) methods, can be used to isolate nucleic acid molecules encoding the desired polypeptide. Examples of such methods include the use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). Nucleic acid-containing materials can be used as starting materials from which nucleic acid molecules encoding desired polypeptides can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, liquid samples (e.g., blood, serum, and saliva), and samples from healthy and/or diseased subjects can be used in amplification methods. can. Sources are generally derived from human sources, but include, but are not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources, as appropriate. can be derived from eukaryotic species that are not Nucleic acid libraries can also be used as a source of starting material. Primers can be designed to amplify the desired polypeptide. For example, primers can be designed based on the expressed sequence from which the desired polypeptide is produced. Primers can be designed based on reverse translation of polypeptide amino acid sequences. Degenerate primers can be used for amplification if desired. Oligonucleotide primers that hybridize to sequences at the 3' and 5' ends of a desired sequence can be used as primers to amplify the sequence by PCR from a nucleic acid sample. Primers can be used to generate nucleic acids encoding the entire full-length polypeptide, or truncated sequences thereof, including, for example, any of the TNFR1 antagonist and TNFR1 agonist polypeptides and TNFR1 antagonist/TNFR2 agonist polypeptide constructs provided herein. etc. can be amplified. Nucleic acid molecules produced by amplification can be sequenced to confirm that they encode the desired polypeptide or construct.

Mutagenesis techniques can be used to generate further modified forms of TNFR1 antagonist or TNFR2 agonist antibodies or fragments thereof to produce modified forms of activity modifiers such as the Fc and hinge regions and linker moieties. DNA can also be modified. For example, gene synthesis and routine molecular biology techniques can be used to insert, delete, add or replace/substitute nucleotides. Additional nucleotide sequences, including linker sequences containing restriction endonuclease sites, for the purpose of cloning the synthetic gene into a vector, such as a protein expression vector or a vector designed for the amplification of a DNA sequence encoding a core polypeptide. can be attached to a nucleic acid molecule encoding a polypeptide. Additional nucleotide sequences that specify functional DNA elements such as promoters, enhancers, and IRES sequences can be operably linked to the polypeptide-encoding nucleic acid molecule. Examples of such sequences include promoter sequences designed to promote intracellular protein expression, and secretion sequences, such as heterologous signal sequences, designed to promote protein secretion, including Not limited. Such sequences are known to those skilled in the art. Additional nucleotide sequences can also be linked to the polypeptide-encoding nucleic acid molecule, such as sequences specifying protein binding regions. Such regions include, but are not limited to, sequences that facilitate uptake of the polypeptide into specific target cells or that otherwise alter or enhance the pharmacokinetics of the synthetic gene product.

For example, tags and/or other moieties can be added to aid in detection or affinity purification of the polypeptide. Additional nucleotide sequences can also be linked to the polypeptide-encoding nucleic acid molecule, such as, for example, a sequence of bases that identifies an epitope tag or other detectable marker. Examples of such sequences include nucleic acid sequences encoding SUMO tags, or His tags, or Flag tags.

Any of the amino acid sequences provided herein have been reverse-translated (also referred to as back translated) using standard methods commonly used by those of ordinary skill in the art, and the corresponding It is understood that one can generate an encoding nucleic acid sequence, clone it into a vector, and express it to produce constructs including the polypeptides, antibodies, and antibody fragments provided herein. For example, bioinformatics.org/sms2/rev_trans.html; biophp.org/minitools/protein_to_dna/demo.php; vivo.colostate.edu/molkit/rtranslate/; ebi.ac.uk/Tools/st/emboss_backtranseq/; molbiol. There are several online tools to convert protein sequences into coding DNA sequences, such as ru/eng/scripts/01_19.html; and geneinfinity.org/sms/sms_backtranslation.html. Such reverse translated sequences can be inserted into any of the expression vectors provided herein for expression and production of the provided antibodies or fragments. Anti-TFR1 and anti-TNFR2 antibodies, such as TNFR1 antagonist and TNFR2 agonist constructs, can be expressed as full-length or less than full-length proteins. For example, antibody fragments can be expressed, including, but not limited to, single domain antibodies (dAbs), scFv fragments, and Fab fragments.

The identified and isolated nucleic acid can then be inserted into a suitable cloning vector. A number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives, or pBluescript vectors (Stratagene, La Jolla, Calif.). Insertion into a cloning vector can be achieved, for example, by ligating the DNA fragment into a cloning vector with complementary cohesive ends. Insertions can be made using the TOPO cloning vector (Invitrogen, Carlsbad, CA).

If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecule can be enzymatically modified. Alternatively, any desired site can be created by ligating nucleotide sequences (linkers) to the DNA ends; these ligating linkers contain specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. can do. In an alternative method, the truncated vector and polypeptide gene can be modified by homopolymer tailing.

Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, resulting in the production of many copies of the gene sequence. In certain embodiments, transformation of a host cell with a recombinant DNA molecule that incorporates an isolated polypeptide gene, cDNA, or synthetic DNA sequence allows for the production of multiple copies of the gene. Therefore, the gene can be obtained in large quantities by growing the transformant, isolating the recombinant DNA molecule from the transformant, and, if necessary, removing the inserted gene from the isolated recombinant DNA.

For expression of antibodies and fragments thereof, generally a nucleic acid molecule encoding the antibody heavy chain is cloned into a vector, and a nucleic acid molecule encoding the antibody light chain is cloned into a vector. Methods for producing antibodies and portions thereof are well known (e.g., U.S. Pat. Nos. 4,816,567, 6,331,415, and 7,923,221, and numerous other important (see patent). A gene can be cloned into a single vector for its dual expression or into separate vectors. If desired, the vector can also contain additional sequences encoding additional constant or hinge regions to produce other antibody forms. The vector can be transfected into host cells for expression. Expression can be any cellular expression system known to those skilled in the art. For example, host cells include cells that do not otherwise produce immunoglobulin proteins to obtain antibody synthesis within recombinant host cells. For example, host cells include monkey COS cells, Chinese hamster ovary (CHO) cells, such as CHO-DG44 (DHFR-) and FreeStyle™ CHO-S cells (Invitrogen), 293FS cells, HEK293 cells, NSO cells or Including, but not limited to, other myeloma cells. Other expression vectors and host cells are described herein.

Constructs provided herein, including TNFR1 antagonists, TNFR2 agonists, and TNFR1 antagonist/TNFR2 agonist constructs, include, for example, single domain antibodies (dAbs), Fabs, Fab's, Fab hinges, F(ab')2 , single chain Fv (scFv), scFv tandem, Fv, dsFv, scFv hinge, scFv hinge (ΔE), diabodies, as full-length constructs, including antigen-binding fragments such as Fd and Fd' fragments, or as full-length can be produced or expressed as a construct. There are various techniques for producing antibody fragments. For example, fragments can be derived by proteolytic digestion of intact antibodies (e.g., Morimoto and Inouye (1992) Journal of Biochemical and Biophysical Methods 24:107-117; Brennan et al. (1985) Science 229:81- 83). Fragments can also be produced directly by recombinant host cells. For example, dAb, Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from host cells such as E. coli, CHO cells, or HEK293 cells, whereby the Mass production is promoted. F(ab')2 fragments can be generated by chemically coupling Fab'-SH fragments (see, e.g., Carter et al. (1992) Bio/Technology, 10:163-167). ), or they can be isolated directly from recombinant host cell culture. In some instances, the TNFR1 antagonist construct is a single domain antibody (dAb; e.g., International Application Publication Nos. 8/ Enever et al., (2015) Protein Engineering, Design & Selection 28(3):59- 66) , U.S. Patent Application Publication Nos. 2006/0083747, 2010/0034831, and 2012/0107330; and U.S. Pat. Fv fragments (scFv) (see e.g. International Application Publication Nos. WO2017/174586 and WO2008/113515; Richter, F. Thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB," Universitat Stuttgart , 2015; source: pdfs.semanticscholar.org/d8e7/8b87d76dce36225c1d497939ef37445cfa8a.pdf), or Fab fragments (see, e.g., International Application Publications Nos. WO2017/174586 and WO2008/113515; Richter, F Thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB," Universitat Stuttgart, 2015; source: pdfs.semanticscholar.org/d8e7/8b87d76dce36225c1d497939ef37445cfa8a.pdf). dAb, Fv, and scFv fragments have intact binding sites but lack constant regions; therefore, they are suitable for reducing nonspecific binding during in vivo use. dAb and scFv fusion proteins can be constructed to attach an effector protein (eg, IgGFc) to either the amino or carboxy terminus of the dAb or scFv. Antibody fragments may be linear antibodies (see, eg, US Pat. No. 5,641,870). Such linear antibody fragments may be monospecific or bispecific. Other techniques for the production of antibody fragments are known to those skilled in the art.

Upon expression, the heavy and light chains of an antibody, or fragments thereof, pair through interchain disulfide bonds to form a full-length antibody or fragment thereof. For example, for expression of full-length Ig, the sequence encoding VH-CH1-hinge-CH2-CH3 is cloned into a first expression vector, and the sequence encoding the VL-CL domain is cloned into a second expression vector. be able to. When coexpressed, the full-length heavy chain and full-length light chain are linked by disulfide bonds, producing a full-length antibody. In another example, to generate a Fab, sequences encoding fragments containing the VH and CH1 regions are cloned into a first expression vector, and sequences encoding the VL-CL domain are cloned into a second expression vector. can do. When coexpressed, heavy chains pair with light chains to generate Fab monomers. The sequences of the CH1, hinge, CH2 and/or CH3 regions of the various IgG subtypes are known to those skilled in the art (see, e.g., US Patent Application Publication No. 2008/0248028; SEQ ID NOs: 9, 11, 13 and 15). Similarly, CL, lambda, or kappa sequences are known (see, eg, US Patent Application Publication No. 2008/0248028; see also SEQ ID NOS: 17-22).

In addition to recombinant production, the TNFR1 antagonist polypeptides, TNFR2 agonist polypeptides, and TNFR1 antagonist/TNFR2 agonist polypeptide constructs provided herein can be produced by direct peptide synthesis using well-known solid phase techniques. Can be done. In vitro protein synthesis can be performed by manual techniques or automatically. Automated synthesis can be accomplished, for example, using an Applied Biosystems 431A Peptide Synthesizer (PerkinElmer; Foster City, Calif.) according to instructions provided by the manufacturer. Various fragments of a polypeptide can be chemically synthesized separately and combined using chemical methods.

2. Generation of Mutant or Modified Nucleic Acids and Encoded Polypeptides The modifications provided herein can be made by standard recombinant DNA techniques, such as those routine to those skilled in the art. Any method known in the art that results in mutation of any one or more amino acids in a target protein or polypeptide can be used. Methods include standard site-directed mutagenesis of encoding nucleic acid molecules (eg, using kits such as the QuikChange kit available from Stratagene), or solid phase polypeptide synthesis methods.

3. Vectors and Cells For recombinant expression of one or more desired polypeptides, such as any of the TNFR1 antagonist or TNFR2 agonist polypeptides described herein, or the TNFR1 antagonist/TNFR2 agonist polypeptide constructs, A nucleic acid molecule containing all or part of an encoding nucleotide sequence can be inserted into a suitable expression vector, ie, a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence. Vectors containing nucleic acid molecules encoding polypeptides are also provided. After insertion of the nucleic acid molecule, the vector is typically used to transform a host cell, eg, to amplify the nucleic acid for its replication and/or expression. In such instances, vectors suitable for high level expression are used. In other cases, a vector is selected that is compatible with displaying the expressed polypeptide on the cell surface. The choice of vector may depend on the desired use. Many expression vectors are available and known to those skilled in the art for the expression of anti-TNFR1 and anti-TNFR2 antibodies or portions thereof, such as antigen-binding fragments. Such selection is well within the level of skill of those skilled in the art. Generally, expression vectors can include a transcription promoter and optionally an enhancer, translation signals, and transcription and translation termination signals. Expression vectors used for stable transformation typically have selectable markers that allow selection and maintenance of transformed cells. In some cases, a high copy number origin of replication can be used to amplify the copy number of a vector within a cell. Vectors also generally can contain additional nucleotide sequences (eg, His tags, Flag tags) operably linked to the linked nucleic acid molecules. For antibody applications, vectors typically include sequences encoding constant regions. Thus, antibodies or portions thereof can also be expressed as protein fusions. For example, fusion proteins can be generated to add additional functionality to a polypeptide. Examples of fusion proteins include signal sequences, epitope tags for localization, such as His6 tags or myc tags, or tags for purification, such as GST tags, and/or to induce protein secretion and/or membrane association. These include, but are not limited to, fusions of sequences for. Fusion proteins herein also include fusions of TNFR1 antagonists and/or TNFR2 agonists to modified Fc regions, IgG hinge regions, and/or peptide linkers, such as GS linkers.

A variety of host vector systems can be used to express protein coding sequences. These include mammalian cell lines infected with viruses (e.g. vaccinia virus, adenovirus, and other viruses); insect cell lines infected with viruses (e.g. baculovirus); microorganisms such as yeast containing yeast vectors. including, but not limited to, bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The choice of eukaryotic and bacterial expression systems will depend on the desired post-translational modifications such as glycosylation. The expression elements of vectors differ in their strength and specificity. Depending on the host vector system used, any of a number of suitable transcription and translation elements can be used.

Any method known to those skilled in the art for inserting DNA fragments into vectors, such as, for example, antibody fragments or TNFR1 antagonists or TNFR2 agonists provided herein, as well as appropriate transcriptional/translational control signals. Expression vectors can be constructed that include nucleic acid molecules encoding polypeptides. These methods can include in vitro recombinant DNA and synthetic techniques, and in vivo recombination (genetic recombination). Insertion into a cloning vector can be achieved, for example, by ligating the DNA fragment into a cloning vector with complementary cohesive ends. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecule can be enzymatically modified. Alternatively, any desired site can be created by ligating nucleotide sequences (linkers) to the DNA ends; these ligating linkers contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences. be able to.

For example, expression of the construct polypeptides, such as the TNFR1 antagonist, TNFR2 agonist and TNFR1 antagonist/TNFR2 agonist constructs herein, can be controlled by any promoter/enhancer known in the art. Suitable bacterial promoters are well known in the art and are described herein below. Other promoters suitable for mammalian cells, yeast cells, and insect cells are well known in the art, and some are exemplified below. The choice of promoter used to drive expression of a heterologous nucleic acid will depend on the particular application. Promoters that can be used include the SV40 early promoter (see e.g. Benoist and Chambon (1981) Nature 290:304-310), the promoter contained in the 3' terminal repeat of Rous sarcoma virus (e.g. Yamamoto et al. (1980) Cell 22:787-797), herpes thymidine kinase promoter (see e.g. Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445) , the regulatory sequences of the metallothionein gene (see, e.g., Brinster et al. (1982) Nature 296:39-42), and the cytomegalovirus (CMV) promoter; (see, e.g., Jay et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:5543), or the tac promoter (see, e.g., DeBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful Proteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); the nopaline synthetase promoter (e.g. Herrara-Estrella et al. (1984) Nature 303:209-213), cauliflower mosaic virus 35S RNA promoter (see, e.g., Gardner et al. (1981) Nucleic Acids Res. 9(12):2871-2888). ), and the promoter of the photosynthetic enzyme ribulose diphosphate carboxylase (see, e.g., Herrera-Estrella et al. (1984) Nature 310:115-120); yeast and other fungi Promoter elements derived from, for example, the Gal4 promoter, alcohol dehydrogenase promoter, phosphoglycerol kinase promoter, alkaline phosphatase promoter, as well as the following animal transcriptional control regions that exhibit tissue specificity and are used in transgenic animals, namely the pancreatic acinus. Elastase I gene control region that is active in cells (e.g., Swift et al. (1984) Cell 38:639-646; Ornitz et al. (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald (1987) Hepatology 7:425-515), insulin gene control region active in pancreatic beta cells (see, e.g., Hanahan et al. (1985) Nature 315:115-122), lymphatic system Immunoglobulin gene control regions that are active in cells (e.g. Grosschedl et al. (1984) Cell 38:647-658; Adams et al. (1985) Nature 318:533-538; Alexander et al. (1987) Mol. Cell Biol. 7:1436-1444), mouse mammary tumor virus control region active in testis, mammary, lymphatic system and mast cells (see, e.g. Leder et al. (1986) Cell 45:485-495). ), the alpha-fetoprotein gene control region that is active in the liver (see, e.g., Pinkert et al. (1987) Genes and Devel. 1:268-276), the alpha-fetoprotein gene control region that is active in the liver (See, e.g., Krumlauf et al. (1985) Mol. Cell. Biol. 5:1639-1648; Hammer et al. (1987) Science 235:53-58), an alpha-1 anti- trypsin gene control region (see e.g. Kelsey et al. (1987) Genes and Devel. 1:161-171), beta-globin gene control region active in myeloid cells (e.g. Magram et al. (1985) Nature 315:338-340; see Kollias et al. (1986) Cell 46:89-94), myelin basic protein gene regulatory region active in oligodendrocyte cells of the brain (see, e.g. Readhead et al. (1987) Cell 48:703-712), myosin light chain-2 gene control region active in skeletal muscle (see, e.g., Shani (1985) Nature 314:283-286), and the thalamus. gonadotropin-releasing hormone gene control regions that are active in lower gonadotropin-secreting cells (see, e.g., Mason et al. (1986) Science 234:1372-1378). .

Expression vectors typically contain a transcription unit or expression cassette that contains all additional elements necessary for expression of the construct or a portion thereof in a host cell. Typical expression cassettes are operably linked to nucleic acids encoding constructs such as antibody fragments, domains, derivatives or homologues thereof, or other polypeptides described herein (e.g., TNF muteins and fusion proteins). It contains a promoter, and the signals necessary for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene that provides efficient termination. The termination region can be obtained from the same gene as the promoter sequence or from a different gene. For example, a vector may include a promoter, one or more origins of replication, and optionally one or more selectable markers ( For example, antibiotic resistance genes).

The expression system can have markers that provide for gene amplification, such as thymidine kinase and dihydrofolate reductase. Expression systems that do not involve gene amplification are also suitable, such as the use of baculovirus vectors in insect cells with nucleic acid sequences encoding polypeptides under the induction of the polyhedron promoter or other strong baculovirus promoters.

For purposes herein, the Fc region of an IgG antibody, generally encoding a modified Fc, is operably linked to a nucleic acid encoding a TNFR1 antagonist or TNFR2 agonist polypeptide, with a linker therebetween, such as an IgG hinge sequence and / or short peptide linkers such as GS linkers, e.g. glycine-rich flexible linkers such as (Gly4Ser)n, where n is a positive integer such as from 1 to 5 or more, and Vectors containing sequences of nucleotides encoding linkers known to those skilled in the art are provided. The vector can include one or all of the following sequences: CH1, CH2, hinge, CH3 or CH4, and/or CL. Generally, the vector will contain Ch1 or CL (kappa or lambda light chain) sequences, such as for Fab expression. For example, the VH-CH1 and VL-CL sequences can be inserted into a suitable expression vector for expression of the Fab molecule. The sequences of constant or hinge regions are known to those skilled in the art (see, eg, US Patent Application Publication No. 2008/0248028). Examples of such sequences are provided herein.

Typically, vectors can be plasmids, viral vectors, or others known in the art, used for expression of polypeptides in vivo or in vitro. For example, constructs provided herein, such as nucleic acids encoding TNFR1 antagonist and TNFR2 agonist polypeptide constructs, are expressed in mammalian cells, including, for example, Chinese hamster ovary (CHO) cells.

Exemplary eukaryotic vectors include the well-known vectors such as pCMV (Agilent Technologies), pCDNA3.1 (Invitrogen (Thermo Fisher Scientific)), pCBL (Creative BioLabs, see e.g., Figure 1). easy contains vectors available. Other eukaryotic vectors can be used as eukaryotic expression vectors, such as any containing control elements derived from eukaryotic viruses. These include, for example, SV40 vectors, papillomavirus vectors, and vectors derived from Epstein-Barr virus. Exemplary eukaryotic vectors include, for example, pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus pDSCE, and the CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter. , the Rous sarcoma virus promoter, the polyhedron promoter, or other promoters shown to be effective for expression in eukaryotes.

Viral vectors such as adenovirus, retrovirus or vaccinia virus vectors can be used. In some instances, the vector is a defective or attenuated retrovirus or other viral vector (see, eg, US Pat. No. 4,980,286). For example, retroviral vectors can be used (see, eg, Miller et al. (1993) Meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete retroviral sequences unnecessary for packaging of the viral genome and integration into host cell DNA. In some instances, a virus equipped with a nucleic acid encoding a polypeptide herein can facilitate replication and spread within a target tissue. The virus can also be a lytic or non-lytic virus, which replicates selectively under a tissue-specific promoter. As the virus replicates, co-expression of the polypeptide and viral genes facilitates the spread of the virus in vivo.

Vectors for bacterial expression include the well-known and widely used vectors pBR322, pUC, pSKF, pET23D, and fusion vectors such as vectors containing MBP (Sigma-Aldrich), GST (Sigma-Aldrich), and LacZ. included. Exemplary plasmid vectors for transformation of E. coli cells include, for example, the pQE expression vector (available from Qiagen®, Valencia, Calif.; published by Qiagen®, which describes this system). (see also literature). The pQE vector contains a phage T5 promoter (recognized by E. coli RNA polymerase) and two lac operator suppression modules to provide tightly controlled high-level expression of recombinant proteins in E. coli, for efficient translation. It has a synthetic ribosome binding site (RBSII), a 6XHis tag coding sequence, t0 and T1 transcription terminators, a ColE1 origin of replication, and a beta-lactamase gene that confers ampicillin resistance. In the pQE vector, a 6xHis tag can be placed at the N-terminus or C-terminus of the recombinant protein. Such plasmids include pQE32, pQE30, and pQE31, which provide multiple cloning sites in all three reading frames and result in expression of N-terminally 6xHis-tagged proteins. Other exemplary plasmid vectors for transformation of E. coli cells include, for example, pET expression vectors (see, e.g., U.S. Pat. No. 4,952,496; available from NOVAGEN, Madison, Wis.; (See also literature published by Novagen describing the system). Such plasmids include pET 11a, which contains the T7lac promoter, T7 terminator, inducible E. coli lac operator, and lac repressor gene; pET 12a-c containing the E. coli ompT secretion signal; and the His-Tag® leader sequence used for purification on the His column and the thrombin cleavage site that allows for cleavage after purification on the column, the T7-lac promoter region. , and pET 15b and pET19b (NOVAGEN, Madison, Wis.), which contain the T7 terminator.

Cells containing the vectors are also provided. Generally, any cell type that can be engineered to express heterologous DNA and has a secretory pathway is suitable. Cells include eukaryotic and prokaryotic cells, and vectors are any suitable for use therein. Generally, the cell is one that is capable of glycosylating the encoded protein. Prokaryotic and eukaryotic cells containing the vectors are provided. Such cells include bacterial cells, yeast cells, fungal cells, archaea, plant cells, insect cells, and animal cells, especially mammalian cells. The cells are used to produce the polypeptide by growing the cells under conditions such that the encoded polypeptide is expressed by the cell and recovering the expressed polypeptide. For purposes herein, for example, the polypeptide can be secreted into the culture medium.

Host cell lines can be selected for their ability to modulate the expression of inserted sequences or to process expressed proteins in the desired manner. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing can affect polypeptide folding and/or function. Different host cells such as, but not limited to, Chinese hamster ovary (CHO) cells, such as DG44, FreeStyle™ CHO-S cells (Invitrogen), DXB11, CHO-K1), HeLa, MCDK, HEK293 and WI38 cells, Having specific cellular machinery and characteristic mechanisms for such post-translational activity can be selected to ensure correct modification and processing of the introduced protein. Generally, the selection of cells is one that is capable of introducing N-linked glycosylation into the expressed polypeptide. Accordingly, eukaryotic cells containing the vector are provided. An example of a eukaryotic cell is a mammalian Chinese hamster ovary (CHO) cell. For example, CHO cells lacking dihydrofolate reductase (DHFR-), such as DG44 cells, are used to produce the polypeptides provided herein.

4. Expression The polypeptide constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, TNFR1 antagonist/TNFR2 agonist multispecific constructs, and portions thereof, can be prepared using in vivo and in vitro methods, recombinant methods, synthetic methods, can be produced by any method known to those skilled in the art for protein production, including and chemical methods. The desired protein can be expressed, for example, in any organism suitable for producing the necessary amounts and forms of the protein necessary for administration and treatment. Expression hosts include prokaryotes and eukaryotes such as E. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts may differ in the level of protein production as well as the types of post-translational modifications present on the expressed protein. The choice of expression host can be made based on these and other factors known to those skilled in the art; these factors include regulatory and safety considerations, production costs, purification needs and methods. It will be done. Purification methods and methods of assembling the components are well known to those skilled in the art.

Expression in eukaryotic hosts includes yeasts such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as Drosophila cells and lepidopteran cells, tobacco, maize, rice, algae, and This may include expression in plants and plant cells, such as duckweed. Eukaryotic cells for expression also include mammalian cell lines such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293) cells, or baby hamster kidney (BHK) cells. Eukaryotic expression hosts also include production in transgenic animals, such as production in serum, milk and eggs.

Many expression vectors are available and known to those skilled in the art and can be used to express proteins. The choice of expression vector is influenced by the choice of host expression system. Generally, expression vectors can include a transcription promoter and optionally an enhancer, translation signals, and transcription and translation termination signals. Expression vectors used for stable transformation typically have selectable markers that allow selection and maintenance of transformed cells. In some cases, origins of replication can be used to amplify the copy number of a vector within a cell.

The TNFR1 antagonists, TNFR2 agonists, and bispecific TNFR1 antagonist/TNFR2 agonist constructs herein can also be expressed as protein fusions. For example, fusion proteins can be generated to add additional functionality to a polypeptide. Examples of fusion proteins include signal sequences, e.g. tags for localization such as his6 tags or myc tags, or tags for purification such as GST fusions, and for inducing protein secretion and/or membrane association. These include, but are not limited to, fusions of sequences. Fusion proteins also include fusions with an IgG Fc region and a linker, such as an IgG hinge sequence, and/or a glycine-serine (GS) peptide linker. Alternatively, in some embodiments, the TNFR1 antagonists, TNFR2 agonists, and bispecific TNFR1 antagonist/TNFR2 agonist constructs herein can also be fused to serum albumin.

Stable expression is desired for long-term, high-yield production of recombinant proteins. For example, a cell line that stably expresses a polypeptide can be transformed using a viral origin of replication or an expression vector containing endogenous expression elements and a selectable marker gene. After introduction of the vector, cells can be grown for 1-2 days in enriched media before switching to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant cells of stably transformed cells can be expanded using tissue culture techniques appropriate to the cell type.

Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus (HSV) thymidine kinase (TK) (e.g., Wigler et al., (1977) Cell 11:223-232) gene and the adenine phosphoribosyltransferase (APRT) (e.g., Lowy , I. et al. (1980) Cell, 22:817-23), which can be used in TK cells or APRT cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection. For example, dihydrofolate reductase (DHFR), which confers resistance to methotrexate (see, e.g., Wigler et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77:3567-70); the aminoglycosides neomycin and G -418 (see, e.g., Colbere-Garapin et al. (1981) J. Mol. Biol. 150:1-14); and to chlorsulfuron and phosphinothricin acetyltransferases, respectively. als or pat can be used to confer resistance. Additional selectable genes have been described, such as trpB, which allows cells to utilize indole instead of typtophan, or hisD, which allows cells to utilize histinol instead of histidine (e.g. See Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8047-8051). Visible markers such as, but not limited to, anthocyanins, beta-glucuronidase and its substrate, GUS, and luciferase and its substrate luciferin can also be used to identify transformants, as well as transient or stable proteins due to the particular vector system. It can also be used to quantify the amount of expression (see, eg, Rhodes et al. (1995) Methods Mol. Biol. 55:121-131).

a. Prokaryotes Prokaryotes, especially E. coli, provide a system for producing large amounts of proteins. Prokaryotic expression systems are commonly used to produce non-glycosylated products. Transformation of E. coli protocols are well known to those skilled in the art. E. coli expression vectors can include inducible promoters; such promoters are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cell. be. Examples of inducible promoters include, for example, the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters, and the temperature-regulated λPL promoter.

The polypeptide and fusion protein constructs provided herein, such as any provided herein, can be expressed in the cytoplasmic environment of E. coli. The cytoplasm is a reducing environment, and for some molecules this can lead to the formation of insoluble inclusion bodies. Proteins can be resolubilized using reducing agents such as dithiothreitol and β-mercaptoethanol, and denaturing agents such as guanidine-HCl and urea. An alternative approach is the expression of proteins in the bacterial periplasmic space, which provides an oxidizing environment and chaperonin-like and disulfide isomerases and can result in the production of soluble proteins. Typically, a leader sequence is fused to the protein to be expressed to direct the protein to the periplasm. The leader is then removed by signal peptidases in the periplasm. Exemplary pathways for translocating expressed proteins to the periplasm are the Sec pathway, the SRP pathway, and the TAT pathway. Examples of leader sequences targeted to the periplasm include the pelB, StII, and DsbA leader sequences from the pectate lyase gene, and the leader from the alkaline phosphatase gene. In some cases, periplasmic expression may result in leakage of the expressed protein into the culture medium. Secretion of the protein allows for quick and easy purification from culture supernatants. Non-secreted proteins can be obtained from the periplasm by osmotic lysis. As with cytoplasmic expression, in some cases the protein may become insoluble and denaturing and reducing agents can be used to facilitate solubilization and refolding. The temperature of induction and growth can also affect expression levels and solubility; typically temperatures between 25°C and 37°C are used. Typically, bacteria produce aglycosylated proteins. Thus, if a protein requires glycosylation for function, glycosylation can be added in vitro after purification from the host cell.

b. Yeast cells Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolitica, Kluyveromyces lactis Yveromyces lactis), and yeasts such as Pichia pastoris is a well-known expression host that can be used for the production of proteins such as any described herein. Yeast can be transformed by stable chromosomal integration by episomal replicating vectors or homologous recombination. Inducible promoters are typically used to control gene expression. Examples of such promoters include GAL1, GAL7, and GAL5, and metallothionein promoters such as CUP1, AOX1 or other Pichia or other yeast promoters. Expression vectors often include selectable markers such as LEU2, TRP1, HIS3, and URA3 for selection and maintenance of transformed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bip and protein disulfide isomerase can improve expression levels and solubility. Additionally, proteins expressed in yeast include secretion signal peptide fusions, such as the yeast mating-type alpha factor secretion signal from Saccharomyces cerevisiae, and secretion signal peptide fusions, such as the Aga2p mating adhesion receptor or Arxula adeninivorans glucoamylase. fusions with yeast cell surface proteins can be used to induce secretion. Protease cleavage sites, such as Kex-2 protease, can be engineered to remove fusion sequences from expressed polypeptides as they exit the secretory pathway. Yeast can also glycosylate with Asn-X-Ser/Thr motifs.

c. Insects and Insect Cells Insect cells, particularly using baculovirus expression, are useful for the expression of polypeptides, including antibodies or fragments thereof. Insect cells express high levels of proteins and are capable of most of the post-translational modifications used in higher eukaryotes. Baculoviruses have a restricted host range that improves safety and alleviates control concerns for eukaryotic expression. Typically, expression vectors use promoters for high level expression such as the baculovirus polyhedrin promoter and the p10 promoter. Baculovirus systems include baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV), as well as Spodoptera frugiperda. Insect cell lines such as Sf9 from Trichoplusia ni, TN from Trichoplusia ni, A7S from Pseudaletia unipuncta, and DpN1 from Danaus plexippus. For this purpose, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin start codon of the virus. To generate baculovirus recombinants capable of expressing human antibodies, double Dual-expression transfer can be used. Mammalian secretion signals are precisely processed in insect cells and can be used to secrete the expressed protein into the culture medium. Pseudaletia unipunkta (A7S) and Danaus plexippus (DpN1) produce proteins with glycosylation patterns similar to mammalian cell lines. ” modified to reduce immunogenicity, including those with baculovirus expression vectors and those lacking the enzyme FT3.

An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as Schneider 2 (S2) and Kc cells (Drosophila melanogaster) and C7 cells (Aedes albopictus) can be used for expression. The Drosophila metallothionein promoter can be used to induce high levels of expression in the presence of heavy metal induction using cadmium or copper. The baculovirus immediate early gene promoter IE1 can be used to induce uniform levels of expression. Typical expression vectors include pIE1-3 and pI31-4 transfer vectors (Novagen). Expression vectors are typically maintained through the use of selectable markers such as neomycin and hygromycin.

d. Mammalian Expression Cells Mammalian expression systems are used to produce the constructs herein, including the TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusions thereof provided herein. A polypeptide containing a polypeptide can be expressed. Expression constructs are introduced into mammalian cells by viral infection, such as with adenovirus, or by direct DNA transfer, such as by using liposomes, calcium phosphate, and DEAE-dextran, and by physical means, such as electroporation and microinjection. can be introduced. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translation initiation sequence (Kozak consensus sequence), and a polyadenylation element. Internal ribosome entry site (IRES) elements can also be added to allow bicistronic expression with other genes, such as selectable markers. Such vectors often include transcriptional promoter enhancers for high level expression, such as the SV40 promoter enhancer, the human cytomegalovirus (CMV) promoter, and the long terminal repeats of Rous sarcoma virus (RSV). It will be done. These promoter-enhancers are active in many cell types. Tissue and cell type promoters and enhancer regions can also be used for expression. Exemplary promoter/enhancer regions include elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, myosin light chain 2 and gonadotropin releasing hormone genes. Examples include, but are not limited to, those derived from genes such as regulatory genes.

Selectable markers can be used to select and maintain cells containing the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyltransferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select only cells that express the DHFR gene. Modified anti-TNFR antibodies and antigen-binding fragments thereof can be produced using, for example, the NEOR/G418 system, the dihydrofolate reductase (DHFR) system, or the glutamine synthetase (GS) system. In the GS system, co-expression vectors such as pEE12/pEE6 are used to express both heavy and light chains. Fusion with cell surface signaling molecules such as TCR-ζ and FcεRI-γ can induce expression of the protein in its active state at the cell surface.

Many cell lines are available for mammalian expression, including mouse, rat, human, monkey, chicken, and hamster cells. Exemplary cell lines include BHK (e.g., BHK-21 cells), 293-F, CHO, CHO Express (CHOX; ExcelGene), Balb/3T3, HeLa, MT2, mouse NS0 (non-secreting), and other These include, but are not limited to, myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines are also available that are adapted to serum-free media, which facilitates the purification of secreted proteins from the cell culture media. Examples include CHO-S cells (Invitrogen®, Carlsbad, CA, catalog number 11619-012) and serum-free EBNA-1 cell line (Pham et al. (2003) Biotechnol. Bioeng. 84:332- 342). Cell lines adapted for growth in specific media optimized for maximum expression are also available. For example, DG44 CHO cells are adapted to grow in suspension culture in chemically defined animal product-free media.

e. Plants Transgenic plant cells and plants can be used to express polypeptides and proteins such as any described herein. Expression constructs are commonly introduced into plants using direct DNA transfer, such as biolistic and PEG-mediated transfer into protoplasts, and by Agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcription termination elements and translation control elements. Expression vectors and transformation techniques are commonly divided between dicot hosts such as Arabidopsis and tobacco and monocot hosts such as maize and rice. Examples of plant promoters used for expression include, for example, the cauliflower mosaic virus promoter (CaMV 35S), the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter, and the ubiquitin (e.g., maize ubiquitin-1 (ubi-1)) and An example is the UBQ3 promoter. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue), or can be regenerated into whole plants. Transgenic plant cells also include algae that have been engineered to produce polypeptides. Since plants have different glycosylation patterns than mammalian cells, this may influence the selection of proteins produced in these hosts.

5. Purification Host cells transformed with nucleic acids encoding polypeptide constructs provided herein can be cultured under conditions suitable for expression and recovery of the encoded proteins from cell culture. Proteins produced by recombinant cells are generally secreted, but depending on the sequence and/or vector used, they can be contained intracellularly. As will be understood by those skilled in the art, expression vectors containing nucleic acid molecules encoding polypeptides provided herein may contain signal sequences that facilitate direct secretion of the expressed polypeptide through prokaryotic or eukaryotic cell membranes. can be designed using.

The method of purifying polypeptides from host cells depends on the host cell and expression system chosen. For secreted molecules, the protein is generally purified from the culture medium after removing the cells. For intracellular expression, cells can be lysed and proteins purified from the extract. When transgenic organisms, such as transgenic plants and animals, are used for expression, tissues or organs can be used as starting materials to generate lysed cell extracts. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, and the polypeptides can be collected and, if necessary, protein extracted and processed using standard methods in the art. can be used for further purification.

Polypeptides, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, TNFR1 antagonist/TNFR2 agonist bispecific constructs, and other constructs provided herein, and components thereof, can be prepared using protein purification techniques known to those of skill in the art. It can be used and purified. These include SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, column chromatography, HPLC, dialysis, ion exchange chromatography such as anion exchange, and combinations thereof. , but not limited to. Affinity purification techniques can also be used. The constructs herein can be purified using methods developed for the purification of antibodies and antibody fragments. An example of a method for purifying antibodies and antibody fragments is a method involving column chromatography, where the solid support column material contains Protein G, a cell surface binding protein from Streptococcus that binds with high affinity to immunoglobulins. connected to. Antibodies and antibody fragments can also be purified by methods that include Protein A chromatography; in this method cell surface-bound proteins from Staphylococcus aureus that bind with high affinity to immunoglobulins such as IgG The protein Protein A is bound to a solid support column. Other immunoglobulin-binding bacterial proteins that can be used to purify antibodies and antibody fragments include Protein A/G, a recombinant fusion protein that combines the IgG binding domains of Protein A and Protein G; contains protein L, a surface protein of 12825; see Eliasson et al. (1988) J. Biol. Chem. 263:4323-4327). The construct is substantially pure, typically at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure. . Purity can be assessed by standard methods such as SDS-PAGE and Coomassie Blue staining.

When antibodies and fragments thereof and related polypeptides are expressed in large quantities by transformed bacteria, typically after promoter induction, expression may be constitutive, but the polypeptides may form insoluble aggregates. Protocols for the purification of polypeptide inclusion bodies are known to those skilled in the art. For example, in one method, a cell suspension is centrifuged to pellet inclusion bodies, and the inclusion body-containing pellet is treated with, for example, 20mM Tris-HCL (pH 7.2), 1mM EDTA, 150mM NaCl, and non-ionized Resuspend in a buffer that dissolves contaminants without damaging the inclusion bodies, such as 2% Triton-X 100, a surfactant. The washing steps can be repeated to remove as much cell debris as possible. The remaining pellet of inclusion bodies is resuspended in a suitable buffer such as 20mM sodium phosphate, pH 6.8, 150mM NaCl. Other suitable buffers and alternative purification protocols are known or can be developed by those skilled in the art.

Other methods can be used or developed to purify antibodies and fragments thereof, as well as the constructs provided herein. They can be purified from the bacterial periplasm. For polypeptides that are transported to the periplasm of the bacteria in which they are produced, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock, in addition to other methods known to those skilled in the art. For example, in some methods, bacterial cells are centrifuged to form a pellet to isolate recombinant polypeptides from the periplasm. The pellet is resuspended in buffer containing 20% sucrose. To lyse the cells, centrifuge the bacteria and resuspend the pellet in ice-cold 5mM MgSO4 and keep in an ice bath for approximately 10 minutes. Centrifuge the cell suspension, decant and save the supernatant. Recombinant polypeptides present in the supernatant can be separated from host proteins by standard separation techniques well known to those skilled in the art. These methods include, but are not limited to, the following steps: solubility fractionation, size difference filtration, and column chromatography.

The expression construct can also be engineered to include a nucleic acid encoding an affinity tag, for example, for detection or purification of the expression product operably linked to the encoded polypeptide upon expression. Affinity tags include, for example, small ubiquitin-like modifiers (SUMO), myc antibodies, SUMO tags, myc epitopes, GST fusions, or His6 for affinity purification with glutathione and Ni resins. Nucleic acids encoding such tags can be linked to nucleic acids encoding polypeptide constructs provided herein. Tags can facilitate purification and/or detection of soluble proteins. For example, a TNFR1 antagonist polypeptide construct or portion thereof can be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Purification-enhancing domains include a histidine-tryptophan module that allows purification on immobilized metals, a protein A domain that allows purification on immobilized immunoglobulin, and the FLAGS extension/affinity purification system (Immunex Corp., Seattle, USA). Examples include, but are not limited to, metal chelating peptides such as those utilized in WA). Between the purification domain and the encoded expressed polypeptide there is a factor Purification is facilitated by the inclusion of a nucleic acid encoding a cleavable linker sequence, such as (Invitrogen (Thermo Fisher Scientific), San Diego, Calif.). Exemplary expression vectors encode the expression of fusion proteins containing TNFR1 antagonist and/or TNFR2 agonist polypeptides and enterokinase cleavage sites. The small ubiquitin-like modifier (SUMO) tag facilitates immobilized metal ion affinity chromatography (IMIAC) purification, and the enterokinase cleavage site provides a cleavage site for purifying the polypeptide from the fusion protein. .

Purity can be assessed by any method known in the art, including gel electrophoresis, orthogonal HPLC methods, staining and spectrophotometric techniques. Expressed and purified polypeptides can be analyzed using any assay or method known to those skilled in the art, such as any described herein. These include assays based on the physical and/or functional properties of the polypeptide, including analysis by gel electrophoresis, immunoassays, binding assays, and assays of TNF-mediated TNFR1 and/or TNFR2 activity, including but not limited to.

6. Additional Modifications The modified TNFR1 antagonist constructs, TNFR2 agonist constructs, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and other constructs provided as described above, and their components, have pharmacokinetic and pharmacodynamic properties. Contains components (activity modifiers) that modify pharmacological properties, including clinical properties. Portions of the constructs, such as TNFR1 inhibitor polypeptides and small molecules and TNFR2 agonist polypeptides and small molecules, may be polymers or polymeric moieties, such as, but not limited to, polyethylene glycol (PEG) moieties or dextran, or human serum albumin ( HSA) or sialylated to reduce immunogenicity and/or increase half-life in serum and other body fluids. The polypeptide can also be linked to a purification tag, such as a His tag or a SUMO sequence. Additional modifications include, for example, glycosylation, carboxylation, hydroxylation, phosphorylation, or other known modifications. Glycosylation can be performed in vivo, in vitro using a suitable expression system, such as a mammalian expression system, or, for example, the polypeptide can be expressed in prokaryotic cells and further modified in vitro using enzymatic transglycosylation. can be incorporated through a combination of in vivo and in vitro methods. Other modifications can be made in vitro or, for example, by producing modified polypeptides in a suitable host that produces such modifications.

These modifications or activity modifiers and modifications are such that the modified polypeptide incorporates the functionality of the modification, at least in part, generally at least 50%, 60%, 70%, 80% compared to the unfused or unmodified polypeptide. %, 90%, or 95% activity, e.g., 96%, 97%, 98%, 99% or more activity compared to an active unfused or unmodified polypeptide; selected. For example, a TNFR1 antagonist construct retains the ability to inhibit TNF-mediated signaling through TNFR1.

Linking a polypeptide, such as a TNFR1 antagonist or TNFR2 agonist, to another polypeptide can be done directly or indirectly via a linker. In one example, the linkage can be a chemical linkage, such as through a heterobifunctional agent, or a thiol linkage, or other such linkage. Fusions can also be achieved by recombinant expression. The fusion of a polypeptide, such as a TNFR1 antagonist or TNFR2 agonist, with another polypeptide can be to the N-terminus or C-terminus of the TNFR1 antagonist or TNFR2 agonist polypeptide. Non-limiting examples of polypeptides that can be used in fusion proteins with TNFR1 antagonist or TNFR2 agonist polypeptides provided herein include, for example, GST (glutathione S-transferase) polypeptide, immunoglobulin G derived Fc domains, albumin, heterologous signal sequences, and combinations thereof.

Encoding constructs can be produced by standard recombinant techniques. For example, DNA fragments encoding different polypeptide moieties can be prepared according to conventional techniques, e.g., with blunt or cohesive end termination for ligation, restriction enzyme digestion to provide suitable ends, and cohesive ends if necessary. In-frame ligation can be achieved by using filling-in, alkaline phosphatase treatment to avoid undesired binding, and enzymatic ligation. Fusion genes can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that create complementary overhangs between two consecutive gene fragments, followed by annealing and reamplification to generate chimeric genes. be able to. Many expression vectors are commercially available that already encode fusion moieties (eg, GST polypeptides). The polypeptide-encoding nucleic acid can be linked in-frame to a multispecificity construct, such as a TNFR1 antagonist, TNFR2 agonist, and bispecific construct provided herein, such that the fusion moiety is linked in-frame to such an expression vector. can be cloned into.

a. PEGylation Polyethylene glycol (PEG) is used in biomaterials, biotechnology, and medicine; it is a water-soluble polymer that is biocompatible, nontoxic, and generally nonimmunogenic. . In the field of drug delivery, PEG derivatives are used for covalent attachment to proteins (i.e., "PEGylation") to reduce immunogenicity, proteolysis, and renal clearance, increase serum half-life, and increase solubility. (see, eg, Zalipsky (1995) Adv. Drug Del. Rev. 16:157-182). PEG attaches to low molecular weight, relatively hydrophobic drugs to increase solubility, reduce toxicity, and alter biodistribution. Conjugation to linear or branched PEG moieties increases the molecular weight and hydrodynamic radius of the polypeptide and reduces the rate of glomerular filtration by the kidneys. Typically, PEGylated drugs are administered as a solution, such as by injection. In the constructs herein, PEGylated moieties and other such polymers can be part of the linker portion of the construct.

Related applications include cross-linked degradable PEG networks or drug delivery, as much of the same chemistry used to design degradable and soluble drug carriers can also be used to design degradable gels. (See, eg, Sawhney et al. (1993) Macromolecules 26:581-587). Polymer-polymer complexes can be formed by mixing solutions of two complementary polymers. Such complexes are formed by electrostatic interactions (polyanion-polycation) and/or hydrogen bonding (polyacid-polybase) between the polymers involved and/or by hydrophobic interactions between the polymers in the medium. Stabilize (see, eg, Krupers et al. (1996) Eur. Polym. J. 32:785-790). For example, when solutions of polyacrylic acid (PAAc) and polyethylene oxide (PEO) are mixed under appropriate conditions, a complex is formed that is primarily based on hydrogen bonding. Dissociation of these complexes under physiological conditions has been used to deliver free (ie, non-PEGylated) drugs. Complementary polymer complexes are formed from homopolymers and copolymers.

As well as PEGylating therapeutic proteins, numerous reagents for PEGylation are known. Such reagents include N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl α-methylbutanoate, mPEG succinimidyl propionate, mPEG Succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde , PEG maleimide, PEG hydrazine, p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEG butryaldehyde, branched mPEG2 butryaldehyde, mPEG acetyl, mPEG piperidone, mPEG methyl ketone, mPE G “No linker ” Maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridyl thioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS and biotin PEG -NHS (see Veronese et al. (1997) J. Bioactive Compatible Polymers 12:197-207; and numerous US and world patents). In one example, polyethylene glycol has a molecular weight ranging from about 3 kDa to about 50 kDa, typically from about 5 kDa to about 30 kDa. Covalent attachment of PEG to drugs (known as "PEGylation") can be accomplished by known chemical synthesis techniques. For example, PEGylation of a protein can be accomplished by reacting NHS-activated PEG with the protein under suitable reaction conditions.

Although numerous reactions have been described for PEGylation, those most commonly applicable to proteins are those that provide direction, use mild reaction conditions, and remove toxic catalysts or byproducts. Does not require extensive downstream processing. For example, monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl and thus its use limits some of the heterogeneity of the resulting PEG-protein product mixture. Activation of the hydroxyl group at the end of the polymer opposite the terminal methoxy group is generally necessary to achieve efficient protein PEGylation, with the aim of making the derivatized PEG more susceptible to nucleophilic attack. It is. The aggressive nucleophile is usually the epsilon-amino group of the lysyl residue, but other amines may also react if local conditions are favorable (e.g., the N-terminal alpha amine or the histidine ring amine). More direct binding is possible with proteins containing a single lysine or cysteine. The latter residue can be targeted with PEG-maleimide for thiol-specific modification. Alternatively, PEG hydrazide can be reacted with periodate oxidized protein and reduced in the presence of NaCNBH3. More specifically, PEGylated CMP sugars can be reacted with proteins in the presence of an appropriate glycosyltransferase. One technique is the "PEGylation" technique, in which multiple polymer molecules are coupled to the polypeptide of interest. When using this technique, it becomes difficult for the immune system to recognize epitopes on the surface of the polypeptide that are involved in the formation of antibodies, thereby reducing the immune response. For polypeptides (i.e., drugs) that are introduced directly into the human body's circulatory system to confer a specific physiological effect, typical potential immune responses are IgG and/or IgM responses, but respiratory Polypeptides that are inhaled by the respiratory system (ie, industrial polypeptides) can potentially cause an IgE response (ie, an allergic response). One theory explaining the reduced immune response is that the polymer molecule(s) mask epitope(s) on the surface of the polypeptide that are involved in the immune response leading to antibody formation. Another theory, or at least a partial factor, is that the heavier the conjugate, the lower the immune response.

For example, in the PEGylated polypeptide constructs and polypeptide components provided herein, the PEG moiety is conjugated to the polypeptide via a covalent bond. Techniques for PEGylation include, but are not limited to, the use of specialized linkers and coupling chemistries (see, e.g., Harris (2002) Adv. Drug Deliv. Rev. 54:459-476), single conjugate Attachment of multiple PEG moieties to cation sites (such as by the use of branched PEGs; see e.g. Veronese et al., (2002) Bioorg. Med. Chem. Lett. 12:177-180), site-specific PEGylation and/or mono-PEGylation (see e.g. Chapman et al. (1999) Nature Biotech. 17:780-783), and site-specific enzymatic PEGylation (e.g. Sato (2002) Adv. Drug (See Deliv. Rev. 54:487-504). 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 PEG or PEG derivatives attached to a single protein molecule by methods and techniques described in the art. (see, eg, US Patent Application Publication No. 2006/0104968).

b. Albuminization Polypeptides provided herein, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific TNFR1 antagonist/TNFR2 agonist constructs, are converted into albumin (i.e., "albuminated") for human therapy. Fused to human serum albumin (HSA) to increase the half-life, stability, bioavailability, and distribution of the polypeptide and/or improve pharmacological properties such as pharmacokinetics of the polypeptide. A number of products related to human serum albumin (HSA) have been approved for use as therapeutic agents, including as a cancer treatment and for the treatment of type 2 diabetes (e.g., AlQahtani et al. (2019) Biomed and Pharmacotherapy 113:108750; Roscoe et al. (2018) Mol. Pharmaceutics 151:15046-5047; Strohl, WR (2015) BioDrugs 4:215-239). In some examples, a mature HSA protein lacking signal and activation sequences is fused to a protein of interest. In some examples, serum albumin, such as human serum albumin (HSA), is conjugated to the polypeptide. An exemplary HSA protein is set forth in SEQ ID NO:35.

Provided herein are fusions with HSA. These generally include TNFR1 via short peptide linkers such as, but not limited to, glycine-serine (GS) linkers, such as (GSGS)n or (GGGGS)n, where n=1-5 or 6. Included are fusions of antagonists (eg, dAbs, scFvs, Fabs or other antigen-binding fragments provided herein) or TNFR2 agonists (eg, TNF muteins) to the N-terminus or C-terminus with HSA. Exemplary TNFR1 antagonist-HSA fusions are set forth in SEQ ID NOs: 709, 713, 717, 721, and 725.

c. Purification Tags In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins provided herein can be used to facilitate product purification. can contain tags for Exemplary tags for purification are described elsewhere herein. For example, exemplary polypeptides herein can contain SUMO or His sequences for purification. Generally, the tag is a cuttable tag.

Polypeptide constructs provided herein, including fusion proteins, can include a His purification tag, such as a 6xHis tag. The His-tagged polypeptide can optionally contain fusion partners and/or signals for expression and secretion. For example, exemplary His-polypeptide fusion proteins include a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837). , and HSA (SEQ ID NO: 35). In another example, an exemplary His-tagged polypeptide fusion protein includes a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence (sequence 837), and IgGFc (see, eg, SEQ ID NO: 10, 12, 14, 16, 27, and 30).

In some embodiments, polypeptides and fusion proteins provided herein can include a His tag and/or a SUMO sequence for accumulation in inclusion bodies. For example, the His-SUMO sequence set forth in SEQ ID NO: 838 can be linked to any of the polypeptides or fusion proteins provided herein. The His-SUMO tagged polypeptide can optionally contain fusion partners and/or signals for expression and secretion. For example, a His-SUMO-polypeptide fusion protein includes a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837), and HSA (SEQ ID NO: 35). In another embodiment, an exemplary His-SUMO-polypeptide fusion protein comprises a human immunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 835), a 6xHis tag (SEQ ID NO: 836), a SUMO sequence ( SEQ ID NO: 837), and IgGFc (see, eg, SEQ ID NO: 10, 12, 14, 16, 27, and 30).

7. Nucleic Acid Molecules and Gene Therapy Nucleic acid molecules encoding the fusion protein polypeptide constructs provided herein can be used for gene therapy, such as expression in gene therapy vectors or administration as DNA or RNA constructs. can. Among these, some of the TNFR1 antagonists, TNFR2 agonists, and bispecific TNFR1 antagonist/TNFR2 agonist constructs provided herein are provided as fusion proteins. Nucleic acid molecules encoding these constructs, as well as vectors and other delivery vehicles, are provided herein. The nucleic acid molecule is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% relative to any polypeptide or construct provided herein. , 98%, or 99% sequence identity. In another embodiment, a nucleic acid molecule can include one having a degenerate codon sequence encoding any of the polypeptides or constructs provided herein.

Nucleic acid molecules used in gene therapy are optionally operably linked to nucleic acid control sequences. These include promoters, enhancers, signal sequences and other transport sequences, as well as other such control sequences well known to those skilled in the art. For vectors with specific tissue tropism, the control sequences may be specific to such tissues, such as the liver for long-term gene expression, or the eye for any ophthalmological use. Exemplary promoters include inducible and constitutive promoters for expression in mammalian cells. Such promoters are those recognized in eukaryotes, such as mammals, and include adenoviral promoters such as the CMV and SV40 promoters, the E2 gene promoter that responds to the E7 oncoprotein; the PV promoter that responds to the E2 protein; PV promoters, such as the BPV p89 promoter; and other suitable promoters.

Polypeptides provided herein can also be delivered to cells in gene transfer vectors. The introduction vector may also be used to treat the disease or disorder for which the polypeptide is administered, such as rheumatoid arthritis, or any other chronic inflammatory, autoimmune, neurodegenerative, or can encode additional other therapeutic agents for the treatment of demyelinating diseases or disorders. Introduction vectors encoding polypeptides provided herein can be used systemically by administering the nucleic acid molecule to a subject. For example, the introduction vector can be a viral vector, such as an adenovirus vector. Vectors encoding polypeptides or constructs herein can also be incorporated into stem cells, and such stem cells can be administered to a subject, such as by transplanting or engrafting the stem cells at the treatment site. For example, mesenchymal stem cells (MSCs) can be engineered to express therapeutic polypeptides, and such MSCs can be engrafted at a transplant site for therapy.

Instead of delivering the protein, the nucleic acid can be delivered in vivo, e.g. systemically, or by any other route, or ex vivo, e.g. by removing cells, including lymphocytes, introducing the nucleic acid into the cell, and the host, Alternatively, it can be administered by reintroduction to a compatible recipient.

Polypeptides can be delivered to cells and tissues by expression of nucleic acid molecules. Polypeptides can be administered as nucleic acid molecules encoding the polypeptides, such as by ex vivo techniques and direct in vivo expression. Nucleic acids can be delivered to cells and tissues by any method known to those of skill in the art. The isolated nucleic acid sequences can be incorporated into vectors for further manipulation. Methods of administering polypeptides by expression of encoding nucleic acid molecules include administration of recombinant vectors. Vectors can be designed to remain episomal, such as by containing an origin of replication, or can be designed to integrate into a chromosome within a cell. Nucleic acid molecules encoding polypeptides provided herein can also be used in ex vivo gene expression therapy using non-viral vectors. For example, a cell can be engineered to express a polypeptide operably linked to a control sequence, or a polypeptide can be engineered to place the polypeptide operably linked to a control sequence at a genomic location. . Such cells can then be administered locally or systemically to a subject, such as a patient, in need of treatment.

Gene therapy vectors can remain episomal or can integrate into the chromosome of the treated subject. The polypeptide can be expressed by a virus that is administered to a subject in need of treatment. Viral vectors suitable for gene therapy include adenoviruses, adeno-associated viruses (AAV), retroviruses, lentiviruses, vaccinia viruses, and other viruses mentioned above. For example, viral vectors can be used, including adenoviruses, adeno-associated viruses (AAV), poxviruses, herpesviruses, retroviruses, and others designed for gene therapy. AAV vectors with altered tropism such as hepatocytes are available. AAV vectors consist of a capsid that confers tropism and a nucleic acid encoding a polypeptide flanked by ITRs.

For example, adenovirus expression technology is well known in the art, as are methods for producing and administering adenoviruses. Adenovirus serotypes are available, for example, from the American Type Culture Collection (ATCC, Rockville, MD). Adenoviral vectors can be used ex vivo, eg, cells are isolated from a patient in need of treatment and the cells are transduced with a polypeptide-expressing adenoviral vector. After a suitable culture period, the transduced cells are administered to the subject locally and/or systemically. Alternatively, the polypeptide adenoviral particles are isolated and placed in a pharmaceutically acceptable carrier for delivery of a therapeutically effective amount to prevent, treat, or ameliorate a disease or condition in a subject. Formulate. Typically, adenoviral particles are delivered at doses ranging from 1 particle to 10 14 particles per kilogram of subject weight, generally from 10 6 or 10 8 particles to 10 12 particles per kilogram of subject weight. In some cases, it is desirable to provide the nucleic acid source with a cell-targeting agent, such as an antibody specific for a cell surface membrane protein or target cell, or a ligand for a receptor on the target cell. Polypeptides or constructs provided herein can also be targeted for delivery to specific cell types. For example, adenoviral vectors encoding polypeptides or constructs provided herein can be used for stable expression in nondividing cells such as hepatocytes (e.g., Margaritis et al. (2004)). J. Clin. Invest. 113:1025-1031). In another example, a viral or non-viral vector encoding a polypeptide or construct herein can be transduced into isolated cells for subsequent delivery. Additional cell types for expression and delivery include, but are not limited to, fibroblasts and endothelial cells.

Nucleic acid molecules can be introduced into artificial chromosomes and other non-viral vectors. Artificial chromosomes such as ACES (see Lindenbaum et al., (2004) Nucleic Acids Res. 32(21):e172) can be engineered to encode and express isoforms. Briefly, mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into cells in an autonomously replicating, non-integrated format. Unique among MACs, mammalian satellite DNA-based artificial chromosome expression (ACE) can be produced de novo in a reproducible manner in cell lines of various species and is easily purified from host cell chromosomes. can do. The purified mammalian ACE can then be reintroduced into various recipient cell lines, where it is maintained stably for long periods of time in the absence of selective pressure using the ACE system. Using this approach, specific loading of one or two gene targets was achieved in LMTK(-) and CHO cells.

Another method for introducing nucleic acids encoding polypeptides is the complete adenoviral genome (Ad2; Ketner et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91: 6186-6190) and a two-step genetic assay in yeast starting from a plasmid containing adenoviral sequences targeting specific regions of the YAC clone, an expression cassette for the gene of interest, and positive and negative selection markers. It is a replacement technology. YACs are of particular interest because they allow for the integration of larger genes. This approach can be used to construct adenovirus-based vectors carrying nucleic acids encoding any of the polypeptides or constructs described herein for gene transfer into mammalian cells or whole animals. can.

Nucleic acids can be encapsulated in vehicles such as liposomes, or introduced into cells such as bacterial cells, especially attenuated bacteria, or introduced into viral vectors. For example, when using liposomes, proteins that bind to cell surface membrane proteins involved in endocytosis can be used, e.g., capsid proteins or fragments thereof, which are directed against a particular cell type, in a cycle within a cell. Antibodies to proteins that undergo translocation and target intracellular localization and prolong intracellular half-life can facilitate targeting and/or uptake of proteins.

In ex vivo and in vivo methods, nucleic acid molecules encoding polypeptides or constructs herein are introduced into cells from a suitable donor or subject to be treated. Cells into which nucleic acids can be introduced for therapeutic purposes include, for example, any desired available cell type appropriate to the disease or condition being treated, including, but not limited to, epithelial cells; endothelial cells; keratinocytes; fibroblasts; myocytes; hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; and various stem or progenitor cells, especially hematopoietic cells. Included are stem cells or progenitor cells, such as stem cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and other sources thereof.

In ex vivo treatment, cells from a donor that is compatible with the subject being treated or cells from the subject being treated are removed, nucleic acids are introduced into these isolated cells, and the modified cells are administered to the subject. Treatment includes direct administration, e.g., encapsulated within a porous membrane that is implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). , each of which is incorporated herein by reference in its entirety). Suitable techniques for introducing nucleic acids into mammalian cells in vitro include the use of liposomes and cationic lipids (e.g., DOTMA, DOPE, DC-Chol), electroporation, microinjection, cell fusion, DEAE-dextran. , and calcium phosphate precipitation methods. Methods of DNA delivery can be used to express polypeptides or constructs provided herein in vivo. Such methods include liposomal and naked DNA delivery of nucleic acids, including local and systemic delivery, such as using electroporation, ultrasound, and calcium phosphate delivery. Other techniques include microinjection, cell fusion, chromosome-mediated gene transfer, micronucleated cell-mediated gene transfer, and spheroplast fusion.

In vivo expression of the polypeptides or constructs herein can be linked to the expression of additional molecules. For example, expression of the polypeptide can be associated with the expression of a cytotoxic product, such as in a genetically engineered virus, or can be expressed in a cytotoxic virus. Such viruses can target specific cell types for therapeutic efficacy.

In vivo expression of a polypeptide or construct provided herein can involve operably linking a polypeptide-encoding nucleic acid molecule to specific control sequences, such as a cell-specific or tissue-specific promoter. Polypeptides can also be expressed from vectors that specifically infect and/or replicate in target cell types and/or tissues. Inducible promoters can be used to selectively control expression of a polypeptide or construct.

Nucleic acid molecules such as naked nucleic acids, or vectors, artificial chromosomes, liposomes and other vehicles can be administered to a subject by systemic, topical, local and other routes of administration. Systemically and in vivo, nucleic acid molecules or vehicles containing nucleic acid molecules can be targeted to cells. Administration includes intravenous administration and direct injection into tissues such as direct injection into the liver, including methods of direct injection into compartmentalized organs such as the liver or parts thereof (e.g., U.S. Pat. No. 9,821,114).

Administration can also be direct, such as by administration of vectors or cells, typically targeted to cells or tissues. Cells used for in vivo expression of polypeptides or constructs herein also include cells that are autologous to the patient. Such cells can be removed from a patient, introduced with a nucleic acid for expression of the polypeptide, and then administered to the patient, such as by injection or transplantation.

J. Compositions, Formulation and Dosage Polypeptides and constructs provided herein, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific TNFR1, such as bispecific, in a pharmaceutically acceptable vehicle. Pharmaceutical compositions are provided that include either an antagonist/TNFR2 agonist construct, such as a fusion protein, or a nucleic acid molecule encoding a polypeptide or construct. Such compositions contain an amount of polypeptide, construct, or nucleic acid that can be diluted to a therapeutically effective amount or formulated for direct administration undiluted. The particular concentration of construct or nucleic acid depends on various parameters within the skill of those in the art, including, for example, the indication being treated, the construct or nucleic acid, the route of administration, and the regimen. Routes of administration include systemic and topical, oral, rectal, intravenous, intramuscular, subcutaneous, mucosal, intraperitoneal, and any suitable route known to those skilled in the art.

Selected amounts, such as therapeutically effective amounts, can be used in constructs provided herein, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, such as bispecific, TNFR1 antagonist/constructs, as discussed above. Depending on various parameters, the TNFR2 agonist construct, such as a fusion protein, or a nucleic acid molecule encoding the construct, is formulated in a suitable vehicle for administration. Pharmaceutical compositions can be formulated in any conventional manner by mixing a selected amount of the construct or mixture thereof with one or more physiologically acceptable carriers or excipients or vehicles. Can be done. Pharmaceutical compositions can be used for therapeutic, prophylactic, and/or diagnostic uses. The concentration of active compound, i.e., construct or nucleic acid, in a composition depends on a variety of factors, including those mentioned above, as well as the absorption, inactivation, and excretion rates of the active compound, the dosing schedule, and the amount administered, the subject depending on the age and size of the person, as well as other factors known to those skilled in the art.

Pharmaceutical carriers or vehicles suitable for administering the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. Pharmaceutical compositions containing a therapeutically effective amount of a construct or nucleic acid molecule described herein can also be provided as a lyophilized powder for reconstitution with sterile water, etc. immediately prior to administration.

1. Formulations Pharmaceutical compositions comprising any of the constructs and nucleic acids provided herein are prepared by mixing a selected amount of the active compound with one or more physiologically acceptable carriers or excipients. can be formulated in any conventional manner. The choice of carrier or excipient is within the skill of the administering professional and may depend on a number of parameters. Parameters include, for example, the mode of administration (ie, systemic, oral, nasal, pulmonary, topical, topical, or any other mode) and the disorder being treated. Generally, pharmaceutical compositions include ingredients that do not significantly impair the biological properties of the construct or nucleic acid or encoded polypeptide, or that enhance or improve the pharmacological properties thereof. The formulation may also be in combination with other active agents for combination therapy.

The pharmaceutical compositions provided herein can be in a variety of forms including, but not limited to, solid, semi-solid, liquid, emulsion, powder, aqueous, and lyophilized forms. The pharmaceutical compositions provided herein can be formulated for single dose (direct) administration, or dilution, or other regimens. The concentration of the compound in the formulation, after dilution or admixture with another composition, or for direct administration, is effective to deliver an amount effective for the intended treatment upon administration. The compositions can be formulated for direct administration in single or multiple doses. The compounds can be suspended in micronized or other suitable forms, or can be derivatized to produce more soluble active products. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the chosen carrier or vehicle. The resulting mixtures are solutions, suspensions, emulsions and other such mixtures, non-aqueous or aqueous mixtures, creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes. can be formulated as a foam, aerosol, drench, spray, suppository, bandage, or other formulation suitable for systemic, topical, or topical administration. For local internal administration, such as intramuscular, parenteral, or intraarticular administration, the constructs and nucleic acids can be formulated as a suspension in an aqueous medium, such as isotonic buffered saline, or injected into the body. Combine with a biocompatible support or bioadhesive for administration. The effective concentration is sufficient to improve the target condition and can be determined empirically. To formulate a composition, weight fractions of the compound are dissolved, suspended, dispersed, or mixed in the selected vehicle at an effective concentration such that the target condition is alleviated or ameliorated.

Generally, pharmaceutically acceptable compositions are prepared with consideration for regulatory or other agency approval, and/or in accordance with generally accepted pharmacopoeias for animal and human use. Pharmaceutical compositions can include carriers such as diluents, adjuvants, excipients, or vehicles with which the polypeptide is administered. Such pharmaceutical carriers can be sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition contains, together with the active ingredient, diluents such as lactose, sucrose, dicalcium phosphate, and carboxymethylcellulose; lubricants such as magnesium stearate, calcium stearate, and talc; and binders such as starch, natural Gums such as gum acacia, gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose and its derivatives, povidone, crospovidone, and other binders known to those skilled in the art may be contained. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene glycol. , water, and ethanol. The composition may, if desired, contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and Other such agents may also be included. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, granules, and sustained release formulations. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a mixed powder of the therapeutic compound and a suitable powder base, such as lactose or starch. can. The compound can be formulated as a suppository, with traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and other such agents. Formulations for oral administration may also be suitably formulated with protease inhibitors such as Bowman-Birk inhibitors, conjugated Bowman-Birk inhibitors, aprotinin and camostat. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, generally in purified form, together with a suitable amount of carrier to provide the compound in the form for proper administration to the subject or patient.

The pharmaceutical compositions provided herein may contain other additives, particularly, for example, antioxidants, preservatives, antibacterial agents, analgesics, binders, disintegrants, colorants, diluents, excipients, Bulking agents, glidants, solubilizers, stabilizers, tonicity agents, vehicles, thickeners, flavoring agents, emulsifying agents such as oil-in-water or water-in-oil emulsions, emulsifying agents and suspending agents, such as acacia, agar. , alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, octoxynol-9, oleyl alcohol, povidone, propylene glycol monostearate , sodium lauryl sulfate, sorbitan ester, stearyl alcohol, tragacanth, xanthan gum and its derivatives, solvents and other ingredients, such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, (See generally Alfonso R. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins). Such carriers and/or excipients can be formulated by conventional methods and administered to a subject at a suitable dose. Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can protect the composition from degradation within the body.

The formulation should be compatible with the mode of administration. For example, the active compound can be formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Injectable compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,4-butanediol. . Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, synthetic mono- or diglycerides, fatty acids (including oleic acid), natural vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil, and other oils, or synthetic fatty vehicles such as ethyl oleate, etc. Any bland fixed oil can be used, including but not limited to. Buffers, preservatives, antioxidants, and suitable ingredients can optionally be incorporated or otherwise constitute the formulation.

The active compounds, such as the constructs and nucleic acids provided herein, can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients. Active compounds can be targeted for delivery, such as by conjugation to targeting agents such as antibodies. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared by methods well known to those skilled in the art, such as those described in US Pat. No. 4,522,811. Liposomal delivery can also include sustained release formulations, including pharmaceutical matrices such as collagen gels and liposomes modified with fibronectin (e.g., Weiner et al. (1985) J. Pharm. Sci. 74(9): 922-925). The compositions provided herein can further contain one or more adjuvants to facilitate delivery, such as, but not limited to, inert carriers or colloidal dispersions. Representative, non-limiting examples of such inert carriers include water, isopropyl alcohol, gaseous fluorocarbons, ethyl alcohol, polyvinylpyrrolidone, propylene glycol, gel formers, stearyl alcohol, stearic acid, spermaceti, monoolein. It can be selected from acid sorbitan, methylcellulose, and suitable combinations of two or more thereof.

The active compound is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects in the subject being treated. Therapeutically effective concentrations can be determined empirically by testing compounds in known in vitro and in vivo systems, such as the assays described herein. Determination of a therapeutically effective amount is well within the ability of those skilled in the art. A therapeutically effective dose can be determined using the in vitro and in vivo methods described herein. Thus, the active compounds provided herein or mixtures thereof, when in a pharmaceutical preparation, can be presented in unit dosage form for administration.

2. TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific, constructs, and administration of nucleic acids Constructs provided herein, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and bispecific, etc. The active compounds, including multispecific TNFR1 antagonist/TNFR2 agonist constructs, such as fusion proteins, and nucleic acid molecules encoding the constructs, can be administered by any suitable route. These include by contacting a mixture, such as a body fluid or other tissue sample, with an active compound provided herein in vitro, ex vivo, or in vivo. For example, when administering a compound ex vivo, a body fluid such as a vitreous or tissue sample from a subject can be contacted with a polypeptide coated on a tube or filter, eg, a tube or filter in a bypass machine. When administered in vivo, the active compound may be administered by any suitable route, such as oral, nasal, pulmonary, parenteral, intravenous, intradermal, intravitreal, intraretinal, subretinal, periocular, subcutaneous, intraarticular. Intracisternal, intraocular, intraventricular, intrathecal, intramuscular, intraperitoneal, intratracheal, rectal, or topical injection, or directly into an organ in liquid, semiliquid, or solid form; They can be administered in any combination of two or more thereof, formulated in a manner suitable for each route of administration.

The route of administration is according to known methods, for example, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional route, local, or continuous administration. Depends on the release system. The antibody or fragment thereof is typically administered continuously by infusion or bolus injection. The active compounds provided herein can be prepared in admixture with a pharmaceutically acceptable carrier, as discussed above. Techniques for formulating and administering compounds are known to those skilled in the art. The therapeutic composition can be administered intravenously or through the nose or lungs, for example as a liquid or powder aerosol (lyophilized). The compositions can also be administered parenterally or subcutaneously, if desired. When administered systemically, the therapeutic compositions are sterile, pyrogen-free, and can be administered parenterally, taking due account of pH, isotonicity, and stability, and other conditions known to those skilled in the art. The solution must be acceptable.

Therapeutic formulations can be administered in many conventional dosage formulations. Dosage formulations of active compounds provided herein are prepared for storage or administration by mixing the compound with the desired purity with physiologically acceptable carriers, excipients, or stabilizers. . Such substances are non-toxic to recipients at the dosages and concentrations used, such as TRIS HCl, phosphates, citrates, acetates, and other organic acid salts. buffers; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) peptides such as polyarginine; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine, glutamic acid , aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates, such as cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; Mention may be made of counterions such as sodium and/or those sold under the trademarks TWEEN and PLURONICS, and nonionic surfactants such as polyethylene glycol (PEG).

Pharmaceutical compositions can contain stabilizers. Stabilizers can be amino acids, amino acid derivatives, amines, sugars, polyols, salts or surfactants. In some instances, a stable combination contains a single stabilizer. In other examples, the stable combination contains 2, 3, 4, 5, or 6 different stabilizers. For example, the stabilizer can be a sugar or polyol such as glycerol, sorbitol, mannitol, inositol, sucrose or trehalose. In a particular example, the stabilizing agent is sucrose. In other examples, the stabilizing agent is trehalose. The concentration of sugar or polyol is inclusive, 100mM to 500mM, 100mM to 400mM, 100mM to 300mM, 100mM to 200mM, 200mM to 500mM, 200mM to 400mM, 200mM to 300mM, 250mM to 500mM, 250mM to 400mM. , 250mM ~ 300mM, 300mM to 500mM, 300mM to 400mM, or about 400mM to 500mM.

In examples, stabilizers can be surfactants that are polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamers, and polysorbates. For example, the surfactant can be polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer or polysorbate, such as poloxamer 188, polysorbate 20 and polysorbate 80. In a particular example, the stabilizer is polysorbate 80. The concentration of surfactant as % of the mass concentration (w/v) in the formulation is between 0.005% and 1.0%, between 0.01% and 0.5%, respectively, inclusive. Between 0.01% and 0.1%, between 0.01% and 0.05%, or between 0.01% and 0.02%, or between about 0.005% and 1.0% , between about 0.01% and 0.5%, between about 0.01% and 0.1%, between about 0.01% and 0.05%, or between about 0.01% and 0.02 It is between %.

For in vivo administration, the formulation must be sterile. This is easily accomplished, such as by filtration through a sterile filter membrane, before or after lyophilization and reconstitution. The formulation can be stored in lyophilized form or in solution. Other vehicles can be used, such as naturally occurring vegetable oils such as sesame oil, peanut oil, cottonseed oil, or synthetic fatty vehicles such as ethyl oleate. Buffers, preservatives, antioxidants, etc. can be incorporated according to accepted pharmaceutical practice.

Determination of dosage is within the skill of the physician and may depend on the particular disorder, route of administration, and subject. Exemplary dosages include, for example, 0.1 to 100 mg/kg, such as 1 to 10 mg/kg, or an appropriate amount based on the mass of the subject being treated; ) is approximately 70-75 kg. The polypeptide can be administered one or more times, such as two, three, four, or any number of times necessary to achieve a therapeutic effect. Multiple administrations can be by any route or combination of routes and can be administered every hour, every two hours, every three hours, every four hours, or more.

The active compound may be, for example, 0.1 to 10 mg/mL or about 0.1 to 10 mg/mL, such as at least or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0 .6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 , 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg/mL or more. Can be done. The volume of the solution is 0.1 to 100 mL or more, or about 0.1 to 100 mL or more, such as at least or at least about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, It can be 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL or more. In some instances, the active compound is provided in phosphate buffered saline. For example, the composition can be supplied as a 50 mL vial or other container containing 100 mg of the polypeptide or fusion protein at a concentration of 2 mg/mL in phosphate buffered saline.

The active compounds provided herein can be lyophilized for storage and reconstituted with a suitable carrier before use. This technique has been shown to be effective for conventional immunoglobulin and protein preparations, and art-known lyophilization and reconstitution techniques can be used.

The active compounds provided herein can be provided as controlled release or sustained release compositions. Polymeric materials are known in the art for the formulation of pills and capsules that can achieve controlled or sustained release (e.g., Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New See York (1984); also Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71 :105; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; and International Application Publication Nos. WO99/015154 and WO99/020253). Examples of polymers used in sustained release formulations include poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), Polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. Generally, polymers used in sustained release formulations are inert, free of leachable impurities, storage stable, sterile, and biodegradable. Sustained release formulations can be manufactured using any technique known in the art for manufacturing sustained release formulations.

Constructs and nucleic acids, and their physiologically acceptable forms of salts and solvates, can be administered by inhalation (either orally or nasally), or for example by oral, transdermal, pulmonary, parenteral or rectal administration. can be formulated for administration by other routes of administration, including. For administration by inhalation, the active compounds can be aerosolized from pressurized packs or nebulizers by the use of a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. It can be delivered in a form that presents. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a mixed powder of the therapeutic compound and a suitable powder base, such as lactose or starch. I can do it.

For pulmonary administration to the lung, the construct can be delivered from a nebulizer, turbonebulizer, or microprocessor-controlled metered dose oral inhaler in a form that presents an aerosol spray using a suitable propellant. Generally, the particle size of an aerosol is small, such as in the range of 0.5 to 5 microns. For pharmaceutical compositions formulated for pulmonary administration, detergent surfactants are typically not used. Pulmonary drug delivery is a promising non-invasive method of systemic administration. The lung has become an attractive route for drug delivery, primarily due to its high surface area for absorption, thin alveolar epithelium, extensive vascularization, lack of hepatic first-pass metabolism, and relatively low metabolic activity. There is.

For oral administration, the pharmaceutical compositions contain pharmaceutically acceptable excipients, such as binders (e.g. pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose). or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulfate). , tablets, pills, liquid suspensions, or capsules prepared by conventional means. Tablets may be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. . Such liquid formulations may contain pharmaceutically acceptable excipients such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hardened edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous media (e.g. , almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl-p-hydroxybenzoate or propyl-p-hydroxybenzoate, or sorbic acid); be able to. The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as desired.

Preparations for oral administration may be formulated for controlled release of the active compound. For buccal administration, the compositions can take the form of tablets or troches formulated in conventional manner. The active compounds can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, therapeutic compounds can be formulated using suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g. as sparingly soluble salts. can do.

The active compounds can be formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powdered, lyophilized form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

Pharmaceutical compositions may be in the form of gels, creams, and lotions for topical or topical application, such as topical application to the skin (transdermal) and mucous membranes, such as the eye, and for application to the eye or large It can be formulated for intracisternal or intraspinal application. Such solutions, particularly ophthalmic solutions, can be formulated with appropriate salts as isotonic solutions of 0.01% to 10% and pH of about 5 to 7. The compounds can be formulated as aerosols for topical application such as by inhalation (e.g., U.S. Pat. No. 4,044, which describes aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma). , No. 126, No. 4,414,209 and No. 4,364,923).

The concentration of active compound in the pharmaceutical composition depends on the absorption, inactivation, and excretion rates of the active compound, the dosing schedule, and the amount administered, as well as other factors known to those skilled in the art. As further described herein, dosages can be determined empirically using comparisons of properties and activities. For example, inhibition of TNF-mediated inflammatory signaling through TNFR1 by the constructs provided herein can be compared to conventional anti-TNF therapies such as adalimumab.

The compositions can, if desired, be presented in a package, kit, or dispenser device that can contain one or more unit dosage forms containing the active ingredient. In some examples, the composition can be coated onto a device, such as a filter in a tube or bypass machine. The package includes, for example, metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions containing active agents can be packaged as products that include packaging materials, an agent provided herein, and a label indicating the disorder for which the agent is provided. Pharmaceutical compositions can be packaged in unit dosage forms containing quantities of the pharmaceutical composition for single or multiple administrations. Packaged compositions include the constructs provided herein, e.g., TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, e.g., fusion proteins, and the present invention. A lyophilized powder of a pharmaceutical composition containing a nucleic acid molecule encoding a construct provided herein can be included, where the lyophilized powder of the pharmaceutical composition is mixed with water or saline (e.g., water or saline) prior to administration. can be reconstituted (using water).

The products provided herein include packaging materials. Packaging materials used in packaging pharmaceutical products are well known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252). (see ). Examples of pharmaceutical packaging materials include blister packs, bottles, tubes, inhalers (e.g. pressurized metered dose inhalers (MDIs), dry powder inhalers (DPIs), nebulizers (e.g. jet or ultrasonic nebulizers) and others. single breath liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging materials suitable for the selected formulation and intended mode of administration and treatment. These include, but are not limited to:

Constructs provided herein include, e.g., TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, e.g., fusion proteins, and nucleic acid molecules encoding the constructs. The active compounds, pharmaceutical compositions, and combinations of active agents and other compositions containing other therapeutic agents can be provided in kits. The kit includes one or more components such as instructions for use, a device, additional reagents (e.g., sterile water or saline for dilution of the composition and/or reconstitution of the lyophilized protein), and Components such as tubes, containers and syringes for practicing the method may be included. Exemplary kits can include the constructs and encoding nucleic acids provided herein, instructions for use, a device for administering a compound to a subject, a device for detecting a compound in a subject, and a device for administering additional therapeutic agents to the subject. The kit may include instructions for use. Instructions for use will typically include the active compound contained in the kit, and optionally other components, as well as methods of administration, such as methods for determining the appropriate condition of a subject, appropriate dosages, administration regimens, and methods of administration. The instructions for use can also include guidelines for monitoring the subject over the course of treatment.

Kits can also include the pharmaceutical compositions and diagnostic items described herein. For example, such kits can include items for determining the concentration, amount, or activity of an administered active compound in a subject. Kits provided herein can also include devices for administering the compounds. Any of a variety of devices known in the art for administering drugs to a subject can be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, and catheters. Typically, the device for administration is compatible with the desired method of administration of the active agent.

3. Administration of nucleic acids encoding polypeptides (gene therapy)
Some pharmaceutical compositions contain nucleic acid molecules encoding polypeptide constructs provided herein. Instead of delivering the protein, the nucleic acid molecule can be delivered in vivo, e.g. systemically or by other routes, or ex vivo, e.g. by removing cells, including lymphocytes, introducing the nucleic acid molecule into the cell, and the host, or Administration can include reintroduction into a compatible recipient.

The polypeptide constructs provided herein, e.g., TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific TNFR1 antagonist/TNFR2 agonist constructs, such as bispecific, e.g., fusion proteins, can be applied to cells by expression of a nucleic acid molecule. and can be delivered to tissues. Nucleic acids can be delivered to cells and tissues by any method known to those of skill in the art. Isolated nucleic acids can be incorporated into vectors for further manipulation. As discussed above, methods of administering polypeptides by expression of encoding nucleic acid molecules include administration of recombinant vectors. Vectors can be designed to remain episomal, such as by containing an origin of replication, or can be designed to integrate into a chromosome within a cell. Polypeptides can also be used in ex vivo gene expression therapy using non-viral vectors. Suitable gene therapy vectors and delivery methods are known to those skilled in the art and are discussed in the sections above.

K. Therapeutic Uses and Methods of Treatment Pharmaceutical compositions such as those described above are prepared and administered to a subject having a disease, disorder, or condition amenable to treatment with constructs that inhibit and/or agonize TNFR1 and TNFR2, respectively. The dosage depends on the particular disorder, disease or condition being treated, as well as the particular subject. Typical doses are similar to known TNF blockers such as etanercept. Exemplary doses for subjects, including humans and other animals, range from about 0.1 mg/kg to 100 mg/kg, such as from 1 mg/kg to about 30 mg/kg, such as from 5 mg/kg to 25 mg/kg. Doses can be determined based on the assumption that the average human mass is approximately 75 kg. Doses can be adjusted for children, infants, and smaller adults.

The TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins provided herein are useful for the treatment of any of the diseases, disorders, and conditions described herein. It can be used for any purpose known to those skilled in the art for the use of such molecules. For example, the TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins provided herein may be used for one or more of therapeutic, diagnostic, industrial, and/or research purposes. can be used. Therapeutic methods provided herein include methods for therapeutic use of the TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins provided herein. . For example, the TNFR1 antagonists described herein antagonize TNFR1 and/or inhibit TNF binding to TNFR1 and/or inhibit TNF-mediated pro-inflammatory signaling through TNFR1. can be used to. TNFR2 agonists are used to agonize TNFR2 to induce protective/anti-inflammatory TNFR2 signaling and/or to induce expansion, proliferation and activation of immunosuppressive TNFR2+ regulatory T cells (Tregs). can do. In some embodiments, the use of a combination of a TNFR1 antagonist and a TNFR2 agonist, or a bispecific TNFR1 antagonist/TNFR2 agonist construct provided herein, as described herein, induces inflammation. provides selective inhibition of TNFR1 activity while maintaining or increasing TNFR2-related protective signaling and Treg immunosuppressive activity, which is useful in the treatment of chronic inflammatory and autoimmune diseases, as well as neurodegenerative and degenerative diseases. Beneficial in the treatment of medullary diseases and disorders.

The TNFR1 antagonists, TNFR2 agonists, multispecific, including bispecific, TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein can have therapeutic activity alone or in combination with other agents. can. The TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins, and encoded nucleic acid molecules provided herein are useful for treating chronic inflammatory and autoimmune diseases and disorders, as well as neurodegenerative diseases. and demyelinating diseases and disorders, including, but not limited to, anti-TNF therapies (e.g., adalimumab, infliximab, etanercept, and the like described herein and/or known in the art). or other disease-modifying antirheumatic drugs (DMARDs; for example, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, abatacept, anakinra, rituximab, tocilizumab, tofacitinib, and others described herein and/or (Others known in the art) may be used to treat any condition for which it applies. For example, the subject to whom the therapeutic molecules provided herein are administered exhibits acute or chronic inflammation of the joints, skin, lungs, and/or intestines, and/or has an autoimmune disease, rheumatoid arthritis (RA). , psoriasis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), spondyloarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), uveitis, fibrotic disease, intrauterine membranous disease, lupus, multiple sclerosis, congestive heart failure, cardiovascular disease, myocardial infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, sepsis, acute respiratory distress syndrome, ( ARDS), Severe Acute Respiratory Syndrome (SARS), COVID-19, influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, Alzheimer's disease, Parkinson's disease, Behcet's disease, Dupuytren's disease, tumors 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), respiratory diseases, Pulmonary inflammation, diseases and conditions of the lungs, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, genetic diseases with TNF/TNFR1 as a pathological mediator, periodic fever syndrome , as well as those suffering from cancer.

The constructs provided herein, when administered, will generally exhibit reduced or alleviated side effects in a subject as compared to side effects that may be observed following administration of anti-TNF therapy. Treatment of diseases and conditions with the polypeptides provided herein, such as TNFR1 antagonists, TNFR2 agonists, and bispecific constructs and fusion proteins thereof, can be performed using suitable formulations described herein. It can be carried out by any suitable route of administration, including, but not limited to, injection, subcutaneous injection, and inhalation, or intramuscular, intradermal, oral, topical and transdermal administration.

As discussed elsewhere herein, existing anti-TNF therapies, such as adalimumab, are immunosuppressive due to blockade of TNF signaling through TNFR1 and TNFR2, e.g., in sepsis and severe infections. Associated with the risk of adverse side effects, such as increased risk of listeriosis, tuberculosis reactivation, hepatitis B/C reactivation, herpes zoster reactivation, as well as invasive It is associated with a risk of adverse side effects, including an increased risk of fungal infections and other opportunistic infections. Anti-TNF agents can also cause exacerbation of severe congestive heart failure and can cause drug-induced lupus, liver damage, psoriasis, sarcoidosis, and central nervous system (CNS) demyelinating disease; It may cause additional autoimmune diseases and increased susceptibility to the development of solid malignancies such as lymphoma and non-melanoma skin cancer. Depending on the anti-TNF agent, approximately 3-33% of treated patients do not respond to treatment, and up to 46% stop responding, leading to discontinuation or dose escalation. Anti-TNF therapies have failed to treat neurodegenerative diseases and CNS conditions such as Alzheimer's disease, Parkinson's disease, stroke, and multiple sclerosis (MS) that are associated with overexpression of TNF. Side effects associated with the use of anti-TNF agents, non-responsiveness in some patients, lack of sustained response in patients who have an initial response, and treatment failure and/or worsening of neurodegenerative diseases such as MS. Therefore, other therapies are required. Such therapies are provided herein.

As described herein, selective inhibition of TNFR1 preserves the potent anti-inflammatory and protective activity of TNFR2 signaling, leading to reductions in opportunistic infections and cancer, and inhibiting TNF-induced Treg function. maintained. Conventional selective TNFR1 antagonists have been shown to reduce immunogenicity, including the formation of anti-drug antibodies (ADA), e.g. short serum half-life, rapid renal clearance, and/or pharmacokinetics, including reduced binding affinity and capacity. Subject to decreased pharmacodynamics. The therapies provided herein overcome the limitations associated with traditional selective TNFR1 antagonists. Examples of therapeutic improvements using polypeptides provided herein, such as TNFR1 antagonists, include, but are not limited to, lower doses, less and/or less frequent administration, reduced side effects, and therapeutic efficacy. An example of this is an increase in

Accordingly, the selective TNFR1 antagonists, TNFR2 agonists, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs and fusion proteins provided herein are associated with reduced side effects and, if desired, more Advanced dosing regimens can be used to improve efficacy and safety. Side effects that can be reduced, alleviated or eliminated compared to those observed with existing anti-TNF therapies, such as adalimumab and others described herein and/or known in the art, include: Any undesirable non-therapeutic effects described herein or known in the art, including, but not limited to, sepsis, serious infections, congestive heart failure/cardiotoxicity, antibody production, development of cancer. or worsening, including autoimmune diseases and/or central nervous system (CNS) demyelinating diseases. In some instances, administration of the TNFR1 antagonists, bispecific constructs, or fusion proteins provided herein may reduce anti-TNF the severity of the one or more side effects of the therapy is at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85% , at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10%.

Dosage levels and regimens can be determined based on known doses and regimens and, if necessary, extrapolated based on changes in the properties of the polypeptides and constructs provided herein. , and/or can be determined empirically based on various factors. Such factors include, for example, the individual's body weight, general health, age, sex, and diet, as well as the activity of the particular compound used, time of administration, rate of excretion, drug combinations, severity of the disease, and These include the course of the disease, as well as the patient's predisposition to the disease, as well as the judgment of the attending physician. The active ingredient is typically combined with a pharmaceutically effective carrier. The amount of active ingredient that may be combined with the carrier materials to produce a single or multiple dosage form may vary depending on the host treated and the particular mode of administration.

Once the patient's condition improves, maintenance doses of the compound or composition can be administered as needed; the dosage, dosage form, or frequency of administration, or combinations thereof, can be modified. In some cases, the subject may require intermittent treatment on a long-term basis if symptoms of the disease recur or based on scheduled dosage.

This section provides exemplary uses and methods of administration of constructs, including the polypeptides and encoding nucleic acid molecules provided herein. These described therapies are merely illustrative and do not limit the uses of the molecules/constructs provided herein. Identifying treatable diseases or conditions using the TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins, and encoded nucleic acid molecules provided herein includes: It is within the skill of the physician performing the procedure.

1. Treatment of Chronic Inflammatory/Autoimmune Diseases and Disorders As described herein, elevated levels or uncontrolled expression of TNF and deregulation of TNF signaling cause chronic inflammation. This can lead to the development of autoimmune diseases and tissue damage. TNF signaling through TNFR1 is primarily pro-inflammatory and promotes the development of chronic inflammatory and autoimmune diseases and disorders. For example, TNFR1 signaling has been linked to the development of arthritis, inflammatory bowel disease (IBD), and respiratory diseases, among others, as well as osteoclast production leading to local bone destruction, and TNFR1 failure and myocardial infarction. It has also been associated with cardiotoxic effects in inducible models. Therefore, selective blockade of TNFR1 is useful in the treatment of chronic inflammatory and autoimmune diseases and conditions. TNF signaling through TNFR2, which is primarily anti-inflammatory, is associated with protective effects on nerves, heart, gut, and bone. The anti-inflammatory and protective effects of TNFR2 signaling have been shown to affect, for example, experimental colitis, heart failure/heart disease, myocardial infarction, inflammatory arthritis, infectious diseases, pancreatic regeneration, stem cell proliferation, destruction of autoreactive T cells, and bone mass. It has been demonstrated in the control of osteoclastogenesis to maintain joint inflammation and protection from erosive destruction. TNFR2 agonism also results in proliferation and expansion of immunosuppressive TNFR2+ Tregs, eliminating autoreactive/effector T cells, preventing tissue destruction, and exerting Treg cell suppressive activity that suppresses inflammatory and autoimmune diseases and conditions. Facilitate. Accordingly, TNFR2 agonism can also be used to treat or alleviate symptoms of chronic inflammatory and autoimmune diseases and conditions.

The TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and encoded nucleic acids provided herein are useful for autoimmune/inflammation associated with elevated TNF levels and deregulated TNF signaling. It can be used to treat or alleviate the symptoms of sexual diseases and disorders. The constructs, fusion proteins, and nucleic acids provided herein are particularly useful, but are not limited to, arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, spondyloarthritis), inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), uveitis, fibrotic diseases (e.g. Dupuytren's disease), Behcet's disease, endometriosis, lupus, ankylosing spondylitis, psoriasis, tumor necrosis factor receptor-related periodic syndrome ( TRAPS), cardiovascular disease, congestive heart failure, myocardial infarction (MI), atherosclerosis, respiratory disease, asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), pancreatitis, type I diabetes, metabolism Sexual diseases, cytokine release syndrome, septic shock, sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), COVID-19, influenza, chronic bronchitis, emphysema, pulmonary inflammation, idiopathic pulmonary fibrosis , including graft rejection, graft-versus-host disease (GvHD), acute fulminant viral or bacterial infections, pneumonia, genetic diseases with TNF/TNFR1 as the causative pathological mediator, and periodic fever syndrome. Can be used to treat diseases, disorders, and conditions.

TNF blockade can also reduce the cytokine storm observed in some viral infections such as the SARS virus and COVID-19 infection. This can prevent ventilator dependence, multi-organ damage, and death in patients with Severe Acute Respiratory Syndrome (SARS) caused by SARS-CoV-2 and other SARS viruses/coronaviruses. TNF-induced viral syndrome (TIVS) is induced by a TNF-driven cytokine storm that is accompanied not only by damage to the lungs but also by multiple organ failure. TIVS is similar to SIRS (Severe Inflammatory Respiratory Syndrome), SARS (Severe Acute Respiratory Syndrome), and sepsis (caused by bacteria). These conditions affect not only lung function but also many other organs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, which causes COVID-19) accesses host cells through angiotensin-converting enzyme, which is expressed in type II surfactant-secreting alveolar cells of the lung. Severe COVID-19 is associated with a major immune inflammatory response with abundant neutrophils, lymphocytes, macrophages, and immune mediators. Deaths due to COVID-19 are primarily due to diffuse alveolar damage with pulmonary edema, hyaline membrane formation, and interstitial mononuclear inflammatory infiltrates consistent with early stage adult respiratory distress syndrome (ARDS). TNF is present in the blood and diseased tissues of COVID-19 patients; TNF is involved in almost all acute inflammatory responses and acts as an amplifier of inflammation. Accordingly, treatment with the constructs herein is provided.

2. Treatment of Neurodegenerative and Demyelinating Diseases and Disorders As discussed elsewhere herein, several neurodegenerative and demyelinating diseases and conditions are Associated with chronically elevated levels of TNF. Elevated TNF levels and TNF signaling through TNFR1 are involved in the initiation and maintenance of neuroinflammation and the promotion of neuronal cell death, demyelination, and cognitive decline. For example, in Alzheimer's disease (AD) patients, TNF promotes microglial activation, synaptic dysfunction, neuronal cell death, and the accumulation of plaques and tangles, and elevated TNF levels are associated with amyloid beta in the brains of AD patients. (Aβ) phagocytosis, which prevents efficient plaque clearance by microglia. In Parkinson's disease (PD) patients, elevated TNF levels lead to neuroinflammation and toxicity of dopaminergic neurons. Elevated TNF levels and polymorphisms in the gene encoding TNFR1 are associated with demyelination and the development of demyelinating disorders such as multiple sclerosis (MS). Accordingly, selective blockade of TNF signaling through TNFR1 can be used to treat or alleviate neurodegenerative and demyelinating diseases and disorders, as well as other conditions of the CNS.

TNF signaling through TNFR2 is associated with anti-inflammatory and neuroprotective effects. For example, activation of TNFR2 by TNF suppresses seizures, attenuates cognitive dysfunction after brain injury, and promotes remyelination and neuron survival. The proliferation, expansion, and activation of immunosuppressive Tregs following TNFR2 agonism also has neuroprotective effects. For example, TNFR2 signaling promotes Treg cell proliferation and suppressive activity in experimental autoimmune encephalomyelitis (EAE), an animal model of inflammatory CNS demyelinating diseases such as multiple sclerosis. Accordingly, TNFR2 agonism can also be used to treat or alleviate neurodegenerative and demyelinating diseases and disorders, as well as other conditions of the CNS.

The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein can be used to treat neurodegenerative and demyelinating diseases, as well as Alzheimer's disease. It can be used to treat or ameliorate the symptoms of other CNS disorders and conditions, including but not limited to Parkinson's disease, multiple sclerosis, and stroke.

3. Treatment of Cancer and Other Immunosuppressive Diseases, Disorders, and Conditions As described herein, tumors contain tumor-killing CD8+ cytotoxic T lymphocytes (CTLs), also known as effector T cells (Teffs). ) are infiltrated by large numbers of immunosuppressive TNFR2+ Tregs that prevent the proliferation of tumors, thereby enabling tumor growth. Antagonism of TNFR2 against lymphocytes in the tumor microenvironment (TME) eliminates Tregs and allows activation and expansion of effector T cells, restoring the balance between the two types of T cells and promoting tumor cell proliferation. (see, e.g., Vanamee et al. (2017) Trends in Molecular Medicine 23(11):P1037-P1046).

TNFR2 is abundantly expressed on the surface of many types of human cancer cells, including, for example, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, multiple myeloma, cutaneous non-Hodgkin's lymphoma, and ovarian cancer. TNFR2 mutations in cancer are associated with gene duplication and constitutive activation. Mouse myeloid-derived suppressor cells (MDSCs) also express TNFR2, and its inhibition has been shown to control metastasis in mouse liver cancer models. Furthermore, immune checkpoint inhibitors cause upregulation of TNFR2 in tumor-infiltrating Tregs, leading to tumor immune escape and drug resistance. Not all patients respond to treatment with immune checkpoint inhibitors, patients may relapse, and severe autoimmune side effects have been observed with checkpoint inhibitor therapy (e.g., Vanamee et al. (2017) Trends in Molecular Medicine 23(11):P1037-P1046). Blocking TNFR2 may therefore be useful in the treatment of certain types of cancer by directly killing tumor cells, through inhibition of immunosuppressive Tregs that allow effector T cell proliferation, and by inhibiting MDSCs. can be used. This prevents the formation of metastases. TNFR2 is also expressed in normal tissues (particularly macrophages; see e.g. proteinatlas.org/ensg00000028137-tnfrsf1b/tissue), so TNFR2 antagonists do not have ADCC activity, but have FcRn activity (or enhanced FcRn activity). activity). As described herein, to be eligible for treatment, a patient's tumor must have significantly higher levels of TNFR2 than adjacent normal tissue, so dosing is individualized. Ru. To this end, TNFR2 antagonists are used in conjunction with other therapies, especially immunomodulatory treatments that otherwise result in the accumulation of regulatory T cells within the tumor.

Accordingly, the TNFR2 agonists, bispecific TNFR1 antagonist/TNFR2 agonist constructs, and fusion proteins provided herein are useful for treating solid cancers, hematologic malignancies, and other hyperproliferative diseases and disorders, such as, but not limited to, It can also be used to treat renal cell carcinoma, colon cancer, Hodgkin's lymphoma, multiple myeloma, cutaneous non-Hodgkin's lymphoma, and ovarian cancer.

4. Combination Therapy Combination therapy includes the use of the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein, radiation, and surgery. This includes administration in combination with another agent or treatment. Additional agents or therapies can be administered simultaneously with, before, after, or intermittently with the treatments provided herein. They may be separate compositions or combinations.

The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, including bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein include, but are not limited to, TNF antagonist/blockers, antibodies, Cytotoxic agents, anti-inflammatory agents, cytokines, growth factors, growth inhibitors, cardioprotective agents, immunosuppressive agents, chemotherapeutic agents, biological or non-biological disease-modifying antirheumatic drugs (DMARDs), infectious disease It can be administered before, after, intermittently, and simultaneously with one or more other therapeutic regimens or agents, including therapeutic agents (including antibodies), or other therapeutic agents. The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs and nucleic acids provided herein can be used as a first-line treatment or for either acute or chronic treatment. As such, it can be administered to patients as a second line treatment if anti-TNF therapy has not been effective. Examples of anti-TNF therapies that can be used in the combination therapy herein include, for example, conventional synthetic DMARDs (e.g., generic names and exemplary trademarks): Methotrexate (MTX), Hydroxychloroquine (HCQ); Plaquenil ( ), sulfasalazine (Azulfidine®), and leflunomide (Arava®); biological DMARDs such as abatacept (Orencia®), anakinra (Kineret®), Rituximab (Rituxan®, Truxima®, MabThera®), tocilizumab (atlizumab, Actemra®, RoActemra®), corticosteroids (e.g. dexamethasone, methylprednisolone, prednisolone) , prednisone, or triamcinolone), tofacitinib (Xeljanz®), and TNF inhibitors/anti-TNF agents such as certolizumab pegol (Cimzia®), infliximab (Remicade®), adalimumab (Humira®), golimumab (Simponi®), and etanercept (Enbrel®). Combination therapy can also include, for example, immunotherapeutic agents such as cyclosporine, methotrexate, adriamycin or cisplatin, and immunotoxins.

Examples of anti-inflammatory drugs and drugs useful in combination therapy include non-steroidal anti-inflammatory drugs (NSAIDs) including salicylates such as aspirin, traditional NSAIDs such as ibuprofen, naproxen, ketoprofen, nabumetone, piroxicam, Diclofenac, or indomethacin, and Cox-2 selective inhibitors, such as celecoxib (sold under the trademark Celebrex®) or lotecoxin (sold under the trademark Vioxx®). Other compounds useful in combination therapy include antimetabolites such as methotrexate and leflunomide; corticosteroids or other steroids such as cortisone, dexamethasone, or prednisone; analgesics such as acetaminophen; aminosalicytes such as mesalamine. and cytotoxic agents such as azathioprine (sold under the trademark Imuran®), cyclophosphamide (sold under the trademark Cytoxan®), and cyclosporine A.

Additional agents that can be used in combination therapy include, for example, biological response modifiers including anti-inflammatory cytokines such as IL-10; B cell targeting agents such as anti-CD20 antibodies (e.g. rituximab); Antigen-targeting compounds; adhesion molecule blockers; chemokine receptor antagonists; kinase inhibitors, such as inhibitors of mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK), or NFκB; and peroxisome proliferation. Factor-activated receptor-gamma (PPAR-γ) ligand. Additional drugs that can be used in combination therapy include immunosuppressants. Immunosuppressive agents may include, for example, tacrolimus or FK-506; mycophenolic acid; calcineurin inhibitors (CNI); CsA; and sirolimus, or other drugs known to suppress the immune system. can.

The polypeptides and constructs provided herein are also useful for agents administered to treat cardiovascular disease and/or administered during procedures to treat cardiovascular disease, such as agents such as anticoagulants. Can be used in combination with Exemplary anticoagulants include heparin, warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate, and direct thrombin inhibitors such as argatroban, lepirudin, bivalirudin, and ximelagatran; but not limited to.

The polypeptides and constructs provided herein can be administered with antibodies for the treatment of autoimmune or inflammatory diseases, graft rejection, or GvHD. Examples of such antibodies include anti-α4β7 integrin antibodies such as LDP-02; anti-beta2 integrin antibodies such as LDP-01; anti-complement (C5) antibodies such as 5G1.1; BTI-322 and MEDI-507. anti-CD2 antibodies such as OKT3, SMART anti-CD3; anti-CD4 antibodies such as IDEC-151, MDX-CD4, and OKT4A; anti-CD11a antibodies; anti-CD14 antibodies such as IC14; anti-CD18 antibodies; IDEC152, etc. anti-CD23 antibodies; anti-CD25 antibodies such as daclizumab; anti-CD40L antibodies such as 5c8, luplizumab, and IDEC-131; anti-CD64 antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114, anti-CD80 antibodies such as ABX-CBL; CD147 antibody, anti-E-selectin antibody such as CDP850, anti-gpIIb/IIIa antibody such as ReoPro (registered trademark)/Abcixima, anti-ICAM-3 antibody such as ICM3, anti-ICE antibody such as VX-740, anti-ICE antibody such as MDX-33, etc. Anti-FcγR1 antibodies, anti-IgE antibodies such as rhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodies such as SB-240563 and SCH55700, anti-IL-8 antibodies such as ABX-IL8, Interferon gamma antibodies; and anti-TNFα antibodies such as CDP571, CDP870, D2E7, infliximab, and MAK-195F, anti-VLA-4 antibodies such as Antegren®. Examples of other Fc-containing molecules that can be co-administered to treat autoimmune or inflammatory diseases, graft rejection, and GVHD include the TNFR2-Fc fusion protein Enbrel® (etanercept). ) and Regeneron's IL-1 Trap.

Examples of antibodies that can be co-administered to treat infections include anti-anthrax antibodies such as ABthrax; anti-CMV antibodies such as CytoGam and sebilumab; anti-Cryptosporidium antibodies such as CryptoGAM, Sporidin-G; Pyloran, etc. anti-Helicobacter antibodies; anti-hepatitis B antibodies such as HepeX-B, and Nabi-HB; anti-HIV antibodies such as HRG-214; anti-RSV antibodies such as Felbizumab, HNK-20, Palivizumab, and RespiGam; and Aurexis, Aurograb , BSYX-A110, and SE-Mab.

In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein can be combined with one or more chemical Administered with therapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; calsterone, dromostanolone propionate, epithiostanol, Androgens such as mepithiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; aclacinomycin, actinomycin, anthramycin, azaserine , bleomycin, cactinomycin, calicheamicin, carbicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin Antibiotics such as , marcellomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, pepromycin, porphyromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin antiestrogens including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifen, keoxifene, LY117018, onapristone, and toremifene (Fareston); methotrexate and 5-fluorouracil (5- antimetabolites such as denopterin, methotrexate, pteropterin, and trimetrexate; aziridines such as benzodepa, carbocone, metredepa, and uredepa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylene Ethyleneimine and methylmelamine, including thiophosphoramide and trimethylolmelamine; folic acid supplements such as folic acid; chlorambucil, chlornafadine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melamine Nitrogen mustards such as faran, novembichin, fenesterine, prednimustine, trophosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine, 6-mercaptopurine, thiamipurine, and thioguanine; ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine, and pyrimidine analogs such as 5-FU; paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.C.); J. ) and taxanes such as docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); topoisomerase inhibitors such as RFS2000; thymidylate synthase inhibitors such as Tomudex; additional chemotherapeutic agents including aceglatone; Famidoglycoside; aminolevulinic acid; amsacrine; bestrabcil; bisanthrene; edatrexate; defosfamide; demecolcin; diazicon; difluoromethylornithine (DMFO); eflornithine; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; Santhrone; mopidamole; nitraculine; pentostatin; phenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide K (PSK, Krestin); razoxane; schizophyllan; spirogermanium; tenuazonic acid; triazicon; ”-Trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobroman; gacytocin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil; gemcitabine; 6-thioguanine; Etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; and topoisomerase inhibitors such as irinotecan. Pharmaceutically acceptable salts, acids or derivatives of any of the above may also be used.

Chemotherapeutic agents can be administered as prodrugs. Examples of prodrugs that can be administered with the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein include: However, for example, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxy These include acetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, and 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted to more active cytotoxic free drugs. I can do it. Multispecific TNFR1 antagonist/TNFR2 agonist constructs, such as TNFR1 antagonist constructs, TNFR2 agonist constructs, and bispecifics, can be combined with targeting agents to target specific tissues or disease loci, e.g., by in vivo cleavable linkers. By linking them, they can be provided as prodrugs, thereby releasing the active form of the construct.

In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific TNFR1 antagonist/TNFR2 agonist constructs, such as bispecific, fusion proteins, and nucleic acids provided herein are, but are not limited to, aminoglycoside-based antibiotics (e.g. apramycin, arbekacin, bambermycin, butyrosin, dibekacin, gentamicin, kanamycin, neomycin, netilmycin, paromomycin, ribostamycin, sisomicin, spectinomycin), aminocyclitol (e.g. spectinomycin), Amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin), carbapenems (e.g., imipenem, meropenem) , and panipenem); cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatridine, cefazedon, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefuroxime, cefixime, cephalexin, and cefrazine); cephamycins (e.g., cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan), lincosamides (e.g., clindamycin, and lincomycin), macrolides (e.g., azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, and tobramycin), monobactams (e.g. , aztreonam, carmonam and tigemonam), mupirocin, oxacephems (e.g. flomoxef, latamoxef, moxalactam); penicillins (e.g. amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillic acid, benzylpenicillin sodium, epicillin, fenbenicillin) , floxacillin, penamecillin, penetamate hydroiodide, penicillin o-benetamine, penicillin O, penicillin V, penicillin V benzoate, penicillin V hydrabamine, penimepicycline, and pheneticillin potassium); polypeptides (e.g., bacitracin, colistin) , polymyxin B, teicoplanin, vancomycin); quinolones (amifloxacin, cinoxacin), ciprofloxacin, enoxacin, enrofloxacin, fleroxacin, flumequin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, losoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, and trovafloxacin); rifampin; streptogramins (e.g., quinupristin, dalfopristin); Sulfonamides (sulfanilamide, and sulfamethoxazole); and tetracyclines (e.g., chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, duramycin, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin).

In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein include amphotericin B, ciclopirox , clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, terbinafine, terconazole, and tioconazole. In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific TNFR1 antagonist/TNFR2 agonist constructs, such as bispecific, and nucleic acids provided herein are protease inhibitors, reverse transcriptase inhibitors, etc. , and type I interferons, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir, amantadine, amprenavir, clevudine, enfuvirtide, entecavir, foscarnet, ganciclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin, rimantadine , ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine.

Combining the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids with the growth factor trap constructs described below, It can also be administered in combination with any of the therapeutic anti-TNF agents and treatments described below for combination therapy with growth factor trap constructs. Combination therapies can also include growth factor trap constructs provided herein.

Pharmaceutical compositions containing the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein are described herein. or can be used to treat any diseases, disorders, and conditions known to those skilled in the art. Diseases, disorders, and conditions include one or more chronic inflammatory, autoimmune, neurodegenerative, or demyelinating diseases or disorders. Also provided are combinations of polypeptides and constructs provided herein for the treatment of chronic inflammatory, autoimmune, neurodegenerative or demyelinating diseases or conditions, as well as other treatments or compounds. The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific, TNFR1 antagonist/TNFR2 agonist constructs, fusion proteins, and nucleic acids provided herein, as well as additional agents, may be used together or sequentially. or can be packaged as a separate composition for intermittent administration. Alternatively, they can be provided as a single composition for administration or as two compositions for administration as a single composition. The combination can be packaged as a kit, optionally with additional reagents, instructions for use, vials and other containers, syringes, and other items for use in the treatment.

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
[Example 1]

Expression and Evaluation of Candidate Monovalent TNFR1 Antagonist Molecules Expression and Purification of Anti-TNFR1 Molecules.
Candidate monovalent TNFR1 antagonist molecules are expressed in mammalian cells under the control of the CMV promoter using the expression plasmid shown in Figure 1 (where TE19080L is the insert). For expression of protein therapeutics such as the constructs herein, mammalian cells such as Chinese hamster ovary (CHO) cells or human embryonic kidney 293 (HEK293) cells are used to develop appropriate protein structure, function, and Provide post-translational modifications, including glycosylation, that are important for activity. Expression in bacteria, yeast, or insect cells results in either no glycosylation (in bacterial cells) or a different glycosylation pattern (in yeast or insect cells) compared to expression in mammalian cells. Bacterial expression can also lead to contamination of protein therapeutics with bacterial endotoxins that activate innate immune cells, complicate analysis of cell-based assays, and cause pyrogenic effects upon in vivo administration.

Construction of expression plasmids and transient and stable cell line expression are performed using methods such as those described in Vazquez-Lombardi et al. (2018) Nat. Protoc. 13(1):99-117. and execute it. Five exemplary TNFR1 antagonist molecules are produced for initial evaluation. The TNFR1 antagonist molecule includes the H398-derived scFv (SEQ ID NO: 678), containing one VL domain and one VH domain of H398, linked by a (GGGGS)3 peptide linker; the TNFR1 antagonist domain antibody (dAb) DOM1h -574-16 (SEQ ID NO: 57); TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58); (SEQ ID NO: 704) containing the TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54) from DMS5541 (described elsewhere herein); and (GGGGS) by a 3-peptide linker. , a fusion protein containing an anti-TNFR1 dAb called DOM1h-131-206 (SEQ ID NO: 59) (SEQ ID NO: 705) fused to the antiserum albumin dAb of DMS5541 (albudAb or DOM7h-11-3; SEQ ID NO: 52), is included. Insertion of the (GGGGS)3 linker into the H398 scFv, DOM1h-574-208 and DOM1h-131-206 fusion proteins increases the plasticity between the VH and VL domains and the two dAbs, respectively, leading to molecular stability and denaturation. The manufacturing process is improved. The sequences of each of these TNFR1 antagonist molecules are provided in Table 12 below, some with various linker sequences (dAbs and modifications thereof that can be used for further modification and addition of linkers and modifiers are described). See also Enever et al., (2015) Protein Engineering, Design & Selection 28(3):59-66).

SEQ ID NOs: 53-83 and 503-671, such as SEQ ID NOs: 57-59, and any variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto. Also provided are Nanobodies comprising two heavy chains selected from those described above and constructs containing the Nanobodies. Constructs containing identical heavy chains are provided. An example of these is a TNFR1 dAb called DOM1h-131-206. Also provided are constructs containing any of these dAbs, e.g. linked to human serum albumin, either directly or more commonly via a linker such as a GS linker, or as described herein. either as Fc fusions or in other constructs as described in .

These and other such dAbs and TNFR1 binding molecules can be modified to increase specificity for TNFR1 by eliminating any antagonistic activity toward TNFR2, and/or to increase or add TNFR2 agonist activity. and/or modified to reduce or eliminate immunogenic epitopes and/or activity modifiers, such as Fc units and modified Fc units/modified Fc dimers, and/or It can be linked to a serum half-life extending moiety.

The HEK293 cell line was used for transient expression and in vitro evaluation of expressed antagonists, as well as high affinity for TNFR1 (e.g., Kd<50nM, or <10nM, or <or 5nM) and potent effects of TNFR1 signaling. Following identification of molecules with desired properties such as inhibition (eg, IC50<50 nM, or <10 nM, or <5 nM), stable cell lines are prepared with derivatives of CHO cells. Generally, it will be an affinity of about or about 19 pM or a picomolar (pM) affinity, such as an affinity of 20 pM, 15 pM, 10 pM, 5 pM, 2 pM, or 1 pM.

Transient expression is optimized in CHO DG44 cells (e.g. CHO-DG44 (DHFR-) and FreeStyle™ CHO-S cells, Invitrogen) and transfections screened to identify or select high expressing clones. You will get a pool of No polyhistidine or other purification tags were used. Alternatively, proteins for screening are purified from serum-free media by HPLC in combination with other well-known methods. The matrix used for HPLC is Amsphere™ A3 Protein A chromatography resin (JSR Life Sciences) or other similar resin according to the manufacturer's protocol. If the protein is not at least 95% pure as judged by size exclusion HPLC, further purification is performed (eg, ion exchange or hydrophobic chromatography).

Remove endotoxin (see, eg, Vazquez-Lombardi et al. (2018) for an exemplary protocol). After protein purification, endotoxin levels are determined using a detection kit such as the QCL-1000 Endpoint Chromogenic LAL assay kit (Lonza). For endotoxin removal, the theoretical pI of the purified protein is determined using a sequence analysis tool (e.g., ExPASy ProtParam), and the pH of the low endotoxin PBS buffer is lower than the theoretical pI of the purified protein; Adjust the pH to close to that value. The protein sample is then dialyzed against at least 30 volumes of pH-adjusted PBS for at least 2 hours at 4°C. An additional dialysis step is performed overnight and again the next day for at least 2 hours. The sample is then purified using anion exchange affinity chromatography, retested to determine endotoxin levels, and the process repeated until an acceptable level of endotoxin is achieved. The size and purity of the protein product is determined by SDS-PAGE analysis or other suitable method. Alternatively, other methods can be used, such as the Proteus endotoxin removal kit and accompanying manufacturer's handbook (BIORAD, see bio-rad-antibodies.com/static/uploads/ifu/pur030.pdf). . This step can be repeated until the desired level of endotoxin is reached, typically >0.5 endotoxin units/ml (0.5 endotoxin units/ml or less).

Screening of Purified Proteins Purified TNFR1 antagonist molecule candidates may be screened using methods described above in the Detailed Description, or by, for example, immunoassay (e.g., ELISA), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or Binding affinity for the extracellular domain of TNFR1 is determined using methods known in the art, such as other kinetic interaction assays known in the art. SPR can be performed using several commercially available platforms, such as, for example, the BIAcore system (GE Healthcare Life Sciences). Exemplary assays describe and compare various assays for assessing binding affinity, eg, Lang et al. (2015) J. Biol Chem 291:5022-5037. It is described in. The selected candidates include those with Kd values <5 nM.

TNFR1 antagonists are also screened using methods known in the art, such as SPR, to determine whether binding to TNFR1 is competitive or non-competitive for TNF. When an inhibitor binds to a receptor (e.g., TNFR1) and blocks the binding of a ligand (e.g., TNF), such as by binding to the active site, the inhibitor "competes" for the enzyme's substrate; i.e., the inhibitor Or this is competitive inhibition since only one of the substrates can bind at a given moment. In non-competitive inhibition, the inhibitor does not block the binding of the ligand to the ligand binding site on the receptor. Instead, it binds to another site and blocks the receptor from responding to the bound ligand. This inhibition is said to be "non-competitive" because both inhibitor and substrate can bind simultaneously. Thus, if a ligand is added to a receptor binding assay at a saturating concentration and it does not inhibit antibody binding, the two molecules are independent and non-competitive. The reverse is also valid. If the two are competing, increasing the concentration of antibody will prevent TNF from binding to the receptor. This is valid regardless of whether the binding assay is performed on cells or with surface-bound receptors or ligands. For exemplary assays, see, eg, Frey et al. (2001) Current Protocols in Neuroscience, "Receptor Binding Techniques," available at doi.org/10.1002/0471142301.ns0104s00.

For example, the ability of TNF to bind to human TNFR1 coated on a BIAcore chip is first determined. The TNFR1 surface is then saturated with TNFR1 antagonist molecules, followed by injection of TNF, and the binding of TNF to TNFR1 is reassessed. If the binding is non-competitive, they both bind TNFR1; if the binding is competitive, they interfere with each other. If the binding of TNF is unaffected or only slightly reduced, the antagonist's binding to TNFR1 is non-competitive with respect to TNF; if the binding of TNF is abolished or significantly reduced, the antagonist The binding of is thought to be competitive with respect to TNF. For purposes of this specification, competitive binders are selected.

A TNFR1 antagonist molecule can be determined using methods known in the art, such as McFarlane et al. (2002) FEBS Lett. 515(1-3):119-126 (ie, gene reporter assay). The cells used in these experiments do not express TNFR1 or TNFR2 unless transfected with a TNFR1 or TNFR2 expression plasmid (e.g., myeloma cell lines AMO1, U266, and L363; Rauert et al. (2011) Cell Death (See Dis. 2(8):e194). Alternatively, human cell lines in which the TNFR1 and/or TNFR2 genes have been inactivated or knocked out using TCRISPR vectors, antisense RNA expression, or other methods known in the art can be used. TNFR1 and/or TNFR2 cell lines are available from commercial sources (eg, Genoway and Synthego) and can also be used. These cell lines can then be specifically transfected with TNFR1 and/or TNFR2 expression cassettes. For example, cells expressing TNFR1, TNFR2, or TNFR1 and TNFR2 can be used to assess the selectivity of antagonists for TNFR1 and to determine the efficacy of inhibition of TNF signaling through TNFR1.

To determine inhibition of TNFR1 signaling by TNFR1 antagonists, cells expressing human TNFR1 were transiently transfected with an NF-κB-luciferase reporter construct using Lipofectamine and received 48 h posttransfection. Measuring luciferase transcription due to body stimulation. Cells stably expressing TNFR1 and NF-κB-luciferase are plated in 24-well plates at a density of 1×10 5 cells/ml medium and incubated until reaching 80% confluency (approximately 24 hours). Cells are then incubated for 6 hours with 50 ng/ml TNF and various concentrations of TNFR1 antagonist. NF-κB-stimulated luciferase activity was determined by washing cells twice with ice-cold PBS and adding 200 μl of ice-cold lysis buffer (25mM Tris-phosphate pH 7.8, 8mM MgCl2, 1mM DTT, 1% Triton X-100, 15% glycerol) and incubation on ice for 5 minutes. The cell extract is then scraped into a 1.5 ml Eppendorf tube, centrifuged to pellet cell debris, and 100 μl of supernatant used to measure luciferase induction using a luminometer. The IC50 is then calculated by plotting relative luminescence units (RLU) versus TNFR1 antagonist concentration and using curve fitting software such as GraphPad Prism. Other similar assays to assess inhibition of TNFR1 signaling include measuring induction of phospho-IκBα, indicative of activation of the classical NF-κB pathway, in TNFR1-expressing cells treated with TNF. assays (see, eg, Rauert et al. (2011) Cell Death Dis. 2(8):e194).

Potential TNFR1 antagonists exhibiting at least 80% inhibition of TNFR1 signaling and an IC50 value approximately equal to the Kd (ie, <5 nM), as determined above, are selected for further optimization.
[Example 2]

Optimization of selected candidate monovalent TNFR1 antagonist molecules Optimization of affinity for TNFR1 and efficacy of inhibiting TNFR1 signaling Selection criteria outlined above (i.e., Kd and IC50 values <5 nM, high Candidate TNFR1 antagonist molecules are optimized to increase affinity for TNFR1 and potency of inhibiting TNFR1 signaling. This is achieved by methods including one or more of random mutagenesis, site-directed mutagenesis, molecular modeling, and/or error-prone PCR, and is less than 1 nM, generally at least equal to or less than 100 nM. , less than or equal to 50 nM, less than or equal to 10 nM, or less than or equal to 5 nM. For example, this means that the most important amino acids for binding are conserved, while the remaining amino acids of the VH domain are subjected to mutagenesis and a phage library is prepared and screened for high-affinity binding variants against the selected TNFR1. (see Tiller et al. (2017) Frontiers Immunol. 8:986). According to this method, a phage display library is created to select such mutants. In the first step, computational and experimental alanine scanning mutagenesis identifies sites within the complementarity determining regions (CDRs) that allow mutagenesis while maintaining antigen binding. Based on natural antibody diversity, the most permissive CDR positions are then mutated using degenerate codons that encode the wild-type residues and a small number of residues that occur most frequently at each CDR position. let This mutagenesis approach yields antibody libraries with mutants with a wide range of CDR mutations, including antibody domains with a single mutation and other antibody domains with dozens of mutations. The final step is to screen the yeast surface-displayed library (approximately 10 million variants) to identify the CDR mutations that most increase affinity.

Half-life extension The optimized molecule is then subjected to half-life extension, for example by fusion with an IgG Fc domain, particularly a modified Fc domain, or human serum albumin (HSA), or by PEGylation, as described above in the detailed description. to a phase elongation moiety to achieve an in vivo serum half-life of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or more, such as 10-12 days, in SCID mice. . The modified molecules are then retested in the in vitro assays described above to confirm that high affinity binding to TNFR1 and potent inhibition of TNFR1 signaling is retained. Production of successful candidates will be scaled up for further in vitro and in vivo assays.

In Vitro Assay Phagocytosis Assay As discussed and explained in the detailed description, existing TNF blockers that inhibit TNFR1 activity also inhibit TNFR2. There are two types of black box warnings from regulators for TNF blockers. The first is the susceptibility to infection of patients treated with anti-TNF (TNF blockers). These infections can be bacterial, mycobacterial (e.g., tuberculosis), fungal (e.g., histoplasmosis, aspergillosis, candidiasis, coccidioidomycosis, blastomycosis, pneumocystis), viral (e.g., hepatitis B), and other Potential involvement of various organ systems and sites by opportunistic pathogens (organisms that do not normally cause disease in healthy people, but can cause serious illness if a person's immune system (resistance) is weakened) There is. Another published black box warning is related to pediatric malignancies (see, eg, online.epocrates.com/u/10b3301/Humira/Black+Box+Warnings). As discussed in the detailed description, this vulnerability is due to the complete blockage of signaling by TNFR1 and TNFR2 when TNF, the ligand for TNFR1 and TNFR2, is blocked. This suppression of the innate immune effects of TNF is mediated by TNFR2 (see, e.g., Ahmad et al. (2018) Front. Immunol. 9:2572), and the transmembrane form of TNF preferentially activates TNFR2. (see, e.g., Miller et al. (2015) Journal of Immunology 195(6):2633-2647).

Anti-TNF therapeutics such as adalimumab, infliximab, and etanercept have been shown to have an inhibitory effect on IFN-γ-induced phagosome maturation in human THP-1 cells differentiated with phorbol myristate acetate. Adalimumab and infliximab, but not etanercept, inhibit phagosome maturation in primary human peripheral blood monocyte-derived macrophages in the presence or absence of IFN-γ (e.g., Harris et al. (2008) J. Infect. Dis. 198:1842-1850). In view of the above, an advantage of specific TNFR1 inhibitor constructs is the preservation of TNFR2 function and thus macrophage function; macrophages are important in clearing opportunistic infections from organisms. Opportunistic pathogens include mycobacteria that cause tuberculosis (see, eg, Fraga et al. (2018) Curr. Issues Mol. Biol. 25:169-198). Macrophage function is disrupted in autoimmune diseases in patients treated with TNF blockers. TNFR1-specific antagonists inhibit only TNFR1 function, thus avoiding disruption of TNFR2 function, which is required for normal macrophage function. These TNFR1-specific antagonists can be identified by assaying for anti-TNF effects on macrophage phagocytosis. Some of the identified TNFR1-specific antagonists also stimulate TNFR2 function and thus boost macrophage activity (TNFR1 antagonists and TNFR2 agonists).

Assay to determine the effect of TNFR1 antagonists on macrophage responses to Mycobacterium tuberculosis infection. TNF plays an important role in mediating inflammatory host responses against various pathogens, including Mycobacterium tuberculosis; It also plays a role in the immunopathology of TB. Mycobacterial infection induces the secretion of TNF by macrophages. TNF increases the ability of macrophages to phagocytose and kill mycobacteria. TNF also stimulates macrophage apoptosis and increases killing and presentation of mycobacterial antigens by dendritic cells. TNF is also required for granuloma formation and maintenance; neutralization of TNF in mice chronically infected with M. tuberculosis disrupts granuloma integrity, worsens infection, and increases mortality. TNF blockers, such as adalimumab and infliximab, increase susceptibility to infection with various pathogens, including Mycobacterium tuberculosis, and reduce the risk of reactivation of latent tuberculosis by suppressing the maturation of mycobacteria-containing phagosomes in human macrophages. enhance Phagosome maturation (i.e., phagosome acidification and fusion with lysosomes) is essential for the presentation of mycobacterial antigens to T cells and the initiation of adaptive immune responses (e.g., Harris et al. (2008) J. Infect. Dis. 198:1842-1850).

To identify and/or characterize TNFR1 antagonists, the effect of the TNFR1 antagonists provided herein on macrophage responses to Mycobacterium tuberculosis infection is assessed. We analyzed mycobacteria-containing phagosome maturation in human macrophages using the method described in Harris et al. (2008) J. Infect. Compare with. TNFR1 antagonists that do not increase susceptibility to infection are of interest compared to known TNF blockers such as adalimumab.

Preparation of THP-1 cells and monocyte-derived macrophages (MDM) Human THP-1 cells are cultured in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS; Gibco). Cells are differentiated into macrophage-like cells by treatment with 100 nmol/L phorbol myristate acetate (PMA) for 24 hours and then cultured in regular medium for 3 days. To prepare human monocyte-derived macrophages (MDMs), peripheral blood mononuclear cells (PBMCs) were collected from healthy donor blood using density gradient centrifugation on Histopaque®-1077 (Sigma). isolate Monocytes are isolated by adhesion to gelatin-coated culture dishes and cultured overnight in RPMI 1640 containing 5% human AB serum (Sigma). Adherent cells are removed with 10 mmol/L EDTA in PBS and grown on coverslips in 12-well plates for 10 days. THP-1 cells and MDM are grown on coverslips at a concentration of 2 x 105 cells/well.

Preparation of Mycobacteria Green Fluorescent Protein (GFP) labeled Mycobacterium bovis Bacillus Calmetter-Guerin (GFP-BCG) and attenuated Mycobacterium tuberculosis strain H37Ra and its pathogenic counterpart H37Rv is grown in Middlebrook 7H9 broth containing 0.5% Tween, 0.2% glycerol, and 10% albumin-dextrose-catalase supplement (BD). Mycobacteria are grown to log phase before use and resuspended in RPMI 1640 containing 10% FBS before infection. Mycobacterium tuberculosis H37Ra strain is fluorescently labeled with PKH67 (Sigma) and H37Rv strain is labeled with fluorescein isothiocyanate (FITC, 1 mg/mL; Sigma) according to the manufacturer's protocol.

Determination of phagosome maturation Mycobacteria inhibit phagosome-lysosome fusion, preventing acidification and recruitment of lysosomal hydrolases. This block in phagosome maturation can be overcome by pre-treating macrophages with IFN-γ. Treatment of mycobacteria-infected cells with TNF (5 ng/mL) also promotes phagosome acidification. To determine the effects of TNFR1 antagonists and TNF blockers on mycobacterial phagosome-lysosome fusion in macrophages, PMA differentiated THP-1 cells or human peripheral blood MDM were treated with GFP-BCG, or PKH67-labeled Mycobacterium tuberculosis strain H37Ra, or FITC-labeled Mycobacterium tuberculosis strain H37Rv was infected in the presence of a TNFR1 antagonist or TNF blocker, with or without IFN-γ treatment, and phagosome-lysosome fusion was detected using Lyso Tracker® as a marker for acidified phagosomes. ) Red (LT) and analyzed by confocal microscopy using CD63 and cathepsin D as phagolysosomal markers. Inhibition of IFN-γ-induced phagosome maturation/acidification is determined by colocalization of labeled mycobacteria with Lyso Tracker® Red, CD63, or cathepsin D. For example, a decrease in the percent colocalization of labeled mycobacteria with LT, CD63, or cathepsin D compared to a control is indicative of inhibition of IFN-γ-induced phagosome acidification.

TNFR1 antagonists or TNF blockers (e.g., adalimumab, infliximab, etanercept, etc.; 10 μg/mL) with or without IFN-γ (200 U/mL) were administered to THP-1 cells or Add to MDM. As a control, cells are treated with medium alone or with 10 μg/mL human IgG1 (Calbiochem) from a patient with an IgG1-producing myeloma. Next, the cells were transferred to M. M. bovis GFP-BCG, PKH67-labeled M. tuberculosis H37Ra, or FITC-labeled M. tuberculosis H37Rv are infected for 15 minutes, washed three times with PBS to remove unbound mycobacteria, and incubated for 2 hours. Multiplicity of infection (MOI) is recorded microscopically by acid-fast staining 15 minutes after infection of macrophages. Cells are infected at an MOI of 1-5 bacteria at approximately 70% cells.

After 2 hours of incubation, cells are fixed with 2% paraformaldehyde for 20 minutes at room temperature (RT); for strain H37Rv, cells are fixed with 4% paraformaldehyde overnight. Cells are then permeabilized with 0.1% Triton X-100 in PBS and blocked with 1% bovine serum albumin and 1% goat serum in PBS for 30 minutes at room temperature. Cells were incubated with primary antibodies (1 μg/mL mouse monoclonal antibody against CD63 (LAMP-3; Santa Cruz Biotechnology); or 10 μg/mL mouse monoclonal antibody against cathepsin D (Calbiochem)) for 1 h at room temperature, followed by secondary Incubate with antibody (4 μg/mL Alexa Fluor 488 or 568 labeled goat anti-mouse IgG; Invitrogen) for 1 hour at room temperature. Alternatively, cells are incubated with Lyso Tracker® Red DND-99 (100 nmol/L; Invitrogen) for the last 60 minutes of incubation with mycobacteria prior to fixation. LysoTracker® Red DND-99 is a red fluorescent dye for labeling and tracking acidic organelles (such as acidified phagosomes) in living cells.

Coverslips are mounted on glass slides using fluorescent mounting medium (Dako) and images are recorded with a laser scanning confocal microscope such as an Olympus FluoView® 1000 and a Zeiss LSM 510 laser scanning confocal microscope. Images are analyzed and prepared using appropriate software and Adobe Photoshop.

Measurement of TNF THP-1 cells are prepared as described above and infected with BCG or Mycobacterium tuberculosis H37Ra with or without IFN-γ pretreatment. The level of immunoreactive TNF (secreted in response to mycobacterial infection) in the supernatant is measured using a commercially available ELISA kit (R&D systems) according to the manufacturer's instructions.

Regulatory T cell (Treg cell) and cytokine assays that distinguish between TNF blockade (such as treatment with adalimumab, infliximab, or etanercept) and specific TNFR1 inhibition1. Preservation of FoxP3 Expression TNF blockade (using adalimumab, rituximab, or etanercept) on FoxP3 promoter methylation as a surrogate marker for functional regulatory T cells is compared to specific TNFR1 inhibition. Transgenic mice constitutively expressing human TNFR1 (HuTNFR1) are used to assess the differential effects of TNF blockade versus specific TNFR1 inhibition on FoxP3 promoter methylation. Transgenic mice are produced by standard methods and can be produced by a contractor service such as Cyagen, Genoway, or Polygene or by any method.

This effect is evaluated in transgenic mice with collagen-induced arthritis (CIA), a widely used model of RA. C57/BL6N. Q; Mice with the H-2q/HuTNFR1/Hunt background are generated by Genoway or TaconicLabs. Mice are immunized with bovine type II collagen emulsified in complete Freund's adjuvant (CFA) as described by Tseng et al. ((2019) Proc. Natl. Acad. Sci. U.S.A. 116:21666-21672). FoxP3 methylation assays are also performed as described by Tseng et al. ((2019) Proc. Natl. Acad. Sci. U.S.A. 116:21666-21672). Regulatory T cells from TNF blockade-treated mice have lower levels of FoxP3 than regulatory T cells with specific TNFR1 blockade, as determined by the median fluorescence intensity (MFI) and histogram of FoxP3 in CD4+CD25+ cells. manifest.

2. Specific inhibition of TNFR1 vs. TNF blockers evades regulatory T cells (Spares)
Transgenic (C57/BL6N.Q; H-2q/HuTNFR1/Hunt) CIA mice were generated using the method described by McCann et al. ((2014) Arthritis & Rheumatology 66(10):2728-2738). The numbers of regulatory T cells in lymph nodes and spleen are compared after treatment with TNF blockers and specific inhibitors of TNFR1.

3. Inflammatory cytokines are upregulated with TNF blockade versus specific TNFR1 inhibition in collagen-induced arthritis Transgenic mice with collagen-induced arthritis (CIA) (C57/BL6N.Q; H-2q/HuTNFR1/Hunt) (See, eg, McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738) are treated with a TNF blocker and a TNFR1-specific antagonist. Assess serum inflammatory cytokines (IFN-gamma, IL-12p70, IL-10, RANTES (CCL5); see e.g. McCann et al. (2014) Arthritis & Rheumatology 66(10):2728-2738) . Antagonists specific for blocking TNFR1 induce significantly less one or more of IFN-gamma, IL-12p70, IL-10, or RANTES (CCL5). This is due to evasion of TNFR2 function and regulatory T cell function in the spleen and lymph nodes.

In Vivo Assay The study is performed using humanized (HuTNFR1/HuTNF) transgenic mice. TNFR1 antagonist molecules are evaluated for efficacy in multiple autoimmune disease models. These include the models described above expressing human transgenes for TNFR1 and TNF. Alternative models of autoimmune diseases are known. For example, models, including those for RA, are described in detail in Schinnerling et al. ((2019) Front. Immunol 10:203). Specific TNFR1 antagonist constructs, as well as other constructs provided herein, are tested in multiple models to establish efficacy. Models include at least rheumatoid arthritis (RA), Crohn's disease, and multiple sclerosis (experimental autoimmune encephalitis) models (discussed in the detailed description).

Inflammatory bowel disease (IBD, including ulcerative colitis and Crohn's disease) was the first approved indication for TNF blockers. A number of mouse models of these diseases are available and described by Mueller ((2002) Immunology 105(1):1-8). As discussed in the detailed description, autoimmune neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer's disease are important disease targets. Alzheimer's disease (AD) is the leading cause of dementia worldwide and represents one of the most serious health problems for the elderly. An estimated 5.4 million Americans have AD, and this number is expected to triple by 2050 unless there are medical breakthroughs to halt, prevent, or slow the disease. (See, e.g., Chang et al. (2017) J. Cent. Nerv. Syst. Dis. 9:1179573517709278). Evidence indicates that the TNFR1 antagonist constructs and other constructs provided herein are therapeutic candidates, as subjects who undergo long-term treatment with TNF blockers are less likely to develop disease (e.g., Chou et al. (2016) CNS Drugs 30:1111). Data indicate that upregulated TNF expression is associated with various neurodegenerative diseases and conditions such as Alzheimer's disease, Parkinson's disease, stroke, and multiple sclerosis (e.g., McCoy et al. (2008) J. Neuroinflammation 5(1):45). However, existing TNF blockers do not appear to be effective in treating, ameliorating, preventing, or slowing disease progression (see, e.g., Tortarolo et al. (2015) J. Neurochem. 135:109-124). thing). As described herein, several lines of evidence indicate that TNF blockers do not work in such indications due to simultaneous inhibition of TNFR1 and TNFR2; It has neuroprotective properties that are lost with treatment. Others have attempted to approach this problem with various forms of "TNFR1 inhibitors" or "TNFR2 agonists." None of these studies involved determining whether TNFR1 or TNFR2 are selectively targeted, as opposed to being one of many epitopes in the body that can be targeted. No cross-reactivity was involved.

This problem is solved herein. Initially, a family of anti-TNFR1 antagonists was generated and tested in the models described above and shown to act as expected. Next, use immunochemistry to demonstrate that they are selective. Several contract research organizations offer this service (eg, SinoBiological, Inc., and LSBio).

As discussed in the detailed description, other autoimmune and chronic inflammatory disease conditions are associated with the presence of TNF. These include type II diabetes and endometriosis. Mouse models for these diseases are known, and HuTNFR1/HuTNF transgenic versions of these mice are used to demonstrate the efficacy of the specific anti-TNFR1 antagonists provided herein.

Acute Respiratory Distress Syndrome Viral pathogens of the respiratory tract (eg, influenza, SARS virus/coronavirus) infect airway epithelial cells, and tissue-resident alveolar macrophages are the first responders to viral infections of the lungs. They influence clearance by phagocytosis of opsonized viral particles or infected apoptotic cells and release of excessive inflammatory cytokines and chemokines to initiate immune responses (e.g., Herold et al. (2015) Eur. (See Resp. J. 45:1463-1478). TNF blockers have been shown to prolong the survival of mice infected with influenza (see, eg, Shi et al. (2013) Crit. Care 17:R301). TNF blockers have been used to treat SARS-Cov2 (see, eg, Feldmann et al. (2020) Lancet 395:1407-1409). As noted in the detailed description, a known effect of TNF blockers is to reduce regulatory T cells (Tregs), but this is problematic because Tregs are natural suppressors of inflammation. be. Therefore, the TNFR1-specific inhibitors described and provided herein that do not interact with or agonize TNFR2 are excellent for this purpose. To test the constructs provided herein, mice are engineered to lack endogenous TNFR1 and express HuTNFR1/HuTNF. These mice develop HuTNF/HuTNFR1-induced acute respiratory distress syndrome after influenza infection (as described in Shi et al. (2013) Crit. Care 17:R301). A TNFR1-specific antagonist construct that does not antagonize TNFR2 is administered. Effectiveness is determined by any of several criteria:
1. Circulating inflammatory cytokines (e.g., IFN-gamma, IL-1 alpha, IL1-beta, IL-17) are significantly reduced in response to TNF blockers;
2. Significantly faster recovery is measured by weight gain compared to TNF blockers (see, eg, Shi et al. (2013) Crit. Care 17:R301); and 3. Compared to TNF blockers, survival rates are significantly increased (see, eg, Shi et al. (2013) Crit. Care 17:R301).

Transgenic mice expressing human TNFR1 and human TNFR2 for use in disease models are those described by Atretkhany et al. (2018) Proc. Natl. Acad. Sci. U.S.A. 115(51):13051-13056 etc., produced by standard genetic engineering methods known in the art. Specific in vivo assays for various diseases and conditions are as follows. For example, using a humanized RA mouse model, such as the collagen-induced arthritis (CIA) model of RA, or any other animal model of RA known in the art and/or described herein. The therapeutic effects of the TNFR1 antagonist molecules provided herein are evaluated and compared to the therapeutic effects of anti-TNF therapies such as etanercept or adalimumab. A TNFR1 antagonist molecule provided herein, or an anti-TNF therapy such as etanercept or adalimumab, is administered to the animal daily for a total of 10 days after the onset of clinical arthritis in one or more limbs. The extent of swelling of the initially affected joint is monitored by measuring the thickness of the foot using calipers. Inflammatory cytokines and chemokines, such as granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-10 (IL-10), IL-1β, IL-6, IL-protein 8, RANTES (CCL5), and monocytes Serum is collected from mice for measurements such as chemotactic protein 1 (MCP-1; also known as CCL2). Next, regression of RA in a mouse model will be compared between mice treated with a TNFR1 antagonist molecule and mice treated with etanercept or adalimumab.

The TNFR1 antagonist molecules provided herein are also tested in humanized mouse models of Severe Acute Respiratory Syndrome (SARS) and virus-induced cytokine storm. Mouse models of SARS were generated, for example, by infecting humanized hTNFR1 (or hTNFR1/hTNFR2) knock-in mice with various doses, such as 102, 103, 104, and 105 plaque-forming units (PFU) of SARS-CoV. do. A TNFR1 antagonist molecule is administered to infected mice and survival is assessed and compared to survival of mice administered anti-TNF therapy such as adalimumab.

Virus-induced mouse models of cytokine storm include, for example, the lymphocytic choriomeningitis virus (LCMV)-induced model of cytokine storm syndrome (CSS). An LCMV-induced mouse model of CSS is generated by intraperitoneal administration of 2 x 105 PFU of LCMV-Armstrong, into 8-12 week old perforin-deficient (Prf-/-) or Prf-Tmem178 double knockout mice. To deplete monocytes/macrophages, 100 μl of clodronate-liposomes are injected intravenously into Prf −/− mice 2 days before and 48 and 96 hours after LCMV infection. Alternatively, 1 mg of neutralizing anti-CSF1 antibody (clone 5A1, BioXCell) is administered 2 days before infection and 0.5 mg of antibody 48 and 96 hours later. Animals are bled by submandibular venipuncture to measure serum cytokines on days 3 and 8 post-infection (see e.g. Mahajan et al. (2019) J. Autoimmun. 100:62-74). thing).
[Example 3]

Identification and Removal of Immunogenic Sequences As described herein, immunogenic sequences, such as B-cell and/or T-cell epitopes within a protein therapeutic, can, for example, neutralize and/or or through the formation of anti-drug antibodies (ADA) that facilitate the removal of therapeutic agents from the body, which can adversely affect the activity, efficacy, and in vivo half-life of therapeutic agents. Immunogenic sequences can also induce undesirable immune responses resulting in clinical complications such as delayed infusion-like allergic reactions, anaphylaxis, and even life-threatening autoimmunity. This is detrimental to the safety and tolerability of protein therapeutics. As a result, candidate protein therapeutics are screened for immunogenicity and identified immunogenic sequences are removed/replaced, e.g. by mutagenesis, to improve the in vivo efficacy and safety profile of the therapeutic and its Ensure success is transferred from preclinical research to the clinic.

Immunogenic sequences are identified using methods described in the detailed description or known to those skilled in the art, including the use of in silico immunogenicity prediction tools and in vitro immunogenicity tests. For example, as described elsewhere herein, linear B cell epitopes include, for example, ABCPred, APCPred, BCPRED, BepiPred, LBtope, BcePred, EPMLR, BEST, COBEpro, and SVMTriP, or this Predict using any other available in silico linear B cell epitope prediction tools described herein and/or known to those skilled in the art. Conformational B cell epitopes include, for example, CEP, DiscoTope, BEpro, ElliPro, SEPPA, CBTOPE, EPITOPIA, EPCES, EPSVR, EPMeta, PEASE, EpiPred, 3DEX, PEPOP, PEPOP2.0, and EpiSe. arch, or herein Predict using any other available in silico conformational B cell epitope prediction tools described and/or known to those skilled in the art. T cell epitopes include, for example, EpiMatrix, JanusMatrix, IEDB, SYFPEITHI, MHC Thread, MHCPred, MHCPred2.0, EpiJen, NetMHC, NetCTL, nHLAPred, SVMHC, ProPred, M MBPred, Protean 3D, and Bimas, or as described herein and/or any other available in silico T cell epitope prediction tools known in the art. Below is an exemplary analysis of the human TNFR1 antagonist DMS5541 sequence (SEQ ID NO: 38) for immunogenic linear B cell epitopes.

Analysis of DMS5541 for Immunogenic Linear B Cell Epitopes The sequence of the human TNFR1 antagonist DMS5541 (SEQ ID NO: 38) was analyzed for potential immunogenicity using the SVMTriP algorithm and linear B cell epitopes within the molecule were analyzed. was detected. The algorithm identified three possible epitopes in the sequence of DMS5541, as shown in Table 13 below. The results show that the epitope with the sequence AVKGRFTISRDNSKNTLYLQ, corresponding to residues 63-82 of SEQ ID NO: 38, has a high probability of immunogenicity. The three identified epitopes are then tested for immunogenicity in an in vitro B cell assay. All sequences that are positive for immunogenicity undergo an alanine scan. Positive amino acids/sequences are modified by substituting each amino acid in the sequence one by one with an alanine residue until the immunogenic epitope is destroyed. This produces safer and more effective TNFR1 antagonists.

As a positive control, the SVMTriP algorithm was also used to predict the known high immunogenicity of adalimumab; adalimumab administered without methotrexate is immunogenic in approximately 50% of patients (e.g., Ducourau et al. al. (2020) RMD Open 6:e001047). The SVMTriP algorithm identified at least 10 possible epitopes in the heavy chain of adalimumab, four of which had high probability and correlated well with clinical data, validating the use of this program to predict immunogenicity. Ru.

The results of the SVMTriP analysis of DMS5541 are supported by a second algorithm ABCPred that was used to predict immunogenicity within the sequence of DMS5541. All three B cell epitopes predicted by SVMTriP were also included in the ABCPred epitope prediction results. For example, as shown in Table 14 below, epitopes AVKGRFTISRDNSKNT, TGRWVPFEYWGQGTLV and STDIQMTQSPSSLSAS (see table below for respective residue positions in SEQ ID NO: 38) overlap with three epitopes identified by SVMTriP. Contains the sequence.

[Example 4]

Exemplary TNFR1 Antagonist Constructs Containing Human TNFR1 Antagonist Antibody Fragments (dAbs, scFvs, Fabs) Provided herein are TNFR1 antagonist constructs that selectively inhibit TNFR1 without inhibiting TNFR2. . To avoid TNFR1 receptor clustering that agonizes TNFR1, TNFR1 antagonists are monomeric and monovalent. TNFR1 antagonists include human single domain antibodies (dAbs) specific for TNFR1. dAbs contain variable heavy (VH) or variable light (VL) domains. For example, the dAb may be directed to any dAb whose amino acid sequence is set forth in any one of SEQ ID NOs: 54-672, or to any dAb of SEQ ID NOs: 54-672 that retains binding affinity for TNFR1. Includes dAbs that have at least or at least about 90% or 95% sequence identity. Alternatively, TNFR1 antagonists include scFvs, Fabs, or other antigen-binding fragments, such as those derived from human TNFR1 antagonist antibodies such as H398 or ATROSAB. For example, the TNFR1 antagonist may be an H398-derived scFv set forth in SEQ ID NOs: 677 or 678; or an ATROSAB-derived scFv set forth in any one of SEQ ID NOs: 673-676; or SEQ ID NOs: 679 and 680 (FabATR), respectively. , or ATROSAB-derived Fab fragment light chain and heavy chain set forth in either SEQ ID NO: 681 or 682 (Fab 13.7), respectively; or at least or at least about 90 scFv or Fab fragments having % or 95% sequence identity, or Fab light and heavy chains of either SEQ ID NO: 679 and 680 (FabATR), respectively, or SEQ ID NO: 681 or 682 (Fab13.7), respectively. and retains affinity for TNFR1.

TNFR1 antagonists include serum half-life extenders, such as IgG Fc, particularly Fc modified to eliminate or reduce ADCC, ADCP, and/or CDC, human serum albumin (HSA), and/or poly(ethylene ) It is fused to glycol (PEG) molecules, etc. For example, the C-terminus of a human anti-TNFR1 dAb, scFV, Fab or other antigen-binding fragment is fused via a linker to the N-terminus of the Fc region of a human IgG1 or IgG4 antibody. An IgG1 Fc region, such as the IgG1 Fc from trastuzumab (see SEQ ID NO: 27), or an IgG4 Fc region, such as the IgG4Fc from nivolumab (see SEQ ID NO: 30), is used. The linker comprises a portion of the hinge sequence of trastuzumab, containing the sequence of amino acid residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26), if the Fc is derived from trastuzumab, or if the Fc is derived from nivolumab. containing the sequence of amino acid residues ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), the hinge sequence of nivolumab, or a portion thereof that provides plasticity or other structural properties. be able to. To confer protease resistance and increase the plasticity of the fusion protein, the SCDKTH or ESKYGPPCPPCP hinge sequence is combined with a short glycine-serine (GS) peptide linker, such as (GSGS) or (GGGGS)n (e.g., SEQ ID NO: 707, respectively). (see residues 199-202, and 116-120 of ), (where n = 1-5 or 1-6), or such as GGGGSGGGGSGGGGS (e.g., residues 116-130 or SEQ ID NO: 707). Replaced by other combinations of Gly and Ser residues. In other embodiments, the C-terminus of the human anti-TNFR1 dAb, scFv, Fab or other antigen-binding fragment is linked to a GS linker, the GS linker being sufficient to provide plasticity at the N-terminus of the corresponding Fc region. connected to all or part of the hinge sequence of trastuzumab or nivolumab, which is connected to the end. In some embodiments, a second Fc subunit is linked to the first Fc subunit to increase the serum half-life and stability of the molecule. Because there are two Fc regions, the resulting construct contains one discontinuous Fc region and is therefore not a fusion protein. In some embodiments, the N-terminus of the human TNFR1 antagonist dAb, scFV, Fab or other antigen-binding fragment is fused to the C-terminus of the serum half-life extender via a linker, as described above.

Also, an anti-TNFR1 dAb, scFv, fused to human serum albumin (HSA) via a short peptide linker such as (GSGS)n or (GGGGS)n (where n=1 to 5 or 6), e.g. GGGGSGGGGSGGGGS; Also provided herein are TNFR1 antagonist fusion proteins containing Fabs, or other antigen-binding fragments.

Also provided herein are TNFR1 antagonist molecules containing an anti-TNFR1 dAb, scFv, Fab, or other antigen-binding fragment linked to a PEG molecule that is at least 30 kDa in size.

As described herein, these constructs can be modified to reduce or eliminate immunogenicity. TNFR1 antagonist dAbs, scFvs, Fabs, or other antigen-binding fragments are analyzed by in silico, in vitro, and/or in vivo methods to predict or identify immunogenic sequences. Once immunogenic sequences, such as B-cell and/or T-cell epitopes, have been identified, the identified sequences can be isolated by mutagenesis, e.g., by alanine scanning, as described elsewhere herein. Modify and deimmunize epitopes/remove or replace immunogenic sequences.

Below are exemplary constructs of TNFR1 antagonist fusion proteins described and provided herein. In all embodiments that include a trastuzumab Fc or a nivolumab Fc, the Fc region is modified to reduce or eliminate immune effector functions, including ADCC, ADCP, and CDC, and to enhance binding to FcRn. may be modified to increase the serum half-life of the fusion protein, and immunogenic sequences may be substituted or otherwise modified or removed.

Fc modifications that reduce or eliminate immune effector function are summarized in Table 9 above, and Fc modifications that increase FcRn binding are summarized in Table 7 above. Any one or combination of such modifications is included in the Fc region of the fusion proteins provided herein. All of the exemplary constructs provided herein are prepared with the TNFR1 antagonist at the C-terminus rather than at the N-terminus of the fusion protein.

1a) H398scFv-SCDKTH-Trastuzumab Fc
Provided herein is a human TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) comprises at least amino acid residues fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). It is fused to a portion of the hinge sequence of trastuzumab, which contains the sequence of SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). The H398scFv-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 706):
QVQLQESGAELARPGASVKLSCKASGYTFDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGSGS DFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. Alternatively, the SCDKTH hinge sequence comprises the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues thereof. contains or has up to the entire sequence of the hinge region of trastuzumab.

1b) H398scFv-GGGGSGGGGSGGGGS-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). ). H398scFv-GGGGSGGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 707):
QVQLQESGAELARPGASVKLSCKASGYTFDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGSGS DFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent to Substituted by any other suitable short peptide linker known in the art.

1c) H398scFv-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 6; see also SEQ ID NO: 27). fused to a portion of the hinge sequence of trastuzumab, including at least the sequence of amino acid residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). H398scFv-GGGGSGGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 708):
QVQLQESGAELARPGASVKLSCKASGYTFDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGSGS DFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent to Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). up to the entire sequence of the hinge region of trastuzumab, including the groups.

1d) H398scFv-GGGGSGGGGGSGGGGS-HSA
Provided herein is a TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) is fused to the N-terminus of human serum albumin (HSA) (SEQ ID NO: 35) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). , GGGGSGGGGSGGGGS peptide linker. The H398 scFv-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 709):
has.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker (where n=1, 2, 3, 4 or 5), or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1e) H398scFv-GGGGSGGGGSGGGGS-PEG30kDa
Provided herein is a TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) is fused to a GGGGSGGGGSGGGGS peptide linker, which is covalently attached to a PEG molecule of size 30 kDa.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively or additionally, the PEG molecule can have a molecular weight equal to or greater than about 30 kDa, such as 35 kDa, 40 kDa, 45 kDa, or 50 kDa.

1f) DOM1h-574-16-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 has at least the amino acid residue SCDKTH fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). (corresponding to residues 222-227 of SEQ ID NO: 26), is fused to a portion of the hinge sequence of trastuzumab. DOM1h-574-16-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 710):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL Has HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence comprises the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues thereof. containing up to the entire hinge region sequence of trastuzumab.

1g) DOM1h-574-16-GGGGSGGGGGSGGGGS-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). are doing. DOM1h-574-16-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 711):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV Having LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1h) DOM1h-574-16-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). ) is fused to a portion of the hinge sequence of trastuzumab containing at least the sequence of residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). DOM1h-574-16-GGGGSGGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 712):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent to Substituted by any other suitable short peptide linker known in the art. Alternatively or additionally, the SCDKTH hinge sequence comprises 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. up to the entire sequence of the hinge region of trastuzumab containing groups.

1i) DOM1h-574-16-GGGGSGGGGGSGGGGS-HSA
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of human serum albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). are doing. The DOM1h-574-16-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 713):
has.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker (where n=1, 2, 3, 4 or 5), or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1j) DOM1h-574-16-GGGGSGGGGSGGGGS-PEG30kDa
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which is covalently attached to a PEG molecule of size 30 kDa.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker (where n=1, 2, 3, 4 or 5), or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively or in addition, the PEG molecule can have a molecular weight greater than 30 kDa, such as 35 kDa, 40 kDa, 45 kDa, or 50 kDa.

1k) DOM1h-549-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 contains at least the sequence of residues SCDKTH (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27), which is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 222-227 of SEQ ID NO: 26). DOM1h-549-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 714):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence comprises the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues thereof. containing up to the entire hinge region sequence of trastuzumab.

1l) DOM1h-549-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOMlh-549 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). There is. DOM1h-549-GGGGSGGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 715):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1m) DOM1h-549-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to a GGGGSGGGGSGGGGS peptide linker, which is attached to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). fused to a portion of the hinge sequence of trastuzumab containing at least the sequence of residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). DOM1h-549-GGGGSGGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 716):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY Having RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or a portion containing 11 contiguous residues, up to the entire sequence of the hinge region of trastuzumab.

1n) DOM1h-549-GGGGSGGGGGSGGGGS-HSA
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of human serum albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). There is. DOM1h-549-GGGGSGGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 717):
has.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1o) DOMlh-549-GGGGSGGGGSGGGGS-PEG30kDa
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to a GGGGSGGGGSGGGGS peptide linker, which is covalently attached to a PEG molecule of size 30 kDa.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively or additionally, the PEG molecule can have a molecular weight greater than 30 kDa, such as 35 kDa, 40 kDa, 45 kDa, or 50 kDa.

1p) DOM1h-574-208-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 contains at least the residues SCDKTH, which is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). (corresponding to residues 222-227 of SEQ ID NO: 26), fused to a portion of the hinge sequence of trastuzumab. DOM1h-574-208-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 718):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL Has HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or 11 contiguous sequences of trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). Some residues, including up to the entire sequence of the hinge region of trastuzumab, are replaced.

1q) DOM1h-574-208-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). are doing. DOM1h-574-208-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 719):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV Having LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1r) DOM1h-574-208-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). ) is fused to a portion of the hinge sequence of trastuzumab containing at least the sequence of residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). DOM1h-574-208-GGGGSGGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 720):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or replaced by at least a portion comprising 11 contiguous residues up to the entire sequence of the hinge region of trastuzumab.

1s) DOM1h-574-208-GGGGSGGGGGSGGGGS-HSA
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to a GGGGSGGGGGSGGGGS peptide linker, which is fused to the N-terminus of human serum albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). are doing. DOM1h-574-208-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 721):
has.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1t) DOM1h-574-208-GGGGSGGGGSGGGGS-PEG30kDa
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to a GGGGSGGGGSGGGGS peptide linker, which is covalently attached to a PEG molecule of size 30 kDa.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively or additionally, the PEG molecule can have a molecular weight of at least 30 kDa or greater than 30 kDa, such as 35 kDa, 40 kDa, 45 kDa, or 50 kDa.

1u) DOM1h-131-206-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 contains at least the residues SCDKTH, which is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). (corresponding to residues 222-227 of SEQ ID NO: 26), fused to a portion of the hinge sequence of trastuzumab. DOM1h-131-206-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 722):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or 11 of the hinge region of trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). up to the entire sequence, including consecutive residues.

1v) DOM1h-131-206-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). are doing. DOM1h-131-206-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 723):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV Having LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1w) DOM1h-131-206-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). ) is fused to a portion of the hinge sequence of trastuzumab containing at least the sequence of residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). DOM1h-131-206-GGGGSGGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 724):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is another Gly-Ser (GS) linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or as described herein. or any other suitable short peptide linker described in or known in the art. Alternatively, or in addition, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or replaced in whole or in part, up to the entire sequence, containing 11 contiguous residues.

1z) H398scFv-ESKYGPPCPPCP-nivolumab Fc
Provided herein is a human TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) has the sequence ESKYGPPCPPCP (SEQ ID NO: 29 (corresponding to residues 212-223)) to the hinge sequence of nivolumab. The H398scFv-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 726):
QVQLQESGAELARPGASVKLSCKASGYTFDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGSGS DFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or use part of it.

1aa) H398scFv-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) is fused to the GGGGSGGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). It's fused. H398scFv-GGGGSGGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 727):
QVQLQESGAELARPGASVKLSCKASGYTFDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGSGS DFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1ab) H398scFv-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing an scFv derived from human TNFR1 antagonist antibody H398. The scFv contains the VL and VH domains of H398, connected by a (GGGGS)3 peptide linker. The C-terminus of H398scFv (SEQ ID NO: 678) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). ), fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). H398scFv-GGGGSGGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 728):
QVQLQESGAELARPGASVKLSCKASGYTFDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGSGS DFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL Has TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker (where n=1, 2, 3, 4 or 5), or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Use all or part of an array.

1ac) DOM1h-574-16-ESKYGPPCPPCP-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 has the sequence ESKYGPPCPPCP (SEQ ID NO: 29 (corresponding to residues 212-223) of nivolumab. DOM1h-574-16-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 729):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV Having LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or use part of it.

1ad) DOM1h-574-16-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). are doing. DOM1h-574-16-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 730):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker (where n=1, 2, 3, 4 or 5), or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1ae) DOM1h-574-16-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). DOM1h-574-16-GGGGSGGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 731):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker (where n=1, 2, 3, 4 or 5), or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29), up to the entire sequence. nivolumab hinge sequence containing at least a portion of the nivolumab hinge sequence.

1af) DOM1h-549-ESKYGPPCPPCP-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 has the sequence ESKYGPPCPPCP (remaining SEQ ID NO: 29) fused to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). (corresponding to groups 212-223) to the hinge sequence of nivolumab. DOM1h-549-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 732):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or part of it.

1ag) DOM1h-549-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). There is. DOM1h-549-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 733):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV Having LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1ah) DOM1h-549-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). is fused to the hinge sequence of nivolumab, which contains the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). DOM1h-549-GGGGSGGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 734):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

In some embodiments, the GGGGSGGGGSGGGGS linker is a GS linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4, or 5, or a Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Contains all or part of the sequence.

1ai) DOM1h-574-208-ESKYGPPCPPCP-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 has the sequence ESKYGPPCPPCP (SEQ ID NO: 29 (corresponding to residues 212-223) of nivolumab. DOM1h-574-208-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 735):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV Having LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Or you can include some of it.

1aj) DOM1h-574-208-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to a GGGGSGGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). are doing. DOM1h-574-208-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 736):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

1ak) DOM1h-574-208-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). DOM1h-574-208-GGGGSGGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 737):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or any of the linkers described herein or known in the art. be replaced by other suitable short peptide linkers. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Contains all or part of the sequence.

1al) DOM1h-131-206-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 has the sequence ESKYGPPCPPCP (SEQ ID NO: 29 (corresponding to residues 212-223) of nivolumab. DOM1h-131-206-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 738):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGGPWFDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV Having LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or part of it.

1am) DOM1h-131-206-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). are doing. DOM1h-131-206-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 739):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or any of the linkers described herein or known in the art. be replaced by other suitable short peptide linkers.

1an) DOM1h-131-206-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing the human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). fused to the hinge sequence of nivolumab, containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). DOM1h-131-206-GGGGSGGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 740):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or any of the linkers described herein or known in the art. be replaced by other suitable short peptide linkers. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Contains all or part of the sequence.

HSA-linked Vhhs (nanobodies), especially their single-chain forms, were also synthesized, including dAbs such as DOM1h-131-206. Exemplary Vhh constructs were evaluated for binding and inhibition of TNFR1. These will be explained in the examples below.
[Example 5]

Exemplary TNFR1 Antagonist Constructs Containing Human TNFR1 Antagonist TNF Muteins Provided herein are TNFR1 antagonists that selectively inhibit TNFR1 without inhibiting TNFR2. TNFR1 antagonists are monovalent to avoid TNFR1 receptor clustering that agonizes TNFR1. TNFR1 antagonists are dominant-negative inhibitors of TNF that are incapable of signal transduction, also known as TNF muteins, which are engineered variants of TNF that have one or more mutations that abolish signaling through TNFR1. (DN-TNF). For example, a TNF mutein can contain one or more mutations that confer selectivity for TNFR1 but not for TNFR2. TNFR1 selective TNF mutations include L29S, L29G, L29Y, R31E, R31N, R32Y, R32W, S86T, L29S/R32W, L29S/S86T, R32W/ S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, E146R, V1M, R31C, C69V, Y87H, C101A, A145R, V1M/R31C/C69V/Y87H/C101A/A145 R, I97T, Includes any one or more of I97T/A145R, A84S, V85T, Q88N, T89Q, and A84S/V85T/S86T/Y87H/Q88N/T89Q, and combinations thereof.

For example, TNFR1 antagonists include mutations R32W/S86T (SEQ ID NO: 685), V1M/R31C/C69V/Y87H/C101A/A145R (SEQ ID NO: 701; XPro1595, etc.), A84S/V85T/S86T/Y87H/Q88N/T89Q ( SEQ ID NO: 703; R1antTNF) or I97T/A145R (SEQ ID NO: 702; XENP345, etc.).

TNFR1 antagonists have been fused to serum half-life extending agents such as IgG Fc. For example, the C-terminus of a human anti-TNFR1 antagonist TNF mutein is fused via a linker to the N-terminus of the Fc region of a human IgG1 or IgG4 antibody. An IgG1 Fc region, such as the IgG1 Fc from trastuzumab (see SEQ ID NO: 27), or an IgG4 Fc region, such as the IgG4 from nivolumab (see SEQ ID NO: 30), is used. The linker can contain all or part of the hinge sequence of trastuzumab, including at least the sequence of residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) when the Fc is derived from trastuzumab. or, if the Fc is derived from nivolumab, it can contain the hinge sequence of nivolumab, which is ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), or a portion thereof. To confer protease resistance and increase the plasticity of the fusion protein, the SCDKTH or ESKYGPPCPPCP hinge sequence, or a portion thereof, is replaced with a short glycine-serine (GS) peptide linker, such as (GSGS)n or (GGGGS)n (formula medium, n=1 to 5), for example, GGGGSGGGGSGGGGS. In an alternative embodiment, the C-terminus of the human anti-TNFR1 TNF mutein is linked to a GS linker, and the GS linker is connected to the N-terminus of the corresponding Fc region, sufficient to provide plasticity, of trastuzumab or Connected to all or part of the hinge sequence of nivolumab. In some embodiments, a second Fc subunit is linked to the first Fc region, which can increase the serum half-life and stability of the molecule. The resulting construct is not a fusion protein.

The following are exemplary constructs of TNFR1 antagonist fusion proteins containing TNFR1 selective antagonist TNF muteins described and provided herein. In all embodiments containing a trastuzumab Fc or a nivolumab Fc, the Fc region may be modified to reduce or eliminate immune effector functions, including ADCC, ADCP, and CDC, and binding to FcRn. may also be modified to enhance the serum half-life of the fusion protein. Fc modifications that reduce or eliminate immune effector function are summarized in Table 9, and Fc modifications that increase FcRn binding are summarized in Table 7. Any one or combination of such modifications is included in the Fc region of the fusion proteins provided herein.

2a) TNF (R32W/S86T)-SCDKTH-Trastuzumab Fc
Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an R32W/S86T mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO: 685) is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). fused to all or part of the hinge sequence of trastuzumab, containing at least residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). The TNF(R32W/S86T)-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 741):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or 11 of the hinge region of trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). up to the entire sequence.

2b) TNF (R32W/S86T)-GGGGSGGGGSGGGGS-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an R32W/S86T mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO: 685) is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). GGGGSGGGGSGGGGS peptide linker. TNF (R32W/S86T)-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 742):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2c) TNF (R32W/S86T)-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an R32W/S86T mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO: 685) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; (see also SEQ ID NO: 27), fused to a portion of the hinge sequence of trastuzumab containing at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). TNF (R32W/S86T)-GGGGSGGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 743):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, the SCDKTH hinge sequence is the entire sequence of the hinge region of trastuzumab, containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, replaced by a portion containing 9, 10, or 11 consecutive residues.

2d) TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-SCDKTH-Trastuzumab Fc
Provided herein is a human TNFR1 containing TNFR1 selective antagonist TNF mutein with V1M, R31C, C69V, Y87H, C101A and A145R mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. Antagonist fusion protein. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is connected to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; SEQ ID NO: 27 also 26), fused to a portion of the hinge sequence of trastuzumab containing at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 744):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence is the entire sequence of the hinge region of trastuzumab, containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10 , or by a portion containing 11 consecutive residues.

2e) TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGGSGGGGS-Trastuzumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having V1M, R31C, C69V, Y87H, C101A and A145R mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; SEQ ID NO: 27 also GGGGSGGGGGSGGGGS peptide linker. TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 745):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2f) TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having V1M, R31C, C69V, Y87H, C101A and A145R mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is fused to a GGGGSGGGGSGGGGS peptide linker, which is connected to the N-terminus of the Fc region of trastuzumab (the remainder of SEQ ID NO: 26). A portion of the hinge sequence of trastuzumab containing at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) fused to the residues 234-450; see also SEQ ID NO: 27). It is integrated into the department. TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 746):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or by a portion containing 11 consecutive residues, up to the entire sequence.

2g) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-SCDKTH-Trastuzumab Fc
Provided herein are human TNFR1 selective antagonist TNF muteins containing A84S, V85T, S86T, Y87H, Q88N and T89Q mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. Antagonist fusion protein. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is connected to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; SEQ ID NO: 27 also 26), fused to a portion of the hinge sequence of trastuzumab containing at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). The TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 747):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence comprises at least 5, 6, 7, 8, 9, 10, or 11 of the hinge region of trastuzumab containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26). up to the entire sequence, including consecutive residues.

2h) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGGSGGGGS-Trastuzumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having A84S, V85T, S86T, Y87H, Q88N and T89Q mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is connected to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; SEQ ID NO: 27 also GGGGSGGGGGSGGGGS peptide linker. TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 748):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEV Having KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2i) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having A84S, V85T, S86T, Y87H, Q88N and T89Q mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is fused to a GGGGSGGGGSGGGGS peptide linker, which is connected to the N-terminus of the Fc region of trastuzumab (the remainder of SEQ ID NO: 26). A portion of the hinge sequence of trastuzumab containing at least residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26), fused to SEQ ID NO: 27 (corresponding to groups 234-450; see also SEQ ID NO: 27). It is integrated into the department. TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 749):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, the SCDKTH hinge sequence is the entire sequence of the hinge region of trastuzumab, containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, replaced by a portion containing 9, 10, or 11 consecutive residues.

2j) TNF (I97T/A145R)-SCDKTH-Trastuzumab Fc
Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an I97T/A145R mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO: 702) is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). fused to all or part of the hinge sequence of trastuzumab, containing at least residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). The TNF(I97T/A145R)-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 750):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the SCDKTH hinge sequence is the entire sequence of the hinge region of trastuzumab, containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10 , or by a portion containing 11 consecutive residues.

2k) TNF (I97T/A145R)-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an I97T/A145R mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO: 702) is fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). GGGGSGGGGSGGGGS peptide linker. The TNF (I97T/A145R)-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 751):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2l) TNF (I97T/A145R)-GGGGSGGGGSGGGGS-SCDKTH-Trastuzumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an I97T/A145R mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO: 702) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; (see also SEQ ID NO: 27), fused to a portion of the hinge sequence of trastuzumab containing at least the residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26). TNF (I97T/A145R)-GGGGSGGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 752):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, the SCDKTH hinge sequence is the entire sequence of the hinge region of trastuzumab, containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, replaced by a portion containing 9, 10, or 11 consecutive residues.

2m) TNF (R32W/S86T)-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a human TNFR1 antagonist fusion protein construct containing a TNFR1 selective antagonist TNF mutein having an R32W/S86T mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO: 685) is fused to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). , fused to the hinge sequence of nivolumab, containing residues ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). The TNF(R32W/S86T)-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 753):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or part of it.

2n) TNF (R32W/S86T)-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an R32W/S86T mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO: 685) is fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). GGGGSGGGGSGGGGS peptide linker. The TNF (R32W/S86T)-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 754):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2o) TNF (R32W/S86T)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an R32W/S86T mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(R32W/S86T) mutein (SEQ ID NO: 685) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; 30) to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). TNF (R32W/S86T)-GGGGSGGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 755):
VRSSSRTPSDKPVAHVVANPQAEGQLQLQLQWLWANALLLANGVELRDNQLYLYLYLYLIYSQVLFKLTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTKVNLLLSAIKSPEGAIKSPEGAEAKPWYLGVFQ LEKGDRLLSAERPDYLDFAESGQVYFGGGGGGGGGGGGGGGGGGGPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPKPKPKDTLMISRTPKVVDVVDVVDVSRTQEDPEDPEDPFNWYVDVEVHNAKTKPREEQ FnstyrvvSVLHQDWLNGKEKCKVSNKGLPSSSKGAKTISKGAKPRPQVYTLPQVYTKNQVSLTCLTCLTCLTCFYPSDIAVEEWESNGQPENGQPENYKTTPPPPPPPPPPPPPPPPVLDSFFLTVDKSRWQ EGNVFSCSVMHEALHNHNHYTQKSLSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker (where n=1, 2, 3, 4 or 5), or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Contains part of the array.

2p) TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a human TNFR1 containing TNFR1 selective antagonist TNF mutein with V1M, R31C, C69V, Y87H, C101A and A145R mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. Antagonist fusion protein. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). ) and the hinge sequence of nivolumab, containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-ESKYGPPCPPCP-Nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 756):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or part of it.

2q) TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having V1M, R31C, C69V, Y87H, C101A and A145R mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; SEQ ID NO: 30 is also GGGGSGGGGGSGGGGS peptide linker. TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 757):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2r) TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having V1M, R31C, C69V, Y87H, C101A and A145R mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF(V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) is fused to a GGGGSGGGGSGGGGS peptide linker, which is connected to the N-terminus of the nivolumab Fc region (residues of SEQ ID NO: 29). 224-440; see also SEQ ID NO: 30); TNF (V1M/R31C/C69V/Y87H/C101A/A145R)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 758):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Contains all or part of the sequence.

2s) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-ESKYGPPCPPCP-Nivolumab Fc
Provided herein are human TNFR1 selective antagonist TNF muteins containing A84S, V85T, S86T, Y87H, Q88N and T89Q mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. Antagonist fusion protein. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). ) and the hinge sequence of nivolumab, containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 759):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or part of it.

2t) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having A84S, V85T, S86T, Y87H, Q88N and T89Q mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; SEQ ID NO: 30 is also GGGGSGGGGGSGGGGS peptide linker. TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 760):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2u) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein are TNFR1 antagonists containing TNFR1 selective antagonist TNF muteins having A84S, V85T, S86T, Y87H, Q88N and T89Q mutations with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. It is a fusion protein. The C-terminus of the TNF(A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) is fused to a GGGGSGGGGSGGGGS peptide linker, which is connected to the N-terminus of the nivolumab Fc region (residues of SEQ ID NO: 29). 224-440; see also SEQ ID NO: 30); TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 761):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Contains all or part of the sequence.

2v) TNF (I97T/A145R)-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a human TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an I97T/A145R mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO: 702) is fused to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). , fused to the hinge sequence of nivolumab containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). The TNF (I97T/A145R)-ESKYGPPCPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 762):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the entire nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) or part of it.

2w) TNF (I97T/A145R)-GGGGSGGGGSGGGGS-nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an I97T/A145R mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO: 702) is fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). GGGGSGGGGSGGGGS peptide linker. The TNF (I97T/A145R)-GGGGSGGGGSGGGGS-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 763):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art.

2x) TNF (I97T/A145R)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nivolumab Fc
Provided herein is a TNFR1 antagonist fusion protein containing a TNFR1 selective antagonist TNF mutein having an I97T/A145R mutation with respect to the sequence of soluble TNF set forth in SEQ ID NO:2. The C-terminus of the TNF(I97T/A145R) mutein (SEQ ID NO: 702) is fused to a GGGGSGGGGSGGGGS peptide linker, which corresponds to the N-terminus of the nivolumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; 30) to the hinge sequence of nivolumab, containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29). TNF (I97T/A145R)-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-nivolumab Fc fusion protein has the following sequence (SEQ ID NO: 764):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Alternatively, the GGGGSGGGGSGGGGS linker is a Gly-Ser linker, such as a (GSGS)n or (GGGGS)n linker, where n=1, 2, 3, 4 or 5, or a linker described herein or equivalent. Substituted by any other suitable short peptide linker known in the art. Alternatively, or in addition, a nivolumab hinge containing at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) Contains all or part of the sequence.
[Example 6]

Explained in the context of antibodies, difficulties arise in connection with inhibition of target receptors. Bivalent antibodies induce dimerization of the target receptor, which can lead to activation of the target receptor. While this may be desirable in some cases, it is highly undesirable for inhibitors of TNFR1. Even transient activation of TNFR1 can cause cytokine storm and significant toxicity. Therefore, monovalent inhibitors are needed. To accomplish this, monovalent constructs are provided herein (see entire specification and Example 5 above). This example illustrates the activity and properties of an exemplary construct that is an N-terminal fusion protein of a single chain dAb with human serum albumin.

This example provides exemplary nanobodies. Nanobodies, which are Vhh domain-containing proteins, contain only heavy chains and do not require cooperativity from light chains as in humans and mice (Harmsen and De Haard, Appl Microbiol Biotechnol. 77:13-22 (2007 )). Because they are single-chain, they have a short half-life on their own as small proteins (approximately 13-15 KDa) and therefore need to be presented as fusion proteins, such as grafted CDRs in antibody format.

Phage libraries were prepared using the method described in Sabir et al. ((2014) Comptes Rendus Biologies 337:244-249). Phage that bind with high affinity to tumor necrosis factor receptor-1 (TNFR1) were recovered, and the ability to bind to TNFR1 and human tumor necrosis factor for TNFR1 were determined as described in U.S. Patent Application Publication No. 20140112929. The ability to compete with alpha (TNF-α) binding was tested.

Nucleic acids encoding each of the single chain-containing Vhh antibodies were expressed in CHO cells (see Sokolowska-Wedzina et al. (2014) Protein Expression and Purification 99:50-57 for a description of expression vectors). , each purified by HPLC chromatography. Sample 1 was a control anti-TNFR1 antibody (H398 manufactured by ThermoFisher), and Vhh1-4 were Vhh antibodies containing dAbs. The sequences (see SEQ ID NOs: 54, 1478, 58 and 59) are as follows:
Vhh-1:
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSS
Vhh-2:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYRMHWVRQAPGKSLEWVSSIDTRGSSTYYADPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAVTMFSPFFDYWGQGTLV
Vhh-3:
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSS
Vhh-4:
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSS

The highlighted and underlined cysteine (C) in Vhh-4 and the corresponding cysteine in other Vhh chains form a loop whereby Vhh is a constrained polypeptide. Proline at amino acid residue 14 can be replaced with alanine.

Expression cassettes encoding Vhh domain antibody fragments were prepared, expressed, and purified by HPLC chromatography. The table below shows the expression level of each tested Vhh antibody. A His tag was required for purification of the two molecules. The results are shown in the table below:

Subsequently, binding studies using surface plasmon resonance (SPR) methods (Sciences GL. Biacore Assay Handbook. General Electric Company (2012); and Richter et al. (2019) MAbs 11:166-177) showed that Vhh1-4 Each was tested. Competition assays for Vhh inhibition of TNF-α binding to the TNFR1 extracellular domain were also performed as described in Richter et al. (2019). Equivalent assays for inhibition of TNF-induced expression of VCAM or IL8 were described in Lin et al., (2015) J Biomed Sci 22: 53; and Sonnier et al. (2010) Journal of Gastrointestinal Surgery 14:1592-1599, respectively. Performed as described.

A summary of the results is shown in the table below. The results show that Vhh-4 has a very high affinity (6.6 x 10-13 M) for the extracellular domain of TNFR-1; (IC50 approximately 1 nM) and was the most efficient of the four candidates in inhibiting TNF-induced VCAM-1 synthesis (0.3 nM). Of these Vhh dAb antibodies tested, Vhh-4 performed best and a construct containing Vhh-4 and HSA was prepared.

The following sequence represents a construct containing Vhh-4 (SEQ ID NO: 1475): the dAb portion is residues 20-138 of SEQ ID NO: 1475 and the Gly-Ser linker (residues 139-147 of SEQ ID NO: 1475). ) to human serum albumin (HSA; residues 148-732 of SEQ ID NO: 1475).
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSS...HSA....

Constructs containing Vhh-4 antibodies linked to HSA were shown to have activity as TNFR1 antagonists. This construct blocked TNFR1, as shown by a significant decrease in IL-6 and IL-8 gene expression in THP1 cells stimulated with LPS. 3 x 10 THP1 cells were treated with 5 μg and 20 μg and 0 μg of the constructs as a control for 30 min, followed by stimulation with LPS (10 ng/ml) for 4 h. RNA samples were collected and qPCR analysis was performed to analyze IL-6 and IL-8 gene expression using the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) as an internal control. Results show that both doses significantly reduced IL-6 and IL-8 expression (n=3, mean ± SEM; *p<0.05, **p<0.01 , ***p<0.001).

Since modifications will be apparent to those skilled in the art, it is intended that the invention be limited only by the scope of the claims appended hereto.

Claims (286)

A construct that is a tumor necrosis factor receptor 1 (TNFR1) antagonist construct of formula I:
(TNFR1 inhibitor) n-linker p-(activity modifier) q [wherein,
n and q are each integers, each independently 1, 2, or 3;
p is 0, 1, 2, or 3;
A TNFR1 inhibitor is a molecule that binds to TNFR1 and inhibits (antagonizes) the activity of TNFR1;
An activity modifier is a moiety that modulates or alters the activity or pharmacological properties of the construct as compared to the construct in the absence of the activity modifier;
The linker increases the plasticity of the construct and/or alleviates or reduces steric effects of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media]. 2. The construct of claim 1, wherein the linker is selected from chemical linkers, polypeptide linkers, and combinations thereof. 3. The construct according to claim 1 or claim 2, which is a fusion protein. A construct according to any one of claims 1 to 3, wherein the linker comprises multiple linker components. A construct according to any one of claims 1 to 4, wherein the TNFR1 inhibitor comprises a domain antibody (dAb). 6. The construct of any one of claims 1 to 5, when the TNFR1 inhibitor is a domain antibody (dAb), the activity modifier is neither an unmodified single Fc region nor a human serum albumin antibody. A construct according to any one of claims 1 to 6, wherein the TNFR1 inhibitor inhibits TNFR1 signaling. A construct according to any one of claims 1 to 7, wherein the activity modifier increases the serum half-life of the construct. 1 . The activity modifier is albumin or an Fc modified to have reduced or no ADCC activity and/or reduced or no CDC activity. The construct according to any one of items 1 to 8. A construct according to any one of claims 1 to 9, wherein the TNFR1 inhibitor inhibits TNFR1 activity but does not antagonize tumor necrosis factor receptor 2 (TNFR2) activity. 11. The construct of claim 10, wherein the TNFR1 inhibitor inhibits TNFR1 signaling. A construct that is a multispecific construct comprising a TNFR1 inhibitor and a Treg expander, wherein the bispecific construct interacts with two different target receptors or antigens or epitopes on the receptors. 13. The construct of claim 12, which is bispecific for TFNR1 and Treg expanders. 14. The construct of claim 12 or claim 13, wherein the Treg expander is a TNFR2 agonist. A construct according to any one of claims 12 to 14, comprising a linker to provide plasticity, increase solubility, or alleviate or reduce steric hindrance or van der Waals interactions. A construct according to any one of claims 12 to 15, further comprising an activity modifier for altering the activity of the construct. The construct construct according to any one of claims 12 to 16, having the following formula 2:
(TNFR1 inhibitor) n- (activity modifier) r1- (linker (L)) p- (activity modifier) r2- (TNFR2 agonist) q, or (TNFR1 inhibitor) n- (activity modifier) r1- (Linker (L)) p- (activity modifier) r2- (Treg expander) q
[In the formula,
n=1, 2, or 3, p=1, 2, or 3, q=0, 1, or 2, and r1 and r2 are each independently 0, 1, or 2;
The components may be in the particular order or any other order as long as the construct interacts with TNFR1 and TNFR2, antagonizes TNFR1, agonizes TNFR2, or has Treg expander activity. ]. A construct according to any one of claims 1 to 17, wherein the TNFR1 inhibitor moiety inhibits binding of TNFα binding to TNFR1 and/or inhibits signal transduction. Construct of formula 3a or 3b below:
(TNFR2 agonist or Treg expander) n-linker p-(activity modifier) q, formula 3a, or (activity modifier) q-linker p-(TNFR2 agonist or Treg expander) n, formula 3b [wherein
n and q are each integers, each independently 1, 2, or 3; p is 0, 1, 2, or 3;
An activity modifier is a moiety that alters the pharmacological properties of the construct;
TNFR2 agonists interact with TNRFR2 and produce TNFR2 activity;
Treg expanders are molecules that include TNFR2 agonists and result in the expansion of Treg cells;
The linker increases the plasticity and/or alleviates or reduces steric effects of the construct or its interaction with the receptor; and/or alters the solubility of the construct]. the activity modifier is an Fc region or a modified Fc region or a short FcRnBP;
A construct according to any one of claims 1 to 19, wherein the linker comprises a hinge region or is a linker comprising G and S residues. The construct according to any one of claims 1 to 20, wherein the linker has a sequence shown in any of SEQ ID NOs: 812-834 or is a PEG partial linker. A construct according to any one of claims 1 to 21, comprising an activity modifier that is a modified Fc region or a peptide that increases the serum half-life of the construct. A construct according to any one of claims 1 to 22, comprising an Fc region or an Fc dimer. A construct according to any one of claims 1 to 23, comprising an Fc region that has been modified to reduce ADCC and/or CDC activity. 25. The construct of claim 24, wherein the Fc has been modified to have reduced or no ADCC activity. said TNFR1 inhibitor is defined in the Sequence Listing, listed below, or any known in the art;
The Treg expander is any known in the art, is a TNFR2 agonist, or is shown in the sequence listing, listed below, or is any Treg expander known in the art. can be;
the linker is listed in the Sequence Listing or below, or is any known in the art;
the activity modifier is any shown in the sequence listing, known in the art, and/or shown below;
A construct according to any one of claims 1 to 25. A construct that is a TNFR1 antagonist construct, comprising a TNFR1 inhibitor, a single chain antibody or antigen binding portion thereof, that specifically targets and inhibits TNFR1 but does not antagonize TNFR2, thereby inhibiting receptor clustering. Constructs that prevent mediated transient activation of TNFR1. 28. The construct of claim 27, wherein the antibody or antigen binding portion thereof comprises modifications that improve the pharmacological properties and/or structure of the construct. 29. A construct according to any one of claims 1 to 28, which agonizes TNFR2 signaling, thereby increasing the expression of regulatory T cells (Tregs). 30. The construct of any one of claims 27-29, wherein the single chain antibody inhibits TNFR1 by inhibiting TNFR1 signaling. 31. A TNFR1 antagonist construct according to any one of claims 27 to 30, wherein the antibody or antigen binding portion of the construct inhibits binding of TNFα to TNFR1. 31. The construct of any one of claims 27-30, wherein the antibody or antigen-binding portion of the construct does not inhibit TNFα binding to TNFR1, but inhibits TNFR1 signaling. A construct according to any one of claims 28 to 32, wherein the pharmacological property is an increase in serum half-life. A construct according to any one of claims 27 to 33, wherein the TNFR1 construct comprises an Fc modified to eliminate ADCC and/or CDC activity. 35. The construct of any one of claims 27-34, wherein said TNFR1 construct comprises an Fc dimer. 36. The construct of claim 35, wherein one Fc monomer contains a hole and the other a knob to form a heterodimer. knob mutations were selected among S354C, T366Y, T366W, and T394W (according to EU numbering);
hole mutations are selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
This causes the Fc monomer to form a heterodimer,
36. The construct of claim 35. 38. A construct according to any one of claims 1 to 37, comprising Fc, wherein the Fc is derived from trastuzumab. 39. The construct of claim 38, comprising a linker that is a hinge region from the Fc region. 40. The construct of claim 39, wherein the hinge region is derived from trastuzumab and is linked to an Fc region. 41. A construct according to any one of claims 27 to 40, comprising a linker connected to an anti-TNFR1 antagonist antibody or antigen binding portion thereof. 42. The construct of claims 27-41, wherein the anti-TNFR1 antagonist antibody or antigen-binding portion thereof comprises a linker connected to the Fc region directly or via the hinge region. 43. The Fc region or modified Fc region comprising the sequence of amino acids shown in any of SEQ ID NOs: 10, 12, 14, 16, 27, 30, 1469 and 1470, according to any one of claims 1 to 42. construct. 44. A construct according to any one of claims 1 to 43, comprising a hinge region linked to an Fc portion. 45. A construct according to any one of claims 1 to 44, which binds to neonatal Fc receptors (FcRn). the TNFR1 construct comprises a short FcRn binding peptide (FcRnBP);
a short FcRn-binding peptide (FcRnBP) provides interaction of the construct with FcRn and comprises 6-25 or 10-20 amino acid residues;
46. The construct of claim 45. 47. The TNFR1 antagonist construct of claim 46, wherein said FcRnBP comprises 12-20 residues or 15 or 16 residues. 48. The TNFR1 antagonist construct according to claim 46 or claim 47, wherein the FcRn binding peptide (FcRnBP) comprises a peptide of any of SEQ ID NOs: 48-51. 49. The TNFR1 antagonist construct according to any one of claims 46 to 48, wherein the FcRn binding peptide (FcRnBP) consists of a peptide of any of SEQ ID NOs: 48 to 51. 49. The TNFR1 construct comprises an Fc heterodimer, one Fc monomer comprising a hole and the other a knob, and the resulting Fc dimer is a heterodimer. The construct according to any one of the following. TNFR1 inhibitor;
a TNFR1 antagonist construct comprising an Fc dimer; and a Treg expander;
the Fc dimer comprises two complementary Fc monomers;
the TNFR1 inhibitor is linked to one of the Fc monomers and the Treg expander is linked to the other Fc monomer;
A construct according to any one of claims 1 to 50. 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, and 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. 54. The construct of claim 53, wherein the Treg expanders are the same. 56. The construct of any one of claims 51-55, wherein said TNFR1 inhibitor inhibits or blocks TNFR1 signaling. 57. The construct of any one of claims 51-56, wherein the TNFR1 inhibitor binds TNFR1 and blocks or inhibits TNFα binding and TNFR1 signaling. 57. The construct of any one of claims 51-56, wherein the TNFR1 inhibitor binds TNFR1, does not interfere with or interferes with TNFα binding, and blocks or inhibits TNFR1 signaling. 59. The construct of any one 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 one of claims 51 to 60, wherein the Treg expander is a TNFR2 agonist that is an scFv, a VHH single domain antibody, or a Fab of a TNFR2 agonist monoclonal antibody. 62. A construct according to any one of claims 1 to 61, comprising all or part of trastuzumab and dimerized by N-terminal fusion with the C-terminus of trastuzumab. The construct according to any one of claims 7 to 16, wherein the Treg expander is a TNFR2 agonist that is a small molecule, or a nucleic acid aptamer, or a peptide aptamer. The construct construct according to any one of claims 1 to 63, which is a TNFR2 agonist construct of formula 3:
(Treg expander) n-linker p-(activity modifier) q, formula 3a, or (activity modifier) q-linker p-(Treg expander) n, formula 3b [wherein
n and q are each integers, each 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 pharmacological properties of the construct as compared to the construct in the absence of the activity modifier;
The linker increases the plasticity of the construct, and/or alleviates or reduces steric effects of the construct or its interaction with its receptor, and/or increases the solubility of the construct in aqueous media]. 65. The construct of any one of claims 51-64, wherein the Treg expander is a TNFR2 agonist. 66. The construct of claim 65, wherein the TNFR2 agonist stimulates or induces TNFR2 signaling. 67. The construct of any one of claims 51-66, wherein the Treg expander is a TNFR2 agonist that is an scFv, a VHH single domain antibody, or a Fab of a TNFR2 agonist monoclonal antibody. 68. The construct of claim 67, wherein the construct is dimerized by N-terminal fusion with the C-terminus of trastuzumab. 69. The TNFR1 antagonist construct of any one of claims 51-68, wherein the Treg expander is a TNFR2 agonist that is a small molecule, a nucleic acid, or a peptide aptamer. comprising a TNFR1 inhibitor moiety linked to one or more Treg expanders via a central PEG linker, or comprising at least two TNFR1 inhibitors that are the same or different, or comprising two Treg expanders that are the same or different; construct. 71. The construct of claim 70, comprising a branched PEG moiety linking the TNFR1 inhibitor and one or more Treg expanders. The construct according to claim 70 or claim 71, having a structure selected from the following formulas 4A to 4D:
Formula 4A:

[where n is 1 to 5;
R1 is H or CH3, or CH2CH3 or other C1-C5 alkyl;

is a TNFR1 inhibitor (TNFR1 antagonist);

is a Treg expander]; or formula 4B:

[In the formula,

is a TNFR1 inhibitor (TNFR1 antagonist),

is a Treg expander;
n is 1 to 5]; or formula 4C:

[In the formula,

is a TNFR1 inhibitor (TNFR1 antagonist) or a Treg expander;
n is 1 to 5]; or formula 4D:

[In the formula,

are each the same or different, each independently selected from a TNFR1 inhibitor (TNFR1 antagonist), and a TNFR2 agonist;
The activity modifier is optional and may be linked to any suitable locus in the molecule;
n is 1 to 5]. 73. The construct of any one of claims 70-72, wherein the Treg expander is a TNFR2 agonist. Contains activity modifiers,
the activity modifier is an Fc region or an Fc region that includes a hinge region or other linker;
The Fc region or Fc region having a hinge region is an Fc modified to reduce or eliminate ADCC and/or CDC activity,
A construct according to any one of claims 1-73. 75. The construct of claim 74, wherein the Fc or modified Fc is an IgG Fc, or an IgG1 or IgG4 Fc. 76. A construct according to any one of claims 1 to 75, which binds to neonatal Fc receptors (FcRn). said construct comprises a short FcRn binding peptide (FcRnBP);
the short FcRn binding peptide (FcRnBP) provides interaction of the construct with FcRn and comprises 6 to 25, such as 10 to 20 amino acid residues;
77. The construct of claim 76. 78. The construct of claim 77, wherein said FcRnBP comprises 12-20 residues or 15 or 16 residues. 79. The TNFR1 antagonist construct according to claim 78, wherein the FcRn binding peptide (FcRnBP) comprises a peptide of any of SEQ ID NOs: 48-51. 79. The TNFR1 antagonist construct according to claim 78, wherein the FcRn binding peptide (FcRnBP) consists of a peptide of any of SEQ ID NOs: 48-51. a) a TNFR1 inhibitor moiety that is TNFR1 selective;
b) optionally one or more linkers; and c) optionally a half-life extending moiety, the TNFR1 antagonist construct comprising at least one of b) and c). Constructs described in. 82. The construct of claim 81, wherein the TNFR1 selective antagonist selectively binds to and inhibits TNFR1 signaling rather than TNFR2 signaling. 83. The construct of claim 81 or claim 82, wherein the selective antagonist TNFR1 inhibitor comprises an antigen binding fragment that selectively binds to and inhibits TNFR1 signaling rather than TNFR2 signaling. 84. The construct of claim 83, wherein the antigen-binding fragment that selectively binds to and inhibits TNFR1 rather than TNFR2 signaling comprises a domain antibody (dAb), scFv, or Fab fragment. 85. The construct of any one of claims 1-84, wherein the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody. the human anti-TNFR1 antagonist monoclonal antibody is H398 comprising SEQ ID NO: 678, or ATROSAB, or an antigen binding portion thereof, or a sequence having at least 95% of SEQ ID NO: 31 or 32 or 673 or 678; 86. The construct of claim 85, having sequences with identity or antigen binding portions thereof that bind to TNFR1. the TNFR1 inhibitor comprises a domain antibody (dAb) or antigen binding portion thereof, or has a sequence of amino acids set forth in any of SEQ ID NOs: 52-672, or at least 95% sequence identity thereto; 87. A construct according to any one of claims 1 to 86, comprising a sequence that retains TNFR1 inhibitor activity. The TNFR1 inhibitor comprises an scFv shown in any of SEQ ID NOs: 673 to 678, or a variant of these polypeptides having at least 90% or 95% sequence identity thereto and retaining TNFR1 inhibitor activity. 88. A construct according to any one of claims 1-87, comprising: The TNFR1 inhibitor comprises a Fab shown in any of SEQ ID NOs: 679 to 682, or a sequence having at least 90% or 95% sequence identity thereto and retaining TNFR1 inhibitory or binding activity. A construct according to any one of claims 1-88. the TNFR1 inhibitor comprises a Nanobody having the sequence shown in SEQ ID NO: 683 or 684, or a sequence having at least 90% or 95% sequence identity thereto and retaining TNFR1 inhibitory or binding activity; A construct according to any one of claims 1-89. 91. The construct of any one of claims 1-90, wherein the TNFR1 inhibitor comprises dominant-negative tumor necrosis factor (DN-TNF) or a TNF mutein. the DN-TNF or TNF mutein confers selective inhibition of TNFR1;
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, 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/Q88 N/T89Q (Based on the sequence of soluble TNF shown in SEQ ID NO: 2)
92. The construct of claim 91, which is a soluble TNF molecule comprising one or more amino acid substitutions selected from. The TNFR1 inhibitor TNF mutein has a sequence of residues shown in any one of SEQ ID NOs: 701 to 703, or a sequence of residues shown in any one of SEQ ID NOs: 701 to 703, or a TNFR1 inhibitory activity. 93. The construct of claim 91 or claim 92, comprising a sequence that retains at least 90% or 95% or at least about 90% or about 95% sequence identity to the fragment thereof. a linker, the linker comprising all or a portion of the trastuzumab hinge sequence, SCDKTH corresponding to residues 222-227 of SEQ ID NO: 26, or up to the entire sequence of the trastuzumab hinge region, comprising the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 26). or at least 5, 6, 7, 8, 9, 10, or 11 contiguous residues thereof, or ESKYGPPCPPCP of residues 212-223 of SEQ ID NO: 29, or in a linker. 94. A construct according to any one of claims 1 to 93, comprising or having a sequence having at least 98% or 99% sequence identity thereto. 95. A construct according to any one of claims 1 to 94, comprising a linker comprising the sequence SCDKTH corresponding to residues 222-227 of SEQ ID NO: 26. 96. The construct of any one of claims 1-95, comprising a linker comprising a glycine-serine (GS) linker. The GS linker is (GlySer)n (in the formula, n=1 to 10); (GlySer2); (Gly4Ser)n (in the formula, n=1 to 10); (Gly3Ser)n (in the formula, n=1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5);
97. The construct of claim 96, selected from GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSGSGG; GGSSGG; 98. A linker according to any one of claims 1 to 97, wherein the linker comprises a GS linker and all or part of the hinge sequence of trastuzumab corresponding to residues EPKSCDKTHTCPPCP (219-233 of SEQ ID NO: 26). Constructs as described. 99. The construct of any one 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 one of claims 1-99, wherein the linker comprises a GS linker and all or part of the nivolumab hinge sequence corresponding to residues 212-223 of SEQ ID NO: 29. 101. The construct of any one of claims 1 to 100, comprising an activity modifier that is a half-life extending moiety that is an IgG Fc, a polyethylene glycol (PEG) molecule, or a 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 thereto. 103. The construct of claim 101 or claim 102, wherein the IgG4 Fc is the Fc of nivolumab as set forth in SEQ ID NO: 30, or a sequence of amino acids having at least 95% sequence identity thereto. The construct according to any one of claims 101 to 104, wherein the IgG1 Fc is the human IgG1 Fc shown in SEQ ID NO: 10. The construct according to any one of claims 101 to 104, wherein the IgG4 Fc is the human IgG4 Fc shown in SEQ ID NO: 16. 107. A construct according to any one of claims 1 to 106, comprising a TNFR1 inhibitor that is monovalent. 108. The construct of any one of claims 81-107, comprising a linker comprising (Gly4Ser)3. 108. The construct of any one of claims 81-107, comprising a linker comprising (Gly4Ser)3 and SCDKTH (residues 217-222 of SEQ ID NO: 31). (Gly4Ser)3 and a linker comprising the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26. (Gly4Ser)3 and a linker comprising the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29. a sequence of residues set forth in any one of SEQ ID NOs: 704-764, or which inhibits TNFR1 and is at least 95% or at least about 95% of the sequence of residues set forth in any one of SEQ ID NOs: 704-764; 112. A construct according to any one of claims 1 to 111, comprising a construct having a sequence having a sequence identity of . a TNFR1 antagonist construct;
the TNFR1 construct comprises a short FcRn binding peptide (FcRnBP);
A short FcRn binding peptide (FcRnBP) provides interaction of the construct with FcRn and comprises 6 to 25, such as 10 to 20 amino acid residues.
A construct according to any one of claims 1-112. 114. The construct of claim 113, wherein said FcRnBP comprises 12-20 residues or 15 or 16 residues. 114. The construct of claim 113, wherein the FcRn binding peptide (FcRnBP) comprises any of the peptides of SEQ ID NOs: 48-51. 114. The TNFR1 antagonist construct according to claim 113, wherein the FcRn binding peptide (FcRnBP) consists of a peptide of any of SEQ ID NOs: 48-51. a) Domain antibody that inhibits TNFR1;
117. The construct of any one of claims 1-116, comprising: b) a linker that increases plasticity; 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 unmodified Fc. a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG; GGSSGGSSGGGSSGGSSG; and GSSSGS 119. The construct of any one of claims 117 or 118, wherein the construct is a TNFR1 antagonist comprising a selected GS linker; and c) a half-life extending moiety that is an IgG Fc. the GS linker is (GGGGS)3;
the IgG Fc is trastuzumab Fc or nivolumab Fc,
A construct according to any one of claims 117-119. a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) a linker selected from all or a portion of the hinge sequence of trastuzumab and all or a portion of the hinge sequence of nivolumab; and c) a TNFR1 antagonist construct comprising a half-life extending moiety that is an IgG Fc. A construct according to any one of claims 1 to 120. the linker comprises all or part of the hinge sequence of trastuzumab;
the IgG Fc is the Fc of trastuzumab;
122. The construct of claim 121. the linker comprises all or part of the hinge sequence of nivolumab;
the IgG Fc is the Fc of nivolumab;
122. The construct of claim 121. a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG; GGSSGGSSGGGSSGGSSG; and GSSSGS GS linker selected;
c) a second linker selected from all or a portion of the hinge sequence of trastuzumab and all or a portion of the hinge sequence of nivolumab; and d) a TNFR1 antagonist construct comprising a half-life extending moiety that is an IgG Fc. 124. The construct of any one of claims 1-123, wherein: the GS linker is (GGGGS)3;
said second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31);
the IgG Fc is a trastuzumab Fc;
125. The construct of claim 124. the GS linker is (GGGGS)3;
the second linker comprises all or part of the hinge sequence of nivolumab;
the IgG Fc is the Fc of nivolumab;
125. The construct of claim 124. A construct that is a TNFR1 agonist,
a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG; GGSSGGSSGGGSSGGSSG; and GSSSGS a GS linker of choice; and c) a construct comprising 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 greater. a) a domain antibody (dAb) of any of SEQ ID NO: 52-672, or a scFv of any of SEQ ID NO: 673-678, or a Fab of any of SEQ ID NO: 679-682, or a Nanobody of SEQ ID NO: 683 or 684; or a TNF mutein of any of SEQ ID NOs: 685 to 703;
b) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG; GGSSGGSSGGGSSGGSSG; and GSSSGS 130. The construct of any one of claims 1-129, which is a TNFR1 agonist construct comprising a selected GS linker; 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. A construct according to any one of claims 1 to 131, which is a TNFR1 antagonist and is optimized to eliminate immunogenic sequences or epitopes. The IgG Fc has the following modifications:
a) Modification to introduce knob into hole;
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) a modification to reduce or eliminate immune effector function. The construct according to any one of paragraphs. knob mutations were selected among S354C, T366Y, T366W, and T394W (according to EU numbering);
the hole mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
134. The construct of claim 133. Modifications to increase or enhance FcRn recycling are
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering)
135. A TNFR1 antagonist construct according to claim 133 or claim 134, selected from one or more of: the immune effector function is selected from one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP); 136. A TNFR1 antagonist construct according to any one of claims 133-135. Said modification to reduce or eliminate immune effector function comprises:
Among 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 (according to EU numbering); and among IgG4, L235E, F234A/ L235A, S228P /L235E, and S228P/F234A/L235A (according to EU numbering)
137. The TNFR1 antagonist construct of claim 136, selected from one or more of: 138. A construct according to any one of claims 1 to 137, which is a TNFR1 antagonist or multispecific or comprises a central PEG linker moiety and comprises a modified Fc region. The Fc region is a modified IgG Fc, and the modified IgG Fc has the following modifications:
a) Modification to introduce knob into hole,
Knob mutations were selected among S354C, T366Y, T366W, and T394W (according to EU numbering);
a modification in which the hole mutation is selected among Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering)
Modifications selected from one or more of;
c) a modification to increase or enhance one or more immune effector functions,
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to increase or enhance immune effector function are
In IgG1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P 396L; F243L/R292P/Y300L; L234Y/G236W/S298A (in the first heavy chain) and S239D/A330L/I332E (in the second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in the first heavy chain) and D270E/K326D/A330M/K334E (in second heavy chain); A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A /I332E;G236A/ S239D/I332E; G236A/S239D/A330L/I332E; introduction of biantennary glycan at residue N297; introduction of defucosylated 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 (according to EU numbering)
139. The construct of claim 138, comprising one or more of the modifications selected from one or more of the following. 140. The construct of any one of claims 1-139, comprising an Fc region comprising an IgG1 Fc comprising one or more modifications to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. Said modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K (according to EU numbering). 141. The TNFR1 antagonist construct of claim 140, wherein the TNFR1 antagonist construct is selected. A construct that is a Treg expander construct,
a) Treg expander;
b) a linker that increases the plasticity of the construct and/or alleviates or reduces steric effects of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media; and c) activity. A construct comprising an activity modifier that is a moiety that modulates or alters the activity or pharmacological properties of the construct as compared to the construct in the absence of the modifier. 142. The construct of claim 142 or any one of claims 1-141, comprising a Treg expander that is a TNFR2 agonist. 144. The construct of claim 142 or claim 143, further comprising a TNFR1 inhibitor. 145. The construct of claim 143 or claim 144, wherein the TNFR2 agonist is a TNFR2 selective agonist. a) TNFR2 agonist;
b) a linker that increases the plasticity of the construct and/or alleviates or reduces steric effects of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media; and c) activity. 145. Any of claims 1-145, which is a TNFR2 agonist construct, comprising an activity modifier that modulates or alters the activity or pharmacological properties of the construct as compared to the construct in the absence of the modifier. The construct described in Section 1. 147. The construct of claim 146, wherein the TNFR2 agonist is a TNFR2 selective agonist. 148. The construct of any one of claims 142-147, comprising an activity modifier that is a half-life extending moiety. 149. The construct of any one of claims 142-148, wherein the TNFR2 agonist selectively activates or antagonizes TNFR2 without activating or antagonizing TNFR1. 150. The construct of any one of claims 1-149, comprising a TNFR2 agonist that binds to one or more epitopes within TNFR2. 151. The construct of claim 150, wherein said TNFR2 is human TNFR2. 152. The construct of claim 151, wherein the epitope is selected from one or more of the epitopes comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 839-865, 1202, and 1204. 153. The construct of any one 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 an antigen-binding portion thereof, or a single chain form thereof. . 154, wherein the agonist anti-TNFR2 antibody is selected from MR2-1 (also referred to as ab8161; US Patent No. 9,821,010) or MAB2261 (US Patent No. 9,821,010). construct. 155. The construct of any one 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 one of claims 149-155, wherein the TNFR2 agonist is a TNFR2 selective agonist. 157. The construct of any one of claims 1-156, comprising a TNFR2 agonist comprising a TNFR2 agonist TNF mutein. The TNFR2 muteins are all K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A according to SEQ ID NO: 2. 145R, 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, E 146D/S147D, D143V/F144L/A145S, 158. The construct of claim 157, which is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from S95C/G148C, and D143V/A145S, and combinations of any of the foregoing. 158. The construct of claim 157, wherein the TNFR2 agonist is a TNF mutein comprising the mutations D143N/A145R. The linker comprises all or part of the trastuzumab hinge sequence corresponding to residues 219-233 of SEQ ID NO: 26, or all or part of the nivolumab hinge sequence corresponding to residues 212-223 of SEQ ID NO: 29. 160. The construct of any one of claims 142-159, comprising: 161. A construct according to any one of claims 1 to 160, comprising a linker comprising the sequence SCDKTH corresponding to residues 217-222 of SEQ ID NO: 31. 162. The construct of any one of claims 161, comprising a linker comprising a glycine-serine (GS) linker. The GS linker is (GlySer)n (in the formula, n=1 to 10); (GlySer2); (Gly4Ser)n (in the formula, n=1 to 10); (Gly3Ser)n (in the formula, n=1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSSG; SGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS 163. The construct of claim 162, selected from: 164. The construct of any one of claims 146-163, wherein the linker comprises a GS linker and all or part of the trastuzumab hinge sequence corresponding to residues 219-233 of SEQ ID NO:26. 164. The construct of any one 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. 164. The construct of any one of claims 146-163, wherein the linker comprises a GS linker and all or part of the nivolumab hinge sequence corresponding to residues 212-223 of SEQ ID NO:29. 167. The construct of any one of claims 1-166, comprising a half-life extending moiety that 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. 169. The construct of claim 168, wherein the IgG4 Fc is the nivolumab Fc set forth in SEQ ID NO: 30. 169. The construct of claim 168, wherein the IgG1 Fc is the human IgG1 Fc set forth in SEQ ID NO: 10. 169. The construct of claim 168, wherein the IgG4 Fc is a human IgG4 Fc set forth in SEQ ID NO: 16. 173. The construct of any one of claims 146-172, wherein said TNFR2 agonist is monovalent. 174. The construct of any one of claims 1-173, which is a TNFR2 agonist construct, wherein the TNFR2 agonist is bivalent. 174. The construct of any one of claims 1-173, which is a TNFR2 agonist construct, wherein said TNFR2 is trivalent. 176. The construct of any one of claims 1-175, which is a TNFR2 agonist construct and wherein the linker comprises (Gly4Ser)3. 177. The construct of any one of claims 1-176, wherein the TNFR2 agonist construct is TNFR2, and the linker comprises (Gly4Ser)3 and SCDKTH (residues 217-222 of SEQ ID NO: 31). 178. The construct of any one of claims 1-177, which is a TNFR2 agonist construct, wherein the linker comprises (Gly4Ser)3 and the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO: 26. 178. The construct of any one of claims 1-177, which is a TNFR2 agonist construct, wherein the linker comprises (Gly4Ser)3 and the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO: 29. 180. A construct according to any one of claims 1 to 179, comprising an activity modifier that is a half-life extending moiety. 181. The construct of claim 180, wherein the half-life extending moiety is PEG. 181. The construct of claim 180, wherein the PEG has a molecular weight of at least 30 kDa or at least about 30 kDa. 181. The construct of claim 180, wherein the half-life extending moiety is human serum albumin (HSA). a) a TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) GS linker, comprising (GlySer)n (in the formula, n=1 to 10); (GlySer2); (Gly4Ser)n (in the formula, n=1 to 10); (Gly3Ser)n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; SSSGSGSG;GGSSGGSSSGGGSSSGGSSG and GSSSGS; and c) a half-life extending moiety that is an IgG Fc. the GS linker is (GGGGS)3;
the IgG Fc is trastuzumab Fc or nivolumab Fc;
185. The construct of claim 184. a) a TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) a linker selected from all or part of the hinge sequence of trastuzumab and all or part of the hinge sequence of nivolumab; and c) a TNFR2 agonist construct comprising an activity modifier that is a half-life extending moiety that is an IgG Fc. 186. The construct of any one of claims 1-185, wherein: the linker comprises all or part of the hinge sequence of trastuzumab;
the IgG Fc is trastuzumab Fc;
187. The construct of claim 186. the linker comprises all or part of the hinge sequence of nivolumab;
the IgG Fc is the Fc of nivolumab;
188. The construct of claim 187. a) a TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202 and 1204;
b) GS linker, comprising (GlySer)n (in the formula, n=1 to 10); (GlySer2); (Gly4Ser)n (in the formula, n=1 to 10); (Gly3Ser)n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; SSGSGSG;GGSSGGSSSGGGSSGGSSSG and a GS linker selected from GSSSGS;
c) a second linker selected from 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.
184. The construct of any one of claims 1-183, which is a TNFR2 construct comprising: the GS linker is (GGGGS)3;
said second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31);
the IgG Fc is a trastuzumab Fc;
200. The construct of claim 189. the GS linker is (GGGGS)3;
the second linker comprises all or part of the hinge sequence of nivolumab;
the IgG Fc is the Fc of nivolumab;
200. The construct of claim 189. a) a TNFR2 agonist comprising an antigen-binding fragment of an agonistic human anti-TNFR2 antibody selected from MR2-1 or MAB2261;
b)
i) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG;GGSSGGSSSGGGSSSGGSSG; and GSSSGS and/or ii) a linker comprising all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and c) an IgG1 or IgG4 Fc, a PEG molecule, and a human serum albumin ( an activity modifier that is a half-life extending moiety selected from HSA),
IgG1 Fc is human IgG1 Fc shown in SEQ ID NO: 10, or trastuzumab Fc shown in SEQ ID NO: 27;
the PEG molecule has a molecular weight of at least 30 kDa or at least about 30 kDa;
192. A construct according to any one of claims 1 to 191, which is a TNFR2 agonist construct comprising an activity modifier. a) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R according to SEQ ID NO: 2 /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, D 143V/F144L/A145S, S95C/G148C, and a TNFR2-selective TNF mutein, which is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from D143V/A145S;
b)
i) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG; GGSSGGSSGGGSSGGSSG; and GSSSGS a selected GS linker; and/or ii) a linker comprising all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and c) an IgG1 or IgG4 Fc, a PEG molecule, and human serum albumin. an activity modifier that is a half-life extending moiety selected from (HSA),
IgG1 Fc is human IgG1 Fc shown in SEQ ID NO: 10, or trastuzumab Fc shown in SEQ ID NO: 27;
193. The construct of any one of claims 1-192, wherein the PEG molecule is or comprises a TNFR2 agonist construct comprising an activity modifier having a molecular weight of at least 30 kDa or at least about 30 kDa. a) TNFR2 TNF mutein containing mutations D143N/A145R;
b) a (GGGGS)3 linker; and c) a half-life extending moiety that is the Fc of trastuzumab or the Fc of nivolumab. The construct according to any one of paragraphs. a) TNFR2 selective TNF mutein containing mutations D143N/A145R;
b) a (GGGGS)3 linker and a second linker comprising the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and c) a TNFR2 agonist construct comprising an activity modifier that is the Fc half-life extending portion of trastuzumab. 195. The construct of any one of claims 1-194. A construct that is a TNFR2 agonist construct,
a) TNFR2 selective TNF mutein containing mutations D143N/A145R;
b) a (GGGGS)3 linker and a second linker comprising all or part of the hinge sequence of nivolumab; and c) a construct comprising an activity modifier that is the Fc half-life extending portion of nivolumab. a) TNFR2 selective TNF mutein containing 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 portion that is the Fc of trastuzumab. The construct according to any one of 1 to 196. a) TNFR2 selective TNF mutein containing 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) a TNFR2 agonist construct comprising an activity modifier that is the Fc half-life extending portion of nivolumab. 208. A construct according to any one of claims 1 to 197, which is or comprises. 200. The construct of any one of claims 1-198, wherein the IgG Fc is a monomeric or dimeric TNFR1 antagonist construct or a TNFR2 agonist construct or both. 200. A construct according to any one of claims 1-199, comprising a dAb. 201. The construct of claim 200, comprising 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. dAb Dom1h-131-206 of SEQ ID NO: 59, comprising residues 20-732, linked to HSA via a linker as shown in SEQ ID NO: 1475. A construct as described, 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. dAbs 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. 204. The construct of claim 200 or 203, wherein the construct has TNFR1 antagonist activity. 205. Said dAb has a sequence set forth in any of SEQ ID NOs: 57-59, to which the variant has at least 95% sequence identity, whereby the construct has TNFR1 antagonist activity. Constructs described in. 207. The construct of claim 206, wherein the dAb is designated DOM1h-131-206 of SEQ ID NO: 59 and a variant thereof with TNFR1 antagonist activity. 208. A construct according to any one of claims 200 to 207, wherein the sequences of the necessary parts of the dAb or construct for administration to humans are humanized. 209. The construct of any one of claims 1-208, comprising a TNFR2 agonist or being a TNFR2 agonist construct. is or is or comprises a TNFR2 agonist construct, wherein the TNFR2 agonist has been modified to eliminate sequences of amino acids or epitopes that are immunogenic in the subject being treated. 209. The construct of any one of claims 1-208. 201. The construct of claim 200, wherein the subject is a human. 212. The construct of any one of claims 189-211, wherein the TNFR2 agonist is a TNFR2 selective agonist. A TNFR2 agonist construct comprising a modified IgG Fc, wherein said IgG Fc has the following modifications:
a) Modification to introduce knob into hole;
b) modifications to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent Any one of claims 1-212 comprising one or more modifications to reduce or eliminate immune effector function selected from one or more of cell-mediated phagocytosis (ADCP). The constructs described in Section. a) The modification to introduce the knob into hole is
one or more knob mutations selected from S354C, T366Y, T366W, and T394W (according to EU numbering); and selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering). Fc is selected from one or more hole mutations that form dimers;
b) the modification to increase or enhance FcRn recycling is
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y selected from one or more of (according to EU numbering);
c) the modification to reduce or eliminate immune effector function,
Among IgG1, L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G 236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A (according to EU numbering); and among IgG4, L235E, F234A/L 235A, S228P/ L235E, and S228P/F234A/L235A (according to EU numbering)
selected from one or more of
214. The construct of claim 213. A TNFR2 agonist construct comprising a modified IgG Fc, wherein the IgG Fc has the following modifications:
a) one or more modifications to introduce a knob-into-hole,
Knob mutations were selected among S354C, T366Y, T366W, and T394W (according to EU numbering);
a modification in which the hole mutations are selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering); and c) a modification for increasing or enhancing immune effector function,
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to increase or enhance immune effector function are
Among IgG1, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/ P396L; F243L/R292P/Y300L ; L234Y/G236W/S298A (in first heavy chain) and S239D/A330L/I332E (in second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in 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; G236A; /I332E;G236A /S239D/I332E; G236A/S239D/A330L/I332E; introduction of biantennary glycan at residue N297; introduction of defucosylated 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;S 267E/H268F/S324T; G236A/I332E/ S267E/H268F/S324T; E345R; and E345R/E430G/S440Y (according to EU numbering)
214. A construct of any one of claims 1-213, comprising one or more of the modifications selected from one or more of the following. 216. The TNFR2 agonist construct of any one of claims 1-215, wherein the TNFR2 agonist construct is or comprises a modified IgG1 Fc in which the Fc has been modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. Constructs as described. Said modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K (according to EU numbering). 217. The construct of claim 216. is or comprises a TNFR2 agonist construct that selectively activates or agonizes TNFR2 without activating or antagonizing TNFR1;
a) TNFR2 agonist;
b) one or more linkers; and c) an activity modifier that is a half-life extending moiety;
The TNFR2 agonist construct is a fusion protein comprising a single chain TNFR2 selective TNF mutein trimer fused to a multimerization domain and has the formula:
MD-L1-TNFmut-L2-TNFmut-L3-TNFmut (Formula II); or TNFmut-L1-TNFmut-L2-TNFmut-L3-MD (Formula III),
(wherein MD are the same or different multimerization domains; TNFmut is a TNFR2-selective TNF mutein; L1, L2, and L3 can be the same or different linkers), claims 1-217 The construct according to any one of the above. The TNF mutein is 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/S1 47D, 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 ( 220. The construct of claim 218, comprising one or more TNFR2 selective mutations selected from TNFR2 (according to SEQ ID NO: 2). 220. The construct of claim 218, wherein the TNF mutein comprises the TNFR2 selective mutation D143N/A145R. The multimerization domain is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or 221. The construct of any one of claims 218-220, selected from 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 one of claims 218-221, wherein the multimerization domain is an IgG1 Fc or an IgG4 Fc, and the IgG1 Fc or IgG4 Fc is also a half-life extending moiety. The L1, L2 and/or L3 linkers are independently selected from (GGGGS)n, where n=1-5, or all or part of the stalk region of TNF (SEQ ID NO: 812) , the TNFR2 agonist construct of any one of claims 218-222. the linker between the TNFR2 agonist and the half-life extending moiety,
(GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5); ( SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; ;GGSSGGSSSGGGSSGGGSSG; and GSSSGS. or a linker selected from all or part of the trastuzumab hinge sequence and all or part of the nivolumab hinge sequence; or a combination thereof. TNFR2 agonist construct according to item 1. The half-life extension portion is
IgG1 Fc which is human IgG1 Fc shown in SEQ ID NO: 10 or trastuzumab Fc shown in SEQ ID NO: 27;
IgG4 Fc that is human IgG4 Fc shown in SEQ ID NO: 16 or nivolumab Fc shown in SEQ ID NO: 30;
a PEG molecule of at least 30 kDa or at least about 30 kDa in size; and human serum albumin (HSA)
225. The TNFR2 agonist construct of any one of claims 218-224, selected from: a) A construct with the following 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; L1, L2, and L3 are the same or different linkers;
i) the MD is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or human selected from the trimerization domain of TNC (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
ii) L1, L2 and L3 are each (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812), or a mixture thereof;
iii) TNF mutein comprises TNFR2 selective mutation D143N/A145R]
b)
IgG1 Fc which is human IgG1 Fc shown in SEQ ID NO: 10 or trastuzumab Fc shown in SEQ ID NO: 27;
IgG4 Fc that is human IgG4 Fc shown in SEQ ID NO: 16 or nivolumab Fc shown in SEQ ID NO: 30;
a PEG molecule of at least 30 kDa or at least about 30 kDa in size; and human serum albumin (HSA)
a half-life extending moiety selected from; and c) a linker between the TNFR2 selective agonist and the half-life extending moiety,
(GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5); ( SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; ;GGSSGGSSSGGGSSGGGSSG; and GSSSGS. or a linker selected from all or a portion of the hinge sequence of trastuzumab; and all or a portion of the hinge sequence of nivolumab; or a TNFR2 agonist construct comprising a linker comprising a combination thereof; 218. The construct of any one of claims 1-217 comprising. The construct construct of any one of claims 1-217, which is a TNFR2 agonist construct construct comprising the following 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; L1, L2, and L3 are the same or different linkers;
i) said MD is selected from IgG1 Fc or IgG4 Fc;
ii) L2 and L3 in formula II and L1 and L2 in formula III are each independently (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812); or a combination thereof;
iii) Each of L1 of formula II and L3 of formula III is independently (GlySer)n (wherein n=1 to 10); (GlySer2); (Gly4Ser)n (wherein n=1 to 10 ); (Gly3Ser) n (in the formula, n = 1 to 5); (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG ; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSGSSG; GSSSGSGSGGSSSGSGSG; linker ; or independently selected from a combination thereof;
iv) TNF muteins include TNFR2 selective mutations D143N/A145R]. IgG1 Fc in which the MD is the Fc of human IgG1 shown in SEQ ID NO: 10, or the Fc of trastuzumab shown in SEQ ID NO: 27; or the Fc of human IgG4 shown in SEQ ID NO: 16, or the Fc of nivolumab shown in SEQ ID NO: 30. IgG4 Fc that is Fc
228. The construct of claim 227, selected from. 26. The method of claim 1, wherein the MD is a trastuzumab IgG1 Fc, and the linker between the MD and an adjacent TNF mutein is all or part of the trastuzumab hinge sequence corresponding to residues 219-233 of SEQ ID NO: 26. 227 or the construct of claim 228. 229. The method of claim 227 or claim 228, wherein the MD is an IgG1 Fc of trastuzumab and the linker between the MD and an adjacent TNF mutein comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31). construct. 26. wherein the MD is an IgG1 Fc of trastuzumab, and the linker between the MD and an adjacent TNF mutein comprises (Gly4Ser)3 and the hinge sequence of trastuzumab, corresponding to residues 219-233 of SEQ ID NO: 26. The construct according to any one of 227-230. Claim 227 or Claim 228, wherein the MD is an IgG1 Fc of trastuzumab, and the linker between the MD and an adjacent TNF mutein comprises (Gly4Ser)3 and SCDKTH (residues 222-227 of SEQ ID NO: 31). Constructs described in. 29, wherein the MD is an IgG4 Fc of nivolumab, and the linker between the MD and an adjacent TNF mutein comprises all or part of the nivolumab hinge sequence and corresponds to residues 212-223 of SEQ ID NO: 29. 229. A construct according to claim 227 or claim 228. The MD is the IgG4 Fc of nivolumab, and the linker between the MD and the adjacent TNF mutein is (Gly4Ser)3 and all or part of the hinge sequence of nivolumab, residues 212-223 of SEQ ID NO: 29. 229. A construct according to claim 227 or claim 228, corresponding to. 235. The construct of any one of claims 1-234, which is an agonist construct and has been modified to eliminate an immunogenic sequence or epitope that is immunogenic in a subject. 236. The construct of claim 235, wherein the subject is a human. A TNFR2 agonist construct comprising a modified IgG Fc, wherein the IgG Fc has the following modifications:
a) Modification to introduce knob into hole,
the knob mutation is selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering);
a modification in which the hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering); and c) a modification to reduce or eliminate immune effector function,
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to reduce or eliminate immune effector function are
Among IgG1, L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G 236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A (according to EU numbering); and in IgG4, L235E, F234A/L2 35A, S228P/L235E , and S228P/F234A/L235A (according to EU numbering). A TNFR2 agonist construct comprising a modified IgG Fc, wherein the IgG Fc has the following modifications:
a) Modification to introduce knob into hole,
the knob mutation is selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering);
a modification in which the hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering)
and c) a modification for increasing or enhancing immune effector function;
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
The modification to increase or enhance the immune effector function is
Among IgG1, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P 396L; F243L/R292P/Y300L; L234Y/G236W/S298A (in the first heavy chain) and S239D/A330L/I332E (in the second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in the first heavy chain) and D270E/K326D/A330M/K334E (in second heavy chain); A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A /I332E;G236A/ S239D/I332E; G236A/S239D/A330L/I332E; introduction of biantennary glycan at residue N297; introduction of defucosylated 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 (according to EU numbering)
238. The construct of any one of claims 1-237, comprising one or more modifications selected from one or more of: 239. The TNFR2 agonist construct of any one of claims 1-238, wherein the TNFR2 agonist construct comprises an IgG1 Fc modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. The modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K (according to EU numbering) 240. The TNFR2 agonist construct of claim 239. 241. The construct of any one of claims 1-240, which is a multispecific TNFR1 inhibitor/TNFR2 agonist construct and has the 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) n - (TNFR2 agonist) q-linker (L)p, or (TNFR2 agonist) q-(TNFR1 inhibitor) n-linker (L)p, or any of the above constructs [wherein ,
n=1 or 2, p=1, 2, or 3, and q=1 or 2;
the TNFR1 inhibitor interacts with and inhibits the activity of TNFR1;
An activity modifier is a moiety that modulates or alters the activity or pharmacological properties of the construct as compared to the construct in the absence of the activity modifier;
The linker increases the solubility of the construct, increases its plasticity, or alters the steric effect of the construct]. a multispecific TNFR1 inhibitor/TNFR2 agonist construct;
the TNFR1 inhibitor selectively inhibits or antagonizes TNFR1 signaling without inhibiting or antagonizing TNFR2 signaling;
the TNFR1 inhibitor does not interfere with TNFR2 activation or agonism;
the TNFR2 agonist selectively activates or agonizes TNFR2 signaling without activating or agonizing TNFR1 signaling;
the TNFR2 agonist does not prevent inhibition or antagonism of TNFR1;
242. A construct according to any one of claims 1-241. a) the TNFR1 inhibitor is
i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB, or a polypeptide having a sequence having at least 95% sequence identity thereto; or ii) any of SEQ ID NOs: 52-672. A domain antibody (dAb), or a scFv of any of SEQ ID NOs: 673-678, or a Fab of any of SEQ ID NOs: 679-682, or a Nanobody of SEQ ID NOs: 683 or 684, or a TNF of any of SEQ ID NOs: 701-703 a mutein, or a polypeptide having a sequence that has at least 95% sequence identity with any of the aforementioned polypeptides and is a TNFR1 inhibitor; or iii) confers selective inhibition of TNFR1;
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, 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/Q88 N/T89Q (Based on the sequence of soluble TNF shown in SEQ ID NO: 2)
selected among dominant-negative tumor necrosis factor (DN-TNF) or TNF muteins comprising soluble TNF molecules having one or more amino acid substitutions selected from;
b) The linker is
i) (GlySer) n (in the formula, n = 1 to 10); (GlySer2); (Gly4Ser) n (in the formula, n = 1 to 10); (Gly3Ser) n (in the formula, n = 1 to 5) ; (SerGly4) n (in the formula, n = 1 to 5); (GlySerSerGly) n (in the formula, n = 1 to 5); GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSG; GGSSGG; GGSSGGSGGSSSSG; SG;GGSSGGSSSGGGSSSGGSSG; and GSSSGS and/or ii) all or part of the trastuzumab hinge sequence corresponding to residues 219-233 of SEQ ID NO: 26, or all of the nivolumab hinge sequence corresponding to residues 212-223 of SEQ ID NO: 29; or part;
iii) an IgG1 or IgG4 Fc,
the IgG1 Fc is selected from a human IgG1 IgG1 Fc set forth in SEQ ID NO: 10, or a trastuzumab IgG1 Fc set forth in SEQ ID NO: 27;
the IgG4 Fc is selected from a human IgG4 IgG4 Fc set forth in SEQ ID NO: 16, or a nivolumab IgG4 Fc set forth in SEQ ID NO: 30;
Optionally, the Fc is one for introducing knob-into-hole and/or increasing or enhancing neonatal Fc receptor (FcRn) recycling and/or reducing or eliminating immune effector function. Contains one or more modifications;
IgG1 or IgG4 Fc
selected from;
c) the TNFR2 agonist is
i) an antigen-binding fragment that binds to one or more epitopes within human TNFR2 selected from among the epitopes set forth in SEQ ID NOs: 839-865, 1202, and 1204; or ii) selected from MR2-1 or MAB2261 or iii) a TNFR2-selective TNF mutein comprising:
K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q8 8N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E14 6D/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 D 143V/A145S (based on sequence number 2) ); or iv) a TNFR2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2-selective mutations selected from (GGGGS)n, where n=1-5 ), or a single chain TNFR2-selective TNF mutein trimer comprising the mutation D143N/A145R, linked by all or part of the stalk region of TNF (SEQ ID NO: 812); or v) a TNFR2-selective trimer comprising the formula: Agonist:
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; L1, L2, and L3 are linkers that may be the same or different;
The MD can be 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. a trimerization domain (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
L1, L2 and L3 are each (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812), or a mixture thereof;
the TNF mutein comprises the TNFR2 selective mutation D143N/A145R]
243. The construct of claim 241 or claim 242, selected from. a multispecific TNFR1 antagonist/TNFR2 agonist construct;
a) The TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52 to 672, or an scFv of any one of SEQ ID NOs: 673 to 678, or a Fab of any one of SEQ ID NOs: 679 to 682, or SEQ ID NO: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) the linker comprises (GGGGS)3, a polypeptide comprising the sequence SCDKTH (residues 222-227 of SEQ ID NO: 26), and the Fc of trastuzumab;
c) The TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A 145R/S147T, Q88N/T89S /A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E1 46S/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 (according to SEQ ID NO: 2), comprising a TNFR2 selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from A145S (according to SEQ ID NO: 2);
243. A construct according to claim 241 or claim 242. a) The TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52 to 672, or an scFv of any one of SEQ ID NOs: 673 to 678, or a Fab of any one of SEQ ID NOs: 679 to 682, or SEQ ID NO: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) the linker comprises (GGGGS)3, all or part of the hinge sequence of nivolumab, and the Fc of nivolumab;
c) The TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A 145R/S147T, Q88N/T89S /A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E1 46S/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, a soluble TNF variant comprising one or more TNFR2 selective mutations selected from A145S (according to SEQ ID NO: 2);
243. A construct according to claim 241 or claim 242. a) The TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52 to 672, or an scFv of any one of SEQ ID NOs: 673 to 678, or a Fab of any one of SEQ ID NOs: 679 to 682, or SEQ ID NO: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) the linker comprises (GGGGS)3 and the Fc of trastuzumab;
c) The TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A 145R/S147T, Q88N/T89S /A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E1 46S/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 (according to SEQ ID NO: 2), comprising a TNFR2 selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from A145S (according to SEQ ID NO: 2);
243. A construct according to claim 241 or claim 242. a) The TNFR1 inhibitor is a domain antibody (dAb) of any one of SEQ ID NOs: 52 to 672, or an scFv of any one of SEQ ID NOs: 673 to 678, or a Fab of any one of SEQ ID NOs: 679 to 682, or SEQ ID NO: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 701-703, or a sequence having at least 95% or at least about 95% sequence identity thereto;
b) said linker comprises (GGGGS)3 and the Fc of nivolumab;
c) The TNFR2 agonist is K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A 145R/S147T, Q88N/T89S /A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E1 46S/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;
243. A construct according to claim 241 or claim 242. The IgG Fc contains the following modifications:
a) Modification to introduce knob into hole;
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling; and c) a modification to reduce or eliminate immune effector function. The construct according to any one of paragraphs. the Fc comprises a knob-into-hole modification,
the knob mutation is selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering);
the hole mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering);
249. The construct of claim 248. the Fc comprises a modification to increase or enhance FcRn recycling,
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y 249. The construct of claim 248, selected from one or more of (according to EU numbering). said Fc is selected from one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP). 249. The construct of claim 248, comprising an alteration to effector function. Among IgG1, L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G 236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A (according to EU numbering); and in IgG4, L235E, F234A/L2 35A, S228P/L235E , and S228P/F234A/L235A (according to EU numbering)
249. The construct of claim 248, comprising a modification to reduce or eliminate an immune effector function selected from one or more of: The IgG Fc has the following modifications:
a) Modification to introduce knob into hole,
Knob mutations are selected from one or more of S354C, T366Y, T366W, and T394W (according to EU numbering) and hole mutations are selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A, and Y407V (according to EU numbering). ) modification selected from one or more of the following;
b) a modification to increase or enhance neonatal Fc receptor (FcRn) recycling, the modification comprising:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M 252Y/T256Q, M252F/T256D, M252Y/ S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P /N434Y, and T256N/A378V/S383N/N434Y (according to EU numbering)
and c) a modification for increasing or enhancing immune effector function;
the immune effector function is selected from one or more of CDC, ADCC, and ADCP;
Modifications to increase or enhance immune effector function are
Among IgG1, S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/ P396L; F243L/R292P/Y300L ; L234Y/G236W/S298A (in first heavy chain) and S239D/A330L/I332E (in second heavy chain); L234Y/L235Q/G236W/S239M/H268D/D270E/S298A (in 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; G236A; /I332E;G236A /S239D/I332E; G236A/S239D/A330L/I332E; introduction of biantennary glycan at residue N297; introduction of defucosylated 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;S 267E/H268F/S324T; G236A/I332E/ S267E/H268F/S324T; E345R; and E345R/E430G/S440Y (according to EU numbering)
248. The construct of any one of claims 241-247, comprising one or more of the modifications selected from one or more of the following. 254. The construct of any one of claims 241-253, comprising an IgG1 Fc modified to increase binding to the inhibitory Fcγ receptor (FcγR) FcγRIIb. The modification that increases binding to FcγRIIb is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K (according to EU numbering) 255. The construct of claim 254. is a multispecific TNFR1 antagonist/TNFR2 agonist,
the TNFR1 antagonist is monovalent;
the TNFR2 agonist is monovalent;
256. A construct according to any one of claims 1-255. is a multispecific TNFR1 antagonist/TNFR2 agonist,
the TNFR1 antagonist is monovalent;
the TNFR2 agonist is bivalent;
256. A construct according to any one of claims 1-255. is a multispecific TNFR1 antagonist/TNFR2 agonist,
a) the TNFR1 antagonist is
i) an antigen-binding fragment of a human anti-TNFR1 antagonist monoclonal antibody selected from H398 or ATROSAB; or ii) a domain antibody (dAb) of any of SEQ ID NOs: 52-672, or an scFv of any of SEQ ID NOs: 673-678. , or a Fab of any of SEQ ID NOs: 679-682, or a Nanobody of SEQ ID NOs: 683 or 684, or a TNF mutein of any of SEQ ID NOs: 701-703, or at least 95% or at least about 95% sequence identical thereto. or iii) confers selective inhibition of TNFR1;
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, 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/Q88 N/T89Q (Based on the sequence of soluble TNF shown in SEQ ID NO: 2)
selected from dominant-negative tumor necrosis factor (DN-TNF) or TNF mutein, comprising a soluble TNF molecule having one or more amino acid substitutions selected from;
b) said linker is a branched PEG molecule having a size of at least 30 kDa or at least about 30 kDa;
c) the TNFR2 agonist is
i) an antigen-binding fragment that binds to one or more epitopes within human TNFR2 selected from among the epitopes shown in SEQ ID NOs: 839-865, 1202 and 1204; or ii) an antigen-binding fragment selected from MR2-1 or MAB2261. or iii) K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D1 43N/A145R , A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145 N/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, which is a soluble TNF variant comprising one or more TNFR2 selective mutations (according to SEQ ID NO: 2); or iv) TNF A single chain TNFR2 selective comprising the mutation D143N/A145R, in which the mutein is linked by (GGGGS)n (where n=1-5), or all or part 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);
[wherein MD is a multimerization domain; TNFmut is a TNFR2-selective TNF mutein; L1, L2, and L3 are linkers that may be the same or different;
The MD can be 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. a trimerization domain (residues 110-139 of SEQ ID NO: 806, SEQ ID NO: 807);
The L1, L2 and L3 are each (GGGGS)n (wherein n=1-5), or all or part of the stalk region of TNF (SEQ ID NO: 812), or a mixture thereof;
The TNF mutein comprises the TNFR2 selective mutation D143N/A145R]
258. The construct of any one of claims 241-257, selected from: 259. The construct of claim 258, wherein each of the TNFR1 antagonist and TNFR2 agonist is monovalent. 259. The construct of claim 258, wherein the TNFR1 antagonist is monovalent and the TNFR2 agonist is bivalent. For the treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders characterized by overexpression of TNF or deregulation of TNFR1 signaling in its pathogenesis. 261. The construct of any one of claims 1-260, which is a multispecific TNFR1 antagonist/TNFR2 agonist for use in. For the treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders characterized by overexpression of TNF or deregulation of TNFR1 signaling in its pathogenesis. 261. Use of a construct according to any one of claims 1 to 260, which is a multispecific TNFR1 antagonist/TNFR2 agonist for. 261. A composition comprising a construct according to any one of claims 1-260 in a pharmaceutically acceptable carrier or vehicle. For the treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders characterized by overexpression of TNF or deregulation of TNFR1 signaling in its pathogenesis. 264. The composition of claim 263 for use in. said chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder characterized by overexpression of TNF or deregulation of TNFR1 signaling in its pathogenesis;
Rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), spondyloarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), uveitis, fibrosis Sexual diseases, endometriosis, lupus, multiple sclerosis (MS), congestive heart failure, cardiovascular diseases, 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), pulmonary inflammation, pulmonary diseases and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, hereditary with TNF/TNFR1 as a pathological mediator The construct of any one of claims 1-261, or the use of claim 262, or the composition of claim 263 or claim 264, selected from a disease, periodic fever syndrome, or cancer. thing. A construct according to any one of claims 1 to 261, or a use according to claim 262, or a composition according to claim 263 or claim 264, for use in the treatment of rheumatoid arthritis. Use of a construct according to any one of claims 1 to 261 or a use according to claim 262 or a composition according to claim 263 or claim 264 for the treatment of rheumatoid arthritis. A construct that is a TNFR2 antagonist construct comprising a TNFR2 antagonist, an optional linker, and an optional activity modifier. 269. The construct of claim 268, having Formula 5:
(TNFR2 antagonist) n-linker p-(activity modifier) q, or linker p-(activity modifier) q-(TNFR2 antagonist) n [wherein
n and q are each integers, each independently 1, 2, or 3;
p is 0, 1, 2, or 3;
TNFR2 antagonists interact with TNFR2 to inhibit (antagonize) its active TNFR2, thereby inhibiting proliferation and/or inducing death of Tregs, and further inhibiting proliferation and inducing death of TNFR2-expressing tumor cells. obtain, is a molecule;
An activity modifier is a moiety that moderates or alters the activity or pharmacological properties of the construct as compared to the construct in the absence of the activity modifier;
The linker increases the plasticity of the construct, and/or alleviates or reduces steric effects or interactions of the construct with the receptor, and/or increases the solubility of the construct in aqueous media]. 270. The construct of claim 268 or claim 269, wherein each of the activity modifier and linker is as defined and described for the construct of any of claims 1-250. TNFR2 antagonist is
reduce and/or inhibit the proliferation of myeloid-derived suppressor cells (MDSCs); and/or induce apoptosis in MDSCs by binding to TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment; 271. The construct of any one of claims 268-270, which induces the proliferation of T effector cells, including cytotoxic CD8+ T cells, by inhibiting the proliferation and activity of Tregs. The TNRF2 antagonist may contain residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4), or e.g. binds to an epitope within human TNFR2 that includes one or more of the larger epitopes containing contiguous or discontinuous residues, and to an epitope containing residues KCSPG (corresponding to residues 56-60 of SEQ ID NO: 4); or 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); corresponding), 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 residues 75-1 of SEQ ID NO: 4 128, 86-103, 111-128 or 150-190. The construct according to any one of paragraphs 268-271. Where said antibody, fragment thereof, or single chain form thereof is directed against an epitope comprising one or more residues of the KCRPG sequence (SEQ ID No. 840), the same antibody against a peptide comprising the KCSPG sequence of human TNFR2 (SEQ ID No. 839). 273. The construct of any one of claims 268-272, wherein the construct of any one of claims 268-272 binds with an affinity that is at least 10 times greater than the affinity of the antigen-binding fragment. The TNFR2 antagonist is
TNFRAB1 (see SEQ ID NO: 1212 and 1213 for the heavy and light chain sequences of TNFRAB1, respectively), TNFRAB2 and TNFR2A3 (see, e.g., US Patent Publication No. 2019/0144556 for a description of these antibodies);
Antibodies, antibody fragments and single -chain (corresponding to residue of 69 to 112 of array number 1212), TNFRAB2 (ARDGSYSPFDYWG) or TNFR2 or TNFR2) A3 (ARDGSYSPFDYFG; SEQ ID NO: 1223), or CDR-H3 sequence (with at least about 85% sequence identity thereto), TNFRAB1 (e.g., residues of TNFR2 with a 40-fold higher affinity than residues 48-67, including residues KCSPG of TNFR2) specifically binds residues 130-149, including KCRPG),
274. The construct of any one of claims 268-273, which is an antibody or a fragment or single chain form of an antibody selected from among: The TNFR2 antagonist is
an epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851);
one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), positions 91-98 (PECLSCGS; SEQ ID NO: 1248), and/or positions 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR2; epitope containing;
TNFR2A3 selected from the first epitope comprises residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463), an epitope comprising a KCRPG motif, and/or residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 275. The construct of any one of claims 268-274, wherein the construct binds to one or more epitopes in TNFR2 selected from a second epitope comprising 1464). CDR-H1 amino acids having the sequence shown in any of SEQ ID NOs: 1214, 1215, and 1231-1233, wherein said TNFR2 antagonist is substituted with the corresponding CDR sequence of a phenotype-neutral TNFR2-specific antibody, SEQ ID NO: 1216 , 1224, and 1230, the CDR-H3 sequence shown in any of SEQ ID NO: 1217, 1223, and 1225 to 1229, and/or residues 99 to 112 of SEQ ID NO: 1212. Corresponding CDR-H3 of TNFRAB1; CDR-L1 sequence shown in any of SEQ ID NO: 1218 and 1234-1236, and/or CDR-L1 sequence of TNFRAB1 corresponding to residues 24-33 of SEQ ID NO: 1213; SEQ ID NO: 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1 corresponding to residues 49-55 of SEQ ID NO: 1213; and/or SEQ ID NO: 1221, 1222, and 1241- 1244, or the CDR-L3 sequence of TNFRAB1 corresponding to residues 88-96 of SEQ ID NO: 1213; CDR-H1 and CDR-H2 sequences of the human antibody heavy chain variable domain consensus sequence of SEQ ID NO: 1245 substituted with the corresponding CDR sequences of a TNFR2-specific antibody and/or the correspondence of a phenotype-neutral TNFR2-specific antibody an antibody, fragment or single chain thereof, comprising one or more of the CDR-L1, CDR-L2 and CDR-L3 sequences of the human antibody light chain variable domain sequence of SEQ ID NO: 1246, substituted with the CDR sequences of SEQ ID NO: 1246. 276. The construct of any one of claims 268-275, which is in the form of 276. The construct of any one of claims 268-275, wherein the TNFR2 antagonist specifically binds to an epitope within TNFR2 set forth in any one of SEQ ID NOs: 1247-1464. TNFR2 antagonist is
(a) residues KCRPG corresponding to residues 142-146 of SEQ ID NO: 4, or larger epitopes comprising residues 130-149, 137-144 or 142-149, or at least five consecutive or one or more epitopes within human TNFR2 that include one or more of the discontinuous residues and do not bind to an epitope that includes residues KCSPG corresponding to residues 56-60 of SEQ ID NO: 4; b)
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 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 residues 75-128, 86 of SEQ ID NO: 4 specifically for an epitope selected from one or more TNFR2 epitopes comprising a sequence of amino acids comprising an epitope comprising at least 5 contiguous or non-contiguous residues within ~103, 111-128, or 150-190. 278. The construct of any one of claims 268-277, which binds. 279. The construct of any one of claims 268-278, comprising 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 analogs are lenalidomide and pomalidomide. 282. The construct of any one of claims 268-281, comprising a TNFR2 antagonist 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 capable of reducing FoxP3 expression and inhibiting suppressive activity of Tregs. 284. The construct of claim 282 or claim 283, wherein the inhibitor is panobinostat or cyclophosphamide or triptolide. 283. The construct of any one of claims 268-282 for use in the treatment of infectious diseases and cancers expressing TNFR2. The cancer is 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. 286. The construct of claim 285, wherein the construct is a cancer selected from , liver cancer, and lung cancer.