WO2023205333A2

WO2023205333A2 - Tumor necrosis factor receptor 1 antagonist polypeptides and methods of use thereof

Info

Publication number: WO2023205333A2
Application number: PCT/US2023/019264
Authority: WO
Inventors: Benjamin J. Hackel; Jonathan N. Sachs; Nagamani VUNNAM; MaryJane Olivia BEEN
Original assignee: Regents Of The University Of Minnesota
Priority date: 2022-04-21
Filing date: 2023-04-20
Publication date: 2023-10-26
Also published as: WO2023205333A3

Abstract

Certain embodiments of the invention provide helix sequence(s) that have affinity for Tumor Necrosis Factor Receptor 1 (TNFR1). Certain embodiments of the invention provide an isolated TNFR1 binding polypeptide (e.g., anti-TNFRl affibody, or binding fragment thereof) that inhibits the activity of TNFR1. Certain embodiments of the invention provide methods of noncompetitively inhibiting TNFR1 and/or treating TNFR1 related diseases or disorders, including an inflammatory or autoimmune disorder.

Description

TUMOR NECROSIS FACTOR RECEPTOR 1 ANTAGONIST POLYPEPTIDES AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to United States Provisional Application Number 63/333,223 that was filed on 21 April 2022. The entire content of the application referenced above is hereby incorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under EB028274, AI144932, GM13 1814 and EB023339 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Tumor necrosis factor-alpha (TNF-a) is a key regulator of immunity and plays a significant role in initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory or autoimmune diseases, such as Crohn’s disease, ulcerative colitis, and rheumatoid arthritis. Accordingly, anti-TNF drugs have been developed to treat inflammatory or autoimmune diseases. However, despite the clinical success of anti-TNF treatments, the use of these drugs is limited because they target the ligand rather than the specific receptor signaling pathways, and therefore, often induce adverse side effects. Thus, there is a need to develop TNF receptor specific therapeutics.

SUMMARY OF THE INVENTION

Certain embodiments of the invention provide an isolated TNFR1 binding polypeptide, comprising one or more helix domains selected from the group consisting of

(a) a helix 1 domain comprising an amino acid sequence having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 60% sequence identity to an amino acid sequence of SEQ ID NO: 8.

Certain embodiments of the invention provide a composition comprising an isolated TNFR1 binding polypeptide described herein, and a carrier.

Certain embodiments of the invention provide an isolated nucleic acid comprising a nucleotide sequence encoding an isolated TNFR1 binding polypeptide described herein. Certain embodiments of the invention provide a vector comprising the nucleic acid described herein.

Certain embodiments of the invention provide a cell comprising the nucleic acid or the vector described herein.

Certain embodiments of the invention provide a method of inhibiting the activity of TNFR1, comprising contacting TNFR1 with an isolated TNFR1 binding polypeptide described herein.

Certain embodiments of the invention provide an isolated TNFR1 binding polypeptide described herein for use in diagnosis or medical therapy.

Certain embodiments of the invention provide a method for treating a TNFR1 related disorder (e.g., an inflammatory or autoimmune disorder) in a mammal, comprising administering an effective amount of an isolated TNFR1 binding polypeptide described herein to the mammal.

Certain embodiments of the invention provide an isolated TNFR1 binding polypeptide described herein for the prophylactic or therapeutic treatment of a TNFR1 related disorder.

Certain embodiments of the invention provide the use of an isolated TNFR1 binding polypeptide described herein to prepare a medicament for the treatment of a TNFR1 related disorder in a mammal.

Certain embodiments of the invention provide a kit comprising an isolated TNFR1 binding polypeptide described herein, packaging material, and instructions for administering the isolated polypeptide to a mammal to treat a TNFR1 related disorder.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Selection strategy for discovery of TNFR1 -binding ABYs. MACS was used to evolve a naive AB Y library to select against non-specific binding (anti-GFP and LAG3 coated bead) and for TNFR1 binders. The shown selection scheme was iterated through three times before six clones were selected for further analysis and characterization.

Figures 2A-2B. Specificity of naive ABY library enriched for TNFR1. Yeast cells from the final population enriched for TNFR1 binding were labeled with anti-c-myc fluorescent antibody and incubated with lysate from cells overexpressing (Fig.2A) TNFR1ACD-GFP or (Fig.2B) LAG3-GFP for 2 hours at 4°C. Target binding was detected by flow cytometry.

Figures 3A-3C. ABYTNFRI clone shows TNFR1 binding. (Fig.3A) Sequences of the five unique, randomly selected ABY clones with diversified regions shown in gray. (Fig.3B) Production and purification of ABYTNFRI. Gel electrophoresis and Coomassie staining of purified ABYTNFRI-I exhibits high purity. (Fig.3C) Binding of ABYTNFRI variants to TNFR1ACD-GFP- expressing stable cells. HEK293 cells with stable expression of TNFR1ACD-GFP were incubated with 50 nM soluble ABYTNFRI variants. Binding was detected with the AF647-conjugated anti- Hiss antibody using flow cytometry. One clone, ABYTNFRI-I, showed substantial binding (48.6%), indicated by the right shift in the population.

Figures 4A-4D. Affinity titration of ABYTNFRI-I. Fluorescent microscopy images of HEK293T cells transiently transfected with TNFR1ACD-GFP (Fig.4A) or TNFR2ACD-GFP (Fig.4B) plasmids. (Fig.4C) Median fluorescence intensities of TNFR1ACD-GFP or TNFR2ACD-GFP in transfected cells. (Fig.4D) For binding studies HEK293T cells with endogenous expression of TNFR1 or a transient expression of TNFR1 ACD-GFP or TNFR2ACD- GFP were incubated with increasing concentrations of soluble ABYTNFRI-I (0.000001-10 pM). TNFR1 ACD-GFP expressing HEK293 cells were also incubated with a non-binder (0.000001-10 pM). Binding was detected with anti-his antibody, followed by the AF647-conjugated antibody using flow cytometry. Data are presented as the mean ± standard deviation (n=3).

Figures 5A-5F. Direct binding of ABYTNFRI-I to PLAD. Recombinant PL AD was immobilized to a CM5 series S sensor chip by amine coupling and analytes were flowed across the sensor surface while SPR sensorgrams were collected at a rate of 10 Hz using a Biacore S200. Affinity binding curves were fit to a steady state binding model using Biacore S200 evaluation software. Data shown are representative of three repeats. (Fig.5 A, B) ABYTNFRI-I binds PLAD in a dose dependent manner with a KD = 377 ± 66 nM while negative controls (Fig.5C, D) nonbinder and (Fig.5E, F) TNFa do not bind PLAD in the concentration range tested. KD values reported are average KD ± SEM where n = 3.

Figures 6A-6E. ABYTNFRI-I inhibits TNF-induced inflammatory signaling. (Fig.6A) HEK293 cells were treated with ABYTNFRI-I (0.1 nM - 100 pM) or a non-binder (0.1 nM - 100 pM) for 2 hours, followed by the addition of TNF-a (0.6 nM) for 30 minutes. After incubation, cells were lysed and analyzed using western blotting. (Fig.6B) Densitometry analysis of IxBa bands. ABYTNFRI-I inhibits IxBa degradation with an IC50 = 7 ± 6 nM. Data are presented as the mean ± standard deviation (n=2). (Fig.6C) Effect of ABYTNFRI-I on TNF-a-induced NF-KB activation. (Fig.6D) Effect of non-binder on TNF-a-induced NF-KB activation. For NF-KB dependent luciferase reporter assay, HEK293 cells were transfected with luciferase reporter plasmids and treated with a non-binder or ABYTNFRI-I (0.0000001- 10 pM) for 2 hours, followed by the addition of TNF-a (0.6 nM) for 24 hours. ABYTNFRI-I inhibits activation with an IC50 = 0.23 ± 0.07 nM. Luciferase activity values were normalized to the DMSO-only control, and data are mean ± the standard deviation (n=3). (Fig.6E) Cytotoxicity of ABYTNFRI-I. HEK293 cells were treated with increasing concentrations of ABYTNFRI-I (0.00001- 50 pM). Cell viability of ABYTNFRI-I was measured using MTT assay. Data are presented as mean ± standard deviation (n=3)

Figures 7A-7D. Effect of ABYTNFRI-I on TNFR1 knockout cells. (Fig.7A) Surface expression of TNFR1 in WT and TNFR1-KO Hapl cells was determined using flow cytometry. TNFR1-WT showed the highest TNFR1 fluorescent staining, followed by a lower signal of the TNFR1-KO (the peak pointed by arrowhead), followed by baseline signals of unlabeled cells, cells labeled with secondary antibody. Fluorescent signals of cells labeled with TNFR1 antibody (TNFR1-WT and TNFR1-KO) were compared in Fig.7B. Mean fluorescence intensity of Hapl cells labeled with TNFR1 antibody. (Fig.7C) TNFR1-WT or (Fig.7D) TNFR1-KO cells were treated with ABYTNFRI-I (0.0001- 100 pM) for 2 hours, followed by the addition of TNF-a (10 ng/ml) for 30 minutes. After incubation, cells were lysed and analyzed using western blotting for IkB (n=l).

Figures 8A-8E. ABYTNFRI-I does not affect TNFR1-TNF binding. (Fig.8A) ABYTNFRI- i -TNFR1 binding in presence and absence of TNF. HEK293T cells with a transient expression of TNFR1ACD-GFP were incubated with increasing concentrations of soluble ABYTNFRI-I (0.000001-10 pM) in the presence (10 ng/ml) and absence of TNF. Binding was detected with anti-his antibody, followed by the AF647-conjugated antibody using flow cytometry. Data are presented as the mean ± standard deviation (n=3). (Fig.8B) TNF-TNFR1 binding in presence and absence of ABYTNFRI-I or negative controls were assessed by a co-immunoprecipitation assay with anti-flag magnetic beads. Flag-tagged TNF was mixed with anti-flag beads and incubated at 4 °C for 2 hours. The beads were then washed thrice to remove the unbound proteins. Next, TNF-coated beads were incubated with HEK293 lysates (7.5 mg/ml) in the presence and absence of ABYTNFRI- i (100 nM) or a non -binder (100 nM) or with a known TNF competitor, a monoclonal antibody H398 (70 nM). Beads were washed thrice and pulled-down proteins were resolved by SDS-PAGE and immunoblotted with anti -flag and TNFR1 antibodies. (Fig.8C) The TNFR1 recovery for each condition was normalized to the TNF content and then normalized to the TNF only condition. **** p < 0.0001 compared to control by two-tailed unpaired t test. (Fig.8D) Fluorescence lifetime measurements with HEK293T cells transiently expressing TNFR1ACD-GFP only and TNFR1ACD-FRET pair. (Fig.8E) Effect of ABYTNFRI-I on ligand-independent TNFR1-TNFR1 interactions. FRET efficiency did not change with ABYTNFRI-I treatment compared to DMSO control. Data are presented as the mean ± standard deviation (n=3).

Figures 9A-9B. (Fig.9A) Effect of ABYTNFRI-I on TNF-a-induced RelB activation (TNFR2 non-canonical pathway). HUVEC cells were transfected with membrane-TNF for six hours, treated with or without ABYTNFRI-I (10 pM), and analyzed with anti-RelB Western blot. Representative western blot of 3 independent experiments. (Fig.9B) Densitometry analysis of RelB bands.

Figures 10A-10C. Surface expression of TNFR1ACD-GFP or TNFR2ACD-GFP was measured using flow cytometry. (Fig.lOA) Histograms showing the expression of TNFR1ACD- GFP during the ABYTNFRI-I titration (Fig.1 OB) Histograms showing the expression of TNFR1ACD-GFP during the non-binder titration. (Fig.10C) Histograms showing the expression of TNFR2ACD-GFP during the ABYTNFRI-I titration.

Figures 11A-11J. ABYTNFRI-I directly bind TNFR1 and TNFR2. Recombinant TNFR1 and TNFR2 were immobilized to a CM5 series S sensor chip by amine coupling and analytes were flowed across the sensor surface while SPR sensorgrams were collected at a rate of 10 Hz using a Biacore S200. Affinity binding curves for AB YTNFRI-I were fit to a steady state binding model and TNFa sensorgrams were fit to a 1 : 1 kinetic binding model using Biacore S200 evaluation software. Data shown are representative of three repeats. (Fig.l 1A, B) ABYTNFRI-I binds TNFR1 in a dose dependent manner with a KD = 73 ± 1 nM. ABYTNFRI-I also binds TNFR2 (Fig.11C, D) with a KD = 129 ± 28 nM. The negative control non-binder affibody does not bind either (Fig.1 IE, F) TNFR1 or (Fig.11 G, H) TNFR2 at the concentrations tested. TNFa was used as a positive control to ensure that the sensor surface was active. We report the following kinetic values for (Fig.l II) TNFR1 (K_D = 4 ± 1 pM, k_a = 2.08 ± 0.05 x 10⁵ M’ , k_d= 8.69 ± 3.6 x IO'⁷ s’¹; and (Fig.11 J) TNFR2 (KD = 1.9 ± 0.1 nM, k_a =1.31 ± 0.07 x 10⁴ M’ , k_d= 2.5 ± 0.1 x 10’³ s’¹). Affinity and kinetic values reported are average ± SEM where n = 3.

Figures 12A-12C. Affinity and functionality of ABYTNFRI-I in mouse L929 cells. (Fig. l2A) L929 mouse cells were incubated with different concentrations of ABYTNFRI-I and then binding was detected with anti-his antibody, followed by the AF647- conjugated secondary antibody using flow cytometry. (Fig. l2B) For IkB degradation assay, L929 cells were treated with ABYTNFRI-I for 2 hours then were treated with 10 ng/mL mouse TNF for 30 minutes. The cells were lysed and analyzed using western blot for IkBa. (Fig.l2C) Protein bands were analyzed using ImageJ (n=3). IKBO. bands are normalized to total protein content.

Figure 13 Affinity titration of ABYTNFRI-I. HEK293 cells with a stable expression of TNFR1ACD-GFP were incubated with increasing concentrations of soluble ABYTNFRI-I (1 pM -50 pM). Binding was detected by the AF647-conjugated anti-Hiss antibody via flow cytometry. In particular, HEK293 cells with stable expression of TNFR1ACD-GFP or transient expression of LAG3-GFP were lifted using trypsin and washed three times with PBS. Cells were incubated with soluble ABY for 2 hours. After incubation, cells were washed with PBS with 0.1% bovine serum album (PBSA) to remove unbound affibody and labeled with Alexa Fluor 647-conjugated anti -Hiss antibody for 1- 2 hours at 4°C. Fluorescence was analyzed on a BD Accuri C6 flow cytometer. Data are presented as the mean ± standard deviation (n=3). The line represents the minimization of the sum of squared errors for a 1 : 1 binding model.

Figure 14. ABYTNFRI-I is not cross-reactive with TNFR2. HEK293T cells with or without transient expression of TNFR2ACD-GFP were incubated with soluble ABYTNFRI-I (0.1 - 50 pM). Binding was detected by the AF647-conjugated anti-Hiss antibody via flow cytometry. In particular, cells were incubated with soluble ABY for 2 hours. After incubation, cells were washed with PBS with 0.1% bovine serum album (PBSA) to remove unbound affibody and labeled with Alexa Fluor 647-conjugated anti -Hiss antibody for 1- 2 hours at 4°C. Data are presented as the mean ± the standard deviation (n=3). The line represents the minimization of the sum of squared errors for a 1:1 binding model. HEK293 cells have endogenous expression of TNFR1 resulting in moderate binding. No additional binding was observed when cells expressed TNFR2ACD-GFP.

DETAILED DESCRIPTION

Tumor necrosis factor (TNF)-a is a master pro-inflammatory cytokine. One of its receptors, TNFR1, is an important signaling component in numerous disease states (e.g., inflammatory) and is an indirect target of numerous FDA-approved therapies. However, these approved therapies target the ligand, tumor necrosis factor (TNF), rather than the receptor, as a means to inhibit TNFa-TNFR signaling. This conventional approach results in numerous problematic side effects, such as increased risk of infections. Selective inhibition of the receptor (TNFR1) itself avoids impacting other TNF binding partners / receptors (e.g., tumor necrosis factor receptor 2 (TNFR2)) of the native ligand. As described herein, an allosteric, noncompetitive mode of inhibition against TNFR1 was developed to selectively block the TNFa- TNFR 1 signaling pathway.

Certain embodiments of the invention provide an isolated TNFR1 binding polypeptide (e.g., affibody), e.g., comprising a helix 1 and/or helix 2 sequence as described herein, that has affinity to human tumor necrosis factor receptor 1 (TNFR1) and inhibits signaling via a noncompetitive, allosteric mechanism (see, e.g., ABYTNFRI-I and Example 1).

In certain embodiments, an isolated TNFR1 binding polypeptide described herein (e.g., ABYTNFRI-I) binds at a different TNFR1 binding site relative to the TNFR1 binding site for its endogenous ligand TNFa. Thus, in certain embodiments, an isolated TNFR1 binding polypeptide described herein does not affect the native ligand-receptor binding between TNFa and TNFR1. However, in certain embodiments, an isolated TNFR1 binding polypeptide described herein is capable of blocking the TNFa induced TNFR1 signaling cascade. For example, NF-KB activation and/or Ii<Ba degradation may be inhibited by an isolated TNFR1 binding polypeptide described herein. Therefore, the expression of pro-inflammatory gene(s) regulated by NF-KB may be inhibited. Thus, in certain embodiments, an isolated TNFR1 binding polypeptide described herein is an allosteric antagonist that is capable of inhibiting TNFa induced TNFR1 mediated signaling and/or a proinflammatory response.

In certain embodiments, an isolated TNFR1 binding polypeptide described herein selectively binds TNFR1 and does not bind TNFR2 (e.g., cell membrane bound human TNFR2). In certain embodiments, an isolated TNFR1 binding polypeptide described herein is capable of blocking TNFa induced TNFR1 signaling without inhibiting TNFa induced TNFR2 signaling e.g., TNFR2 non-canonical signaling pathway), such as RelB activation. In certain embodiments, a TNFR1 binding polypeptide described herein may bind an isolated soluble extracellular domain (ECD) fragment of TNFR2 but does not bind cell membrane bound TNFR2 expressed on a cell (e.g., and thus does not inhibit TNFa induced TNFR2 signaling in a TNFR2 expressing cell).

As used herein, the term “affibody” or “affibody molecule” refers to an engineered small protein ligand based on the three helical bundle Z domain of the Ig-binding region of protein A. In certain embodiments, an affibody is a small protein of about 58 amino acids in length (about 6-7KDa), which may have a N-terminal segment, helix 1, loop 1, helix 2, loop 2, helix 3 and a C -terminal segment. Affibody molecule libraries can be constructed by randomization of about 13-17 amino acid residues in helices 1 and 2 of the three-helix bundle protein to screen for specific binders, followed by further affinity maturation for more potent binders. Thus, helix 1 and helix 2 are believed to play a role in an affibody’ s binding properties towards a target. For example, Ren, et al. showed smaller 2-helix affibody derivatives have excellent binding affinity (JNuclMed. 2009 Sep; 50(9): 1492-1499).

Additionally, the polypeptides of the invention (e.g., affibody, or binding fragment thereof) may be operably linked to other functional domains. Other function domain(s) may affect the biodistribution and/or circulating half-life of the polypeptides of the invention (e.g., affibody, or binding fragment thereof), and may also confer additional effector function. For example, affibodies or fragments thereof (e.g., binding portions of an affibody), have served as a versatile fusion partner with a variety of other functional domains, such as enzymes, fluorescent proteins, toxins, antibodies or fragments thereof (e.g., Fc domain), albumin binding domains, additional identical affibodies for preparing homodimers or multimers, additional distinct affibodies for generating bi-specificity or multi-specificity, to provide a diverse range of biologic agents (may be larger than 6-7KDa) that contain at least an affibody or a binding portion of an affibody.

Isolated polypeptides having affinity for TNFR1

Thus, certain embodiments of the invention provide an isolated TNFR1 binding polypeptide that comprises a helix 1 domain and/or a helix 2 domain derived from an affibody, or binding fragment thereof, described herein, for example, ABYTNFRI-I. The amino acid sequences of the helix 1 and helix 2 of TNFR1 binder ABYTNFRI-I are set forth in Table 1 below and in Figure 3 A.

In certain embodiments, an isolated TNFR1 binding polypeptide comprises a helix 1 as described in any of the embodiments provided herein, and/or a helix 2 as described in any of the embodiments provided herein.

Thus, certain embodiments of the invention provide an isolated TNFR1 binding polypeptide, comprising one or more helix domains selected from the group consisting of:

(a) a helix 1 domain comprising an amino acid sequence 1) having at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of AKESGYALTEIYC (SEQ ID NO:2); or 2) having up to 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence 1) having at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of VWQLRAFIVALGD (SEQ ID NO:8); or 2) having up to 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO:8. The one or more substitution(s) in a helix domain may be a conservative substitution or non-conservative substitution. In certain embodiments, the one or more substitution(s) in a helix domain is a conservative substitution. In certain embodiments, the one or more substitution(s) in a helix domain is a non-conservative substitution. In certain embodiments, the isolated TNFR1 binding polypeptide comprises a helix 1 and a helix 2 as described above. In certain embodiments, an isolated TNFR1 binding polypeptide comprises a helix 1 domain and a helix 2 domain as described above and further comprises a helix 3 domain as described herein.

For example, certain embodiments of the invention provide an isolated TNFR1 binding polypeptide, comprising one or more helix domains selected from the group consisting of:

(a) a helix 1 domain comprising an amino acid sequence having at least 65% (e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 2, or a sequence having up to 1, 2, 3, or 4 substitutions relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 65% (e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:8, or a sequence having up to 1, 2, 3, or 4 substitutions relative to SEQ ID NO:8.

In certain embodiments, the isolated TNFR1 binding polypeptide comprises one or more helix domains selected from the group consisting of:

(a) a helix 1 domain comprising an amino acid sequence having at least 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 2, or a sequence having up to 1, 2 or 3 substitutions relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 8, or a sequence having up to 1, 2 or 3 substitutions relative to SEQ ID NO:8.

(a) a helix 1 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:2; and (b) a helix 2 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 8.

In certain embodiments, the TNFR1 binding polypeptide comprises one or more helix domains selected from the group consisting of:

(a) a helix 1 domain comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 8.

(a) a helix 1 domain comprising an amino acid sequence having up to 5 substitutions relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having up to 5 substitutions relative to SEQ ID NO: 8.

(a) a helix 1 domain comprising an amino acid sequence having up to 4 substitutions relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having up to 4 substitutions relative to SEQ ID NO: 8.

(a) a helix 1 domain comprising an amino acid sequence having up to 3 substitutions relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having up to 3 substitutions relative to SEQ ID NO: 8.

(a) a helix 1 domain comprising an amino acid sequence having up to 2 substitutions relative to SEQ ID NO:2; and (b) a helix 2 domain comprising an amino acid sequence having up to 2 substitutions relative to SEQ ID NO: 8.

(a) a helix 1 domain comprising an amino acid sequence having up to 1 substitution relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having up to 1 substitution relative to SEQ ID NO: 8.

(a) a helix 1 domain comprising the amino acid sequence of SEQ ID NO:2; and

(b) a helix 2 domain comprising the amino acid sequence of SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 2 (AKESGYALTEIYC), or a sequence having up to 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO:2.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having at least 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:2, or a sequence having up to 1, 2, or 3 substitutions relative to SEQ ID NO:2.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 2, or a sequence having up to 1 or 2 substitutions relative to SEQ ID NO:2.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:2, or a sequence having up to 1 substitution relative to SEQ ID NO:2. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having up to 5 substitutions relative to SEQ ID NO:2. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having up to 4 substitutions relative to SEQ ID NO:2. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having up to 3 substitutions relative to SEQ ID NO:2. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having up to 2 substitutions relative to SEQ ID NO:2. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence having up to 1 substitution relative to SEQ ID NO:2.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising an amino acid sequence of SEQ ID NO:2.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain consisting of an amino acid sequence of SEQ ID NO:2.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 8 (VWQLRAFIVALGD), or a sequence having up to 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having at least 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:8, or a sequence having up to 1, 2, or 3 substitutions relative to SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:8, or a sequence having up to 1 or 2 substitutions relative to SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 8, or a sequence having up to 1 substitution relative to SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having up to 5 substitutions relative to SEQ ID NO:8. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having up to 4 substitutions relative to SEQ ID NO:8. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having up to 3 substitutions relative to SEQ ID NO:8. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having up to 2 substitutions relative to SEQ ID NO: 8. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence having up to 1 substitution relative to SEQ ID NO: 8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain comprising an amino acid sequence of SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 2 domain consisting of an amino acid sequence of SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises two, three, four, five, six, seven, eight, or nine helices as described herein.

In certain embodiments, the TNFR1 binding polypeptide comprises two helices as described herein. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain and a helix 2 domain as described herein.

Certain embodiments provide affibodies and binding portions of affibodies that specifically bind to TNFR1, which comprise a helix 1 and/or helix 2 domain described herein (see, e.g., ABYTNFRI-I). Thus, in some embodiments, a polypeptide described herein is an affibody, or binding fragment thereof, comprising: a helix 1 domain and a helix 2 domain as described in an embodiment provided herein.

In certain embodiments, the isolated TNFR1 binding polypeptide, comprises:

(a) a helix 1 domain comprising an amino acid sequence having at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:2, or a sequence having up to 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO:2; and (b) a helix 2 domain comprising an amino acid sequence having at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:8, or a sequence having up to 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO:8.

In certain embodiments, the isolated TNFR1 binding polypeptide, comprises:

(b) a helix 2 domain comprising an amino acid sequence having at least 65% (e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 8, or a sequence having up to 1, 2, 3, or 4 substitutions relative to SEQ ID NO:8.

In certain embodiments, the isolated TNFR1 binding polypeptide, comprises:

(a) a helix 1 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:2, or a sequence having up to 1 or 2 substitutions relative to SEQ ID NO:2; and (b) a helix 2 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 8, or a sequence having up to 1 or 2 substitutions relative to SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises:

(a) a helix 1 domain comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:2, or a sequence having up to 1 substitution relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO:8, or a sequence having up to 1 substitution relative to SEQ ID NO:8.

In certain embodiments, the isolated TNFR1 binding polypeptide, comprises:

(a) a helix 1 domain comprising an amino acid sequence having up to 2 substitutions relative to SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having up to 2 substitutions relative to SEQ ID NO: 8.

In certain embodiments, the isolated TNFR1 binding polypeptide, comprises: (a) a helix 1 domain comprising an amino acid sequence having up to 1 substitution relative to SEQ ID NO:2; and

In certain embodiments, the TNFR1 binding polypeptide comprises:

(a) a helix 1 domain comprising an amino acid sequence of SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence of SEQ ID NO:8.

In certain embodiments, the TNFR1 binding polypeptide comprises:

(a) a helix 1 domain consisting of an amino acid sequence of SEQ ID NO:2; and

(b) a helix 2 domain consisting of an amino acid sequence of SEQ ID NO: 8.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain, a loop 1 sequence and a helix 2 domain as described herein (e.g., as described in Table 1 below).

In certain embodiments, the polypeptide is a cyclic polypeptide (e.g., a 2-helix polypeptide may comprise helix 1, loop 1 such as SEQ ID NO: 15 and helix 2, while the 2-helix polypeptide may be constrained with a disulfide bridge formed between two homocysteines at the N and C terminals of the polypeptide).

In certain embodiments, the TNFR1 binding polypeptide comprises a N-terminal region, a helix 1 domain, a loop 1 sequence and a helix 2 domain as described herein.

In certain embodiments, the TNFR1 binding polypeptide comprises three helices as described herein. Thus, in certain embodiments, the polypeptide further comprises a helix 3 domain. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 3 domain comprising an amino acid sequence having at least 65% (e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 13, or a sequence having up to 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO: 13. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 3 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 13. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 3 domain comprising an amino acid sequence having up to lor 2 substitutions relative to SEQ ID NO: 13. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain, a helix 2 domain and a helix 3 domain as described herein. In certain embodiments, the TNFR1 binding polypeptide further comprises a helix 3 domain comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 13, or a sequence having up to 1, 2, 3 or 4 substitutions relative to SEQ ID NO: 13. In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising SEQ ID NO:2, a helix 2 domain comprising SEQ ID NO:8, and a helix 3 domain comprising SEQ ID NO: 13.

In certain embodiments, the isolated TNFR1 binding polypeptide comprises:

(a) a helix 1 domain comprising an amino acid sequence having 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO:2;

(b) a helix 2 domain comprising an amino acid sequence having 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO:8; and

(c) a helix 3 domain comprising an amino acid sequence having 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO: 13.

In certain embodiments, the isolated TNFR1 binding polypeptide comprises:

(a) a helix 1 domain comprising an amino acid sequence having 1, 2 or 3 substitutions relative to SEQ ID NO:2;

(b) a helix 2 domain comprising an amino acid sequence having 1, 2 or 3 substitutions relative to SEQ ID NO:8; and

(c) a helix 3 domain comprising an amino acid sequence having 1, 2 or 3 substitutions relative to SEQ ID NO: 13.

In certain embodiments, the isolated TNFR1 binding polypeptide comprises a helix 1 domain comprising SEQ ID NO:2; a helix 2 domain comprising SEQ ID NO:8; and a helix 3 domain comprising SEQ ID NO: 13.

In certain embodiments, a polypeptide described herein is an affibody comprising: a N- terminal region, a helix 1 domain, a loop 1 sequence, a helix 2 domain, a loop 2 sequence, a helix 3 domain and a C-terminal region, as described herein (e.g., Table 1).

In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19. In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 19. In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19. In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity SEQ ID NO: 19. In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19. In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence of SEQ ID NO: 19. In certain embodiments, the TNFR1 binding polypeptide consists of an amino acid sequence of SEQ ID NO: 19.

In certain embodiments, the isolated TNFR1 binding polypeptide comprises an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19, wherein the amino acid sequence comprises a helix 1 domain comprising SEQ ID NO:2 and a helix 2 domain comprising SEQ ID NO:8. In certain embodiments, the TNFR1 binding polypeptide amino acid sequence comprises a helix 3 domain comprising SEQ ID NO: 13.

In certain embodiments, the isolated TNFR1 binding polypeptide comprises an amino acid sequence that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19, wherein the amino acid sequence comprises a helix 1 domain comprising SEQ ID NO:2 and a helix 2 domain comprising SEQ ID NO: 8. In certain embodiments, the TNFR1 binding polypeptide amino acid sequence comprises a helix 3 domain comprising SEQ ID NO: 13.

In certain embodiments, the TNFR1 binding polypeptide comprises a helix 1 domain comprising SEQ ID NO:2, a loop 1 sequence comprising SEQ ID NO: 15, a helix 2 domain comprising SEQ ID NO:8, a loop 2 sequence comprising SEQ ID NO: 16 and a helix 3 domain comprising SEQ ID NO: 13.

In certain embodiments, the TNFR1 binding polypeptide comprises a N-terminal sequence comprising SEQ ID NO: 14, a helix 1 domain comprising SEQ ID NO:2, a loop 1 sequence comprising SEQ ID NO: 15, a helix 2 domain comprising SEQ ID NO:8, a loop 2 sequence comprising SEQ ID NO: 16, a helix 3 domain comprising SEQ ID NO: 13 and a C- terminal sequence comprising SEQ ID NO: 17.

In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19: (AEAKYAKESGYALTEIYCLPNLTVWQLRAFIVALGDDPSQSSELLSEAKKLNDSQAPK). In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19.

In certain embodiments, the TNFR1 binding polypeptide comprises an amino acid sequence that has at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 19.

In certain embodiments, the TNFR1 binding polypeptide comprises the amino acid sequence of SEQ ID NO: 19.

In certain embodiments, the TNFR1 binding polypeptide consists of SEQ ID NO: 19, which is from N-terminal to C-terminal.

In certain embodiments, the TNFR1 binding polypeptide is encoded by a nucleic acid sequence that comprises a sequence that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:24.

In certain embodiments, the TNFR1 binding polypeptide comprises homo or hetero multimer of an affibody as described herein, or a binding fragment thereof. In certain embodiments, the polypeptide comprises two affibodies or two binding fragments thereof. In certain embodiments, the TNFR1 binding polypeptide comprises four helices as described above. In certain embodiments, the TNFR1 binding polypeptide comprises two helix 1 domains (e.g., same or different helix 1) and two helix 2 domains (e.g., same or different helix 2) as described herein.

In certain embodiments, the TNFR1 binding polypeptide comprises six helices as described above. In certain embodiments, the TNFR1 binding polypeptide comprises three helix 1 domains and three helix 2 domains as described above. In certain embodiments, the TNFR1 binding polypeptide comprises two helix 1 domains, two helix 2 domains and two helix 3 domains as described herein.

In certain embodiments, the TNFR1 binding polypeptide is about 25, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 amino acids in length. In certain embodiments, the TNFR1 binding polypeptide is about 25 to 1900, 30 to 1800, 40 to 1700, 50 to 1600, 55 to 1500, 56 to 1400, 57 to 1300, 58 to 1200, 75 to 1100, 100 to 1000, 150 to 900, or 200 to 800 amino acids in length. In certain embodiments, the TNFR1 binding polypeptide is about 45 to about 70 amino acids in length, or about 50 to about 65 amino acids in length, about 53 to about 63 amino acids in length. In certain embodiments, the TNFR1 binding polypeptide is about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids in length. In certain embodiments, the TNFR1 binding polypeptide is a non-immunoglobulin polypeptide. For example, the TNFR1 binding polypeptide is an affibody that is about 58 amino acid residues in length and/or about 6~7KDa in size. In certain embodiments, the TNFR1 binding polypeptide is a 2-helix affibody derivative that is about 32-38 amino acid residues (e.g., 35 aa) in length.

Table 1. Sequences

In certain embodiments, a TNFR1 binding polypeptide as described herein recognizes one or more epitopes within TNFR1 (e.g., human TNFR1). For example, a polypeptide described herein binds one or more epitopes of the extracellular domain of TNFR1.

In certain embodiments, an isolated TNFR1 binding polypeptide as described herein is an inhibitor of TNFR1. In certain embodiments, an isolated polypeptide as described herein does not block the binding between TNFR1 and its endogenous ligand (e.g., TNF-alpha). Hence, in certain embodiments, an isolated TNFR1 binding polypeptide is a non-competitive inhibitor of TNFR1. In certain embodiments, an isolated TNFR1 binding polypeptide is a specific and/or selective inhibitor of TNFR1. For example, a polypeptide described herein is a selective inhibitor of TNFR1 over TNFR2. In certain embodiments, an isolated TNFR1 binding polypeptide as described herein does not bind TNFR2 (e.g., cell membrane bound TNFR2).

In certain embodiments, an isolated polypeptide described herein is capable of inhibiting a TNFR1 mediated pro-inflammatory response. In certain embodiments, an isolated TNFR1 binding polypeptide is capable of inhibiting the activity of TNFR1. For example, in the presence of a TNFR1 binding polypeptide described herein, a TNF-induced, TNFR1 mediated pro- inflammatory response is inhibited; TNF-induced NF-KB activation is inhibited; or TNF-induced IKB degradation is inhibited; as compared to a control or in the absence of an TNFR1 binding polypeptide as described herein.

In certain embodiments, an isolated polypeptide described herein is capable of inhibiting IKB degradation with an IC50 of at least about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, or lOnM, for example, using an IC50 determination method described in Example 1. In certain embodiments, an isolated polypeptide described herein is capable of inhibiting IKB degradation with an IC50 of about 1-13, 2-12, 3-11, 4-10, 5-9, 6-8nM, or 7nM.

In certain embodiments, an isolated polypeptide described herein is capable of inhibiting NF-KB activation with an IC50 of at least about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2nM, for example, using an IC50 determination method described in Example 1. In certain embodiments, an isolated polypeptide described herein is capable of inhibiting NF-KB activation with an IC50 of about 0.2-10, 0.2-5, 0.2-1, 0.2-0.5, or 0.2-0.3nM.

In certain embodiments, a polypeptide (e.g., affibody or binding fragment thereof) as described herein is operably linked to an albumin-binding domain (ABD) or albumin. For example, in certain embodiments, an TNFR1 binding polypeptide as described herein is fused to an albumin-binding domain (ABD), such as albumin-binding domain B2A3 (BA) or Bl A2B2A3 (BABA) from Streptococcal protein G (see, e.g., PNAS December 2, 2014, 111 (48) 17110- 17115; Makrides SC, et al. (1996) J Pharmacol Exp Ther 277(l):534-542; and Exp Mol Med. 2017 Mar; 49(3): e306, which are all incorporated by reference for all purposes). In certain embodiments, a TNFR1 binding polypeptide as described herein is fused to human serum albumin.

In certain embodiments, a polypeptide (e.g., affibody or binding fragment thereof) as described herein is operably linked to an immunoglobulin Fc fragment (e.g., IgGl, IgG2, IgG3, or IgG4 Fc fragment). For example, in certain embodiments, the TNFR1 binding polypeptide as described herein is fused to an immunoglobulin Fc fragment (e.g., to the N terminal and/or C terminal of a IgGl, IgG2, IgG3, or IgG4 Fc fragment or engineered Ig Fc fragment). In certain embodiments, the IgGl Fc fragment comprises an amino acid sequence that has at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of NCBI accession number 1T83 A. In certain embodiments, the IgG4 Fc fragment comprises an amino acid sequence that has at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of NCBI accession number 4D2N_A. In certain embodiments, a TNFR1 binding polypeptide as described herein is fused to a fluorescent protein (e.g., GFP or RFP). In certain embodiments, a TNFR1 binding polypeptide is labeled with a fluorescent moiety (e.g., FITC, or an AlexaFluor dye).

In certain embodiments, a TNFR1 binding polypeptide as described herein is fused to a tag (e.g., affinity tag and/or detectable tag such as HIS tag, FLAG tag, or C-Myc tag).

Certain embodiments of the invention also provide an isolated nucleic acid encoding an isolated polypeptide as described herein.

Certain embodiments of the invention provide an expression cassette comprising a nucleic acid as described herein and a promoter.

Certain embodiments provide a vector comprising a nucleic acid or an expression cassette described herein.

Certain embodiments provide a cell comprising a vector or a nucleic acid, or an expression cassette as described herein. In certain embodiments, the cell is a mammalian cell, or a bacterial cell e.g., E. coli).

Methods of Use

As described herein, in certain embodiments, a TNFR1 binding polypeptide as described herein is a TNFR1 inhibitor. Accordingly, certain embodiments of the invention provide methods of inhibiting the activity of TNFR1 in a cell, comprising contacting TNFR1 with an isolated polypeptide described herein. In certain embodiments, TNFR1 is contacted in vitro. In certain embodiments, TNFR1 is contacted in vivo.

In certain embodiments, the activity of TNFR1 is inhibited by at least about 25% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90% or more) as compared to a control. In certain embodiments, the activity of TNFR1 is inhibited by at least about 45% (e.g., 50%, 55%, 60%, 65%, 70%, 80%, 90% or more) as compared to a control (e.g., as compared to a polypeptide that does not specifically bind to TNFR1 or in the absence of the polypeptide).

Thus, in certain embodiments, TNFa-induced TNFR1 signaling is inhibited by at least about 25% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90% or more) in the presence of a TNFR1 binding polypeptide described herein, as compared to a control. For example, in certain embodiments, TNFa-induced (TNFR1 mediated) NF-KB activation is inhibited by at least about 25% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90% or more) in the presence of a TNFR1 binding polypeptide described herein, as compared to a control. In certain embodiments, TNFa-induced (TNFR1 mediated) iKBa degradation is inhibited by at least about 25% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90% or more) in the presence of a TNFR1 binding polypeptide described herein, as compared to a control.

In certain embodiments, TNFa-induced TNFR2 signaling, such as RelB activation, is not inhibited in the presence of a TNFR1 binding polypeptide described herein as compared to a control.

Certain embodiments of the invention provide an isolated polypeptide as described herein for use in diagnosis or medical therapy.

Certain embodiments of the invention provide a method of treating a TNFR1 related disease or disorder in a mammal (e.g., a mammal in need thereof), comprising administering an effective amount of an isolated polypeptide as described herein to the mammal.

Certain embodiments of the invention provide an isolated polypeptide as described herein for the prophylactic or therapeutic treatment of a TNFR1 related disease or disorder.

Certain embodiments of the invention provide the use of an isolated polypeptide as described herein to prepare a medicament for the treatment of a TNFR1 related disease or disorder in a mammal.

Certain embodiments of the invention provide a method of treating an inflammatory or autoimmune disorder in a mammal (e.g., a mammal in need thereof), comprising administering an effective amount of an isolated polypeptide as described herein to the mammal.

Certain embodiments of the invention provide an isolated polypeptide as described herein for the prophylactic or therapeutic treatment of an inflammatory or autoimmune disorder.

Certain embodiments of the invention provide the use of an isolated polypeptide as described herein to prepare a medicament for the treatment of an inflammatory or autoimmune disorder in a mammal.

In certain embodiments, the TNFR1 related disorder is an inflammatory disorder.

In certain embodiments, the TNFR1 related disorder is an autoimmune disorder.

In certain embodiments, the inflammatory or autoimmune disorder is selected from the group consisting of arthritis, psoriasis, Crohn's disease, ulcerative colitis, and asthma.

In certain embodiments, the inflammatory or autoimmune disorder is rheumatoid arthritis. In certain embodiments, the inflammatory or autoimmune disorder is psoriasis. In certain embodiments, the inflammatory or autoimmune disorder is inflammatory bowel disease. In certain embodiments, the inflammatory or autoimmune disorder is Crohn's disease. In certain embodiments, the inflammatory or autoimmune disorder is ulcerative colitis.

In certain embodiments, the TNFR1 related disorder is a cancer. In certain embodiments, the mammal is human. In certain embodiments, the mammal is a mouse.

Certain embodiments of the invention provide methods of detecting the presence and/or level of TNFR1 in a cell, comprising contacting the cell with an isolated polypeptide as described herein, and detecting whether a complex is formed between the isolated polypeptide and TNFR1. In certain embodiments, the cell is contacted in vitro. In certain embodiments, the cell is contacted in vivo. In certain embodiments, the detecting comprises detecting a fluorescent signal, radionuclide signal or immunohistochemical staining signal.

Administration

Certain embodiments of the invention provide a composition comprising the isolated polypeptide having affinity for TNFR1, and a carrier.

For in vivo use, a polypeptide of the invention is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, one or more polypeptides of the invention may be present as active ingredient(s) (i.e., are present at levels sufficient to provide a statistically significant effect on the symptoms of a relevant disease (e.g., inflammatory or autoimmune condition), as measured using a representative assay). A pharmaceutical composition comprises one or more such polypeptides in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition. Thus, certain embodiments provide a pharmaceutical composition comprising an isolated TNFR1 binding polypeptide as described herein and a pharmaceutically acceptable carrier.

In certain embodiments, the present polypeptides (i.e., isolated TNFR1 binding polypeptides described herein, such as an affibody of the present invention or a binding fragment thereof) may be systemically administered, e.g., intravenously, subcutaneously, intradermally, orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. In certain embodiments, the present polypeptides may be locally administered, e.g., intraarticularly. They may be freeze-dried into lyophilized formulation (e.g., lyophilized cake), may be formulated or reconstituted as a liquid dosage form, may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the polypeptide may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of a polypeptide of the present invention. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of polypeptide in such therapeutically useful compositions is such that an effective dosage level will be obtained.

Lyophilized formulations may also contain bulking agent (e.g., mannitol or glycine) and cryoprotectant/lyoprotectant (e.g., trehalose or sucrose). Lyophilized formulations can be reconstituted into a liquid dosage form using saline, 5% dextrose solution or water before administration. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the polypeptide, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the polypeptide may be incorporated into sustained-release preparations and devices.

The polypeptide may be administered intravenously, intraarticularly, intramuscularly, subcutaneously, intradermally or intraperitoneally by infusion or injection. Additionally, the polypeptide may be administered by local injection, such as by subcutaneous injection or intradermal injection. Solutions of the polypeptide may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the polypeptide that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes or nanoparticles. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be useful to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the polypeptide in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the polypeptide plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present polypeptide may be applied in pure form, z.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present polypeptides can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions that can be used to deliver the polypeptides of the present invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the polypeptides of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

The amount of a polypeptide of the present invention required for use in treatment will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

Polypeptides of the invention can also be administered in combination with other therapeutic agents and/or treatments, such as other agents or treatments that are useful for the treatment of TNFR1 related diseases or disorders. Non-limiting examples of such agents include an anti-TNF antibody or TNF binding protein or anti-inflammatory agent (e.g., Nonsteroidal anti-inflammatory drug). Additionally, one or more polypeptides of the invention may be administered (e.g., a combination of polypeptides may be administered). Accordingly, one embodiment the invention also provides a composition comprising a polypeptide of the invention, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The invention provides a kit comprising a polypeptide of the invention, packaging material, and instructions for administering a polypeptide of the invention to a mammal to treat a TNFR1 related disorder, such as an inflammatory or autoimmune disorder. The invention also provides a kit comprising a polypeptide of the invention, at least one other therapeutic agent, packaging material, and instructions for administering a polypeptide of the invention and the other therapeutic agent or agents to a mammal to treat a TNFR1 related disorder such as an inflammatory or autoimmune disease.

As used herein, the term “therapeutic agent” refers to any agent or material that has a beneficial effect on the mammalian recipient.

Certain Definitions The term “TNFR1 binding polypeptide” as used herein refers to an isolated polypeptide that binds TNFR1 (e.g., specifically binds to TNFR1 through, e.g., its helical domain(s)). “Helix domain” refers to a secondary structure of a polypeptide with a 3D geometry feature having helical turns. A TNFR1 binding polypeptide described herein (e.g., affibody, or binding fragment thereof) may comprise one, two, three, or more helix domains. For example, the polypeptide described herein has at least a Kd of lO M or stronger binding as determined in the binding affinity measurement assay(s) (e.g., cell-based flow cytometry, or surface plasmon resonance (SPR)) in the Example 1 or in Vunnam, et al., Biochemistry 2020 59 (40), 3856-3868, DOI: 10.1021/acs.biochem.0c00529, which is incorporated by reference herein for all purposes. In certain embodiments, the polypeptide described herein has at least a Kd of about IpM or stronger binding affinity. In certain embodiments, the polypeptide described herein has at least a Kd of about 900, 800, 700, 600, 500, 400, 300, 200, 100 nM, or stronger binding affinity. In certain embodiments, the polypeptide described herein has at least a Kd of about 500 nM, or stronger binding affinity. In certain embodiments, the polypeptide described herein has at least a Kd of about 90, 80, 70, 60, 50, 40, 30, 20, 15 nM, or stronger binding affinity. In certain embodiments, the polypeptide described herein has at least a Kd of about 80 nM, or stronger binding affinity. In certain embodiments, the polypeptide has a Kd of about 1-900 nM, 2-500 nM, 3-450 nM, 4-400 nM, 5-380 nM, or 6-350 nM for human TNFR1. For example, in certain embodiments, the polypeptide described herein has a Kd of about 12-20 nM, such as 13-19, 14- 18, or 15-17nM. In certain embodiments, the polypeptide described herein has a Kd of about 16nM. In certain embodiments, the polypeptide described herein has a Kd of about 7-37 nM, such as 12-32, 14-30, or 16-28nM. In certain embodiments, the polypeptide described herein has a Kd of about 20 or 22nM.

In certain embodiments, the polypeptide has a higher binding affinity for the TNFR1 extracellular domain (e.g., isolated soluble ECD fragment of TNFR1, or ECD of cell membrane bound TNFR1) than for the isolated preligand assembly domain (PLAD) fragment of TNFR1. In certain embodiments, the polypeptide may bind TNFR1 on epitope(s) (e.g., conformational epitope) comprising 1) amino acid residue(s) from PLAD, and 2) other non-PLAD amino acid residue(s) that is outside of PLAD. In certain embodiments, the polypeptide has a higher binding affinity for a TNFR1 ECD than for a TNFR1 PLAD fragment (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher, or at least 1, 2, 3, 4, 5-fold higher, or more).

In certain embodiments, the polypeptide also has binding affinity for mouse TNFR1. In certain embodiments, the polypeptide may also inhibit mouse-TNF-induced IkBa degradation mediated by mouse TNFR1. In certain embodiments, an isolated polypeptide described herein is capable of inhibiting mouse IKB degradation with an IC50 of at least about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, or lOnM, for example, using an IC50 determination method described in Example 1. In certain embodiments, an isolated polypeptide described herein is capable of inhibiting mouse IKB degradation with an IC50 of about 60-120, 70-110, 80-100, or 92nM.

The terms “TNFR1 binding fragment” or “TNFR1 binding portion” refer to one or more fragments of a TNFR1 binding polypeptide described herein that retains the ability to bind to TNFR1 (e.g., specifically binds, e.g., through its helical domain(s)). A non-limiting example of such a fragment, is an affibody binding fragment comprising helical domains 1 and 2.

The term “inhibitor of TNFR1” as used herein refers to an isolated TNFR1 binding polypeptide (e.g., an affibody or fragment thereof) that is capable of inhibiting the activity or function of TNFR1 (e.g., inhibits signal transduction activity). For example, in certain embodiments, the isolated TNFR1 binding polypeptide, detectably inhibits the biological activity of TNFR1 as measured, e.g., using an assay described herein. In certain embodiments, the isolated TNFR1 binding polypeptide, inhibits the biological activity of TNFR1 by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In certain embodiments, an isolated TNFR1 binding polypeptide has a higher binding affinity (e.g., 110%, 120%, 130% higher or more) for human TNFR1 (e.g., cell membrane bound TNFR1) than for human TNFR2 (e.g., cell membrane bound TNFR2). In certain embodiments, an isolated TNFR1 binding polypeptide has a higher binding affinity (e.g., 110%, 120%, 130% higher or more) for human TNFR1 (e.g., isolated soluble ECD of TNFR1) than for human TNFR2 (e.g., isolated soluble ECD of TNFR2). In certain embodiments, an isolated TNFR1 binding polypeptide is a selective inhibitor of TNFR1. For example, an affibody of the invention may be at least 5, at least 10, at least 50, at least 100, at least 500, or at least 1,000 fold selective for TNFR1 over another TNFR (e.g., TNFR2) in a selected assay (e.g., an assay described in the Example 1 herein).

In certain embodiments, one or more amino acid residues are mutated within the polypeptide or domain as described herein. For example, the mutation is conducted via error- prone PCR or site directed mutagenesis. In certain embodiments, an amino acid residue is mutated into one that allows the properties of the amino acid side-chain to be conserved. Examples of the properties of amino acid side chains comprise: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising the following side chains: aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containing side-chains (S, T, Y); sulfur atom-containing side-chains (C, M); carboxylic acid- and amide-containing side-chains (D, N, E, Q); base-containing side-chains (R, K, H); and aromatic-containing side-chains (H, F, Y, W). The letters within parenthesis indicate the one- letter amino acid codes. Amino acid substitutions within each group are called conservative substitutions. It is well known that a polypeptide comprising a modified amino acid sequence in which one or more amino acid residues is deleted, added, and/or substituted can retain the original biological activity (Mark D. F. et al., Proc. Natl. Acad. Sci. U.S.A. 81 :5662-5666 (1984); Zoller M. J. and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. et al., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. U.S.A. 79: 6409- 6413 (1982)). The number of mutated amino acids is not limited, but in general, the number falls within 40% of amino acids of each helix, and specifically within 35%, and still more specifically within 30% (e.g., within 25%). The identity of amino acid sequences can be determined as described herein. In certain embodiments, one or more amino acid residue is mutated into one that is a non-conservative substitution.

In certain embodiments, the development of the polypeptides having affinity for TNFR1 involve a display technology (e.g., yeast surface display, phage display, bacterial display, mRNA display or ribosomal display).

The polypeptides obtained can be purified to homogeneity. The polypeptides can be isolated and purified by a method routinely used to isolate and purify proteins. The polypeptides can be isolated and purified by the combined use of one or more methods appropriately selected from column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectro-focusing, for example (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Such methods are not limited to those listed above. Chromatographic methods include affinity chromatography (e.g., metal affinity chromatography), ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography. These chromatographic methods can be practiced using liquid phase chromatography, such as HPLC and FPLC. The polypeptides can also be purified by utilizing target binding, using carriers on which targets have been immobilized.

The polypeptides of the present invention can be formulated according to standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A), and may comprise pharmaceutically acceptable carriers and/or additives. The present invention relates to compositions (including reagents and pharmaceuticals) comprising the polypeptides of the invention, and pharmaceutically acceptable carriers and/or additives. Other exemplary carriers include surfactants (for example, PEG and Tween), excipients, antioxidants (for example, ascorbic acid), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (for example, phosphoric acid, citric acid, and other organic acids), chelating agents (for example, EDTA), suspending agents, isotonizing agents, binders, disintegrators, lubricants, fluidity promoters, and corrigents. However, the carriers that may be employed in the present invention are not limited to this list. In fact, other commonly used carriers can be appropriately employed: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose, polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salt, and so on. The composition may also comprise other low-molecular- weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine, and lysine. When the composition is prepared as an aqueous solution for injection, it can comprise an isotonic solution comprising, for example, physiological saline, dextrose, and other adjuvants, including, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which can also contain an appropriate solubilizing agent, for example, alcohol (for example, ethanol), polyalcohol (for example, propylene glycol and PEG), and non-ionic detergent (polysorbate 80 and HCO-50).

The terms “polypeptide” and “protein” are used interchangeably herein. A protein molecule may exist in an isolated or purified form or may exist in a non-library environment such as, for example, an isolated or purified form as an active ingredient of a drug dosage form or a diagnostic reagent. Fragments and variants of the disclosed proteins or partial-length proteins encoded thereby are also encompassed by the present invention. By "fragment" or "portion" is meant a full length or less than full length of the amino acid sequence of a protein.

The invention encompasses isolated or substantially purified protein compositions. In the context of the present invention, an "isolated" or "purified" polypeptide is a polypeptide that exists apart from its native or library environment. A polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell such as a bacterium or a mammalian expression system for the production of the polypeptide. For example, an "isolated" or "purified" protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of- interest chemicals. Fragments and variants of the disclosed proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

As used herein, "comparison window" makes reference to a contiguous and specified segment of an amino acid or polynucleotide sequence, wherein the sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least about 20 contiguous amino acid residues or nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.

As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, and at least about 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least about 70%, at least about 80%, 90%, or at least about 95%.

The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity or complementarity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

The term "amino acid" includes the residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) in D or L form, as well as unnatural amino acids (e.g., dehydroalanine, homoserine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2,3,4, -tetrahydroi soquinoline- 3 -carboxylic acid, penicillamine, ornithine, citruline, a-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as a (Ci-Ce)alkyl, phenyl or benzyl ester or amide; or as an a-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T.W. Greene, Protecting Groups In Organic Synthesis,' Wiley: New York, 1981, and references cited therein) The term also comprises natural and unnatural amino acids bearing a cyclopropyl side chain or an ethyl side chain.

The term "nucleic acid" and “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA.

"Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

A “vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

"Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

The "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e. further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.

As used herein, the term "operably linked" refers to a linkage of two elements in a functional relationship. For example, “operably linked” may refer to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. “Operably-linked” also refers to the association of two chemical moieties so that the function of one is affected by the other, e.g., an arrangement of elements wherein the components so described are configured so as to perform their usual function.

"Expression" refers to the transcription and/or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

The term “effective amount” or “therapeutically effective amount,” in reference to treating a disease state/condition, refers to an amount of a polypeptide either alone or as contained in a pharmaceutical composition that produces therapeutic effect or is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state/condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial. In the case of inflammatory or autoimmune diseases, the effective amount of the polypeptide may reduce inflammatory response and/or autoimmunity; reduce pro- inflammatory gene(s) expression; and/or relieve to some extent one or more of the symptoms associated with the inflammatory or autoimmune diseases.

The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder, such as inflammatory or autoimmune disease. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

The invention will now be illustrated by the following non-limiting Example.

EXAMPLE 1. Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency that Does not Affect TNFR2 Function

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn’s, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. In this Example, using yeast display, a synthetic affibody ligand (ABYTNFRI-I) with high binding affinity and specificity for TNFR1 was identified. Functional assays showed that the affibody potently inhibits TNF-induced NF-KB activation (IC50 of 0.23 nM) and does not block TNFR2 function. Additionally, ABYTNFRI-I acts non-competitively — it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand assembled dimers — thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases. INTRODUCTION

Tumor necrosis factor (TNF) is a primary pro-inflammatory cytokine that plays a significant role in the initiation and maintenance of inflammation.¹ Upregulation of TNF is observed in autoimmune and neurodegenerative diseases, and dysregulation of TNF signaling through TNFR1 plays a role in the pathology of many inflammatory diseases.²'⁴ Consequently, therapeutics that specifically neutralize the biological function of TNF were developed.⁵ Currently, there are five FDA approved anti-TNF therapeutics (infliximab, adalimumab, certolizumab pegol, golimumab, and etanercept) and are used to treat autoimmune diseases, including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease.⁶'⁷ Yet TNF also plays a multifaceted, beneficial role in healthy immune response. For example, TNF-TNFR2 signaling leads to the activation and proliferation of CD4+FOXP3+ regulatory T (Treg) cell, which plays essential role in immune homeostasis and in the prevention of autoimmune responses.⁸'¹⁰ Thus, despite their clinical success, these anti-TNF therapeutics come with serious side-effects, including common and opportunistic infections, tuberculosis, demyelination diseases, lymphoma, and induction of new autoimmune diseases.¹¹'¹⁴ Moreover, clinical evaluation of an anti-TNF therapy in juvenile RA resulted in development of multiple sclerosis and demyelinating lesions in some patients.¹⁵ These side effects are due to global blockade of TNF biological activity, including inhibition of the immunosuppressive function of TNFR2.^16-17 To overcome these limitations, new strategies that specifically target TNFR1 warrants exploration.¹⁸

TNFR1 is a transmembrane receptor that regulates the inflammatory pathways. Binding of TNF to the extracellular domain of TNFR1 leads to IKBO. degradation and NF-KB activation, which is responsible for the transcription of pro-inflammatory genes. The dominating strategy of therapeutics for inflammatory disease is the sequestration of TNF. However, this prevents TNF from interacting with its other binding partners, specifically TNFR2 which is also responsible for cell proliferation and regeneration, contributing to various detrimental off-target side effects mentioned previously.

There have been considerable efforts to develop antagonistic anti-TNFRl antibodies and antibody derivatives.^19-22 All of these approaches act competitively, either by blocking TNF binding to TNFR1, ^23-24 or by inhibiting receptor-receptor interactions in pre-ligand assembled dimers.^25-30 While competitive inhibition is a reasonable approach, we have hypothesized herein that an even more effective strategy will be to develop allosteric or non-competitive TNFR1 antagonists that don’t require competition with TNF binding. TNF has a picomolar affinity for TNFR1, so orthosteric, competitive inhibitors of TNFR1 require either a molecule that has a higher affinity than TNF, which is difficult to attain, or much higher doses, which may increase the risk of side effects. Additionally, because competitive inhibitors bind to the TNF binding site on TNFR1, a region with high sequence homology to the TNF binding site on TNFR2, there is a higher likelihood of binding to TNFR2 and inhibiting its function. Non-competitive antagonists, on the other hand, have a higher potential for blocking TNFR1 activity without interfering with TNFR2 function and TNF availability.

While therapeutic antibodies have revolutionized the landscape of autoimmune disease treatments,³¹'³⁴ there are important drawbacks: 1) they are large (approximately 150 kDa, reducing tissue penetration and limiting biodistribution and efficacy); 2) they are expensive; 3) they can be challenging to produce; and 4) their multivalency can drive receptor clustering, which is not desirable in all scenarios.²⁰’²²’³⁵'³⁶ To overcome the limitations of monoclonal antibodies, the protein engineering field has developed small non-antibody protein scaffolds as a promising alternative. While there is only one non-antibody protein scaffold approved by the FDA,³⁷ these scaffolds benefit from small size, high-affinity binding, high thermal stabilities, and efficient pharmacokinetic modulation.³⁷'⁴³ Affibody molecules are such small protein scaffolds derived from the three-helix bundle Z domain of the Ig-binding region of protein A. The small size, monomeric structure (to avoid target crosslinking-induced activation), high-affinity binding ability to protein targets, and relatively easy production procedures make them attractive targeting agents for therapeutics and diagnostics. Recently, non-antibody protein scaffolds with picomolar potencies have been discovered from affibody⁴⁴ libraries to various targets, including TNF,⁴⁵ amyloid-P peptide,⁴⁶ B7-H3,⁴⁷ and human epidermal growth factor receptor 2.⁴⁸ Several preclinical studies have been reported on the diagnostic and therapeutic use of these protein scaffolds, and some have reached early clinical studies.^{40, 49-52}

In this Example, we used an affibody library displayed on the surface of yeast and directed evolution to discover a high affinity antagonist of TNFR1. Utilizing magnetic and fluorescence- activated cell sorting selection methods, an affibody variant (named ABYTNFRI-I) that binds to human TNFR1 with high affinity was identified. Cell-based functional and biochemical assays showed that ABYTNFRI-I is apotent, non-competitive inhibitor of TNF -induced IKBOL degradation and NF-KB activation that does not block TNF binding or alter TNFR2 function. These results suggest that the affibody specifically inhibits TNFR1 signaling. Thus, ABYTNFRI-I is a promising compound that acts through a novel mechanism of action for TNFR1 -specific inflammatory diseases.

MATERIALS AND METHODS Identification and Evolution of TNFRl-Specific ABY Binders Using Yeast Surface Display

A previously reported naive affibody library with 2xl0⁹ variants was generated by diversifying 17 solvent-exposed amino acids on helix 1 and 2 of the Z domain of the Ig -binding region of protein A within a yeast display system.⁵ The affibody yeast library was grown in SD- CAA selection medium (16.8 g/L sodium citrate dihydrate, 3.9 g/L citric acid, 20.0 g/L dextrose, 6.7 g/L yeast nitrogen base, and 5.0 g/L casamino acids) at 30 °C while being shaken. After ~20 h of incubation, the growth medium was replaced with SG-CAA induction medium (10.2 g/L sodium phosphate dibasic heptahydrate, 8.6 g/L sodium phosphate monobasic monohydrate, 19.0 g/L galactose, 1.0 g/L dextrose, 6.7 g/L yeast nitrogen base, and 5.0 g/L casamino acids) and incubated overnight to induce the display of the affibody on the yeast surface. Magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) were used to select affibody molecules that specifically bind to TNFR1. Lysate from HEK293T cells stably expressing TNFR1 with the cytoplasmic domain replaced with GFP (TNFR1 ACD-GFP) was used to perform the sorts to target native receptor with appropriate post-translational modifications. For magnetic sorts, target and control proteins were coated on GFP -trap magnetic beads (ChromoTek, gtma-20). HEK293T cells expressing either TNFR1ACD-GFP or lymphocyte activation gene 3 (LAG3 ACD-GFP) were washed with phosphate-buffered saline (PBS) and lysed (2 mM ethylene diamine tetra acetic acid, l%Triton X-100, and IX protease inhibitor in PBS) on ice for 30 min. The cell lysates were centrifuged at 13000 rpm for 15 min at 4 °C to separate insoluble debris. GFP-trap beads were incubated with the soluble supernatants for 1-2 hours at 4 °C and washed three times with PBS. The yeast library was depleted of non-specific binders via exposure to bare beads and then beads with LAG3-GFP. TNFR1 binders were then enriched by incubating the remaining yeast with TNFRIACD-GFP-coated beads and isolating the yeast that bound. The collected population was grown and induced for two more rounds of MACS with higher stringency by increasing the number of washes from one to three. The population was depleted via bare bead and LAG3-GFP binding, then enriched for TNFR1 ACD-GFP binding with TNFR1ACD-GFP lysate to reduce avidity. FACS was used to further enrich for TNFR1 binding. The resulting yeast population was grown, induced, labeled with anti-c-Myc antibody (9E10, BioLegend) and anti-mouse antibody conjugated to Alexa Fluor 647 to detect full-length ABY expression. Yeast cells were then incubated with cell lysate from TNFRIACD-GFP-expressing HEK293T cells as described earlier. The yeast cells that were GFP⁺/Alexa Fluor 647⁺ were collected with BD FACS Aria II. The sorted yeast population were grown, and plasmid DNA was extracted via Zymoprep Yeast Plasmid Miniprep Kit II (Zymo Research Corp.).

Generation of Randomly Mutagenized ABY Library Sorted TNFR1 affibody binders were further engineered to enhance binding affinity using random mutagenesis to the full gene and the helices of ABY in parallel by error-prone polymerase chain reaction (PCR) using nucleotide analogues 2'-deoxy-P-nucleo-side-5 '-triphosphate and 8- oxo-2'-deoxyguanosine-5'- triphosphate as outlined previously.⁵³ Linearized pCT vector with mutagenized gene fragments were transformed into yeast. The resulting mutant ABY population underwent two rounds of MACS and a FACS against mammalian cell lysates expressing TNFR1 ACD-GFP or LAG3-GFP for comparative control. Plasmid DNA was isolated from these yeast cells using zymo prep. Clonal plasmids were obtained by transforming extracted DNA into Escherichia coli (One Shot TOP 10, Invitrogen). Individual colonies were picked and grown in lysogeny broth (LB) medium. Plasmids were extracted using the QIAGEN Miniprep Kit and sequenced by AC GT, Inc.

Cell Cultures and Molecular Biology

HEK293 cells were cultured in phenol red-free Dulbecco’s modified Eagle medium supplemented with 2 mM L-glutamine, heat-inactivated 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 pg/ml). HUVEC cells were cultured on 0.2% gelatin-coated dishes in EGM-2 medium supplemented with heat-inactivated 2% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 pg/ml). Mouse L929 cells were cultured in ATCC-formulated Eagle's minimum essential medium, 10% horse serum, penicillin (100 U/ml), and streptomycin (100 pg/ml). Cell cultures were incubated at 37 °C in a humid atmosphere of 5 % CO2. EGFP and TagRFP vectors were a kind gift from D. D. Thomas (University of Minnesota). cDNA encoding TNFR1ACD (1-240) was inserted at the N-terminus of the EGFP and Tag RFP vectors using standard cloning techniques. To prevent the dimerization and aggregation of EGFP, we mutated alanine at site 206 to lysine (A206K).⁵⁴ All mutations were introduced by QuikChange mutagenesis and sequenced for confirmation.

Production of TNFR1 Affibody Binders

For overexpression and purification of evolved TNFR1 -binding ABY variants, DNA sequences of mutants were transferred from the pCT vector into a pET expression vector⁵⁵ with a C-terminal Hise tag using Nhel and BamHI restriction enzymes. Recombinant soluble TNFR1 affibody binders were overexpressed in E. coli and purified by immobilized nickel affinity chromatography. The purity of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (SDS-PAGE) under reducing conditions followed by Coomassie staining. Determination of TNFR1-ABY Variants Binding using Flow Cytometry

Binding affinity of ABY variants to TNFR1 ACD-GFP on HEK293T cells was determined by flow cytometry. HEK293 cells with stable expression of TNFR1 ACD-GFP or transient expression of LAG3-GFP were lifted using trypsin and washed three times with PBS. Cells were incubated with soluble ABY variants for 2 hours. After incubation, cells were washed with PBS with 0.1% bovine serum album (PBSA) to remove unbound affibody and labeled with Alexa Fluor 647-conjugated anti -Hiss antibody for 1-2 hours at 4°C. Fluorescence was analyzed on a BD Accuri C6 flow cytometer.

Binding Affinity Measurements of ABYTNFRI-I

Binding affinity of ABYTNFRI-I to TNFR1ACD-GFP or TNFR2ACD-GFP on HEK293T cells was determined by flow cytometry. Untransfected HEK293T cells alone or cells with transient expression of TNFR1ACD-GFP or TNFR2ACD-GFP were lifted using trypsin and washed three times with PBS. Cells were incubated with soluble ABYTNFRI-I (0.000001-10 pM) for 2 hours at 4 °C. TNFR1 ACD-GFP expressing cells were also incubated with an affibody that binds to Cluster of Differentiation 276 protein (negative control, hereinafter referred to as a nonbinder) (0.000001-10 pM). After incubation, cells were washed with PBS with 0.1% bovine serum album (PBSA) to remove unbound affibody and labeled with anti -his antibody, followed by the AF647-conjugated secondary antibody. Fluorescence was analyzed on a BD Accuri C6 flow cytometer.

Determination of Direct Binding of ABYTNFRI-I Using Surface Plasmon Resonance

SPR experiments were carried out using a Biacore S200 with PBS-P+ (20 mM phosphate buffer, pH 7.4 with 2.7 mM KC1, 137 mM NaCl, and 0.05% Surfactant P20) as running buffer and equipped with a CM5 series S sensor chip. Commercially available recombinant Fc-tagged TNFR1-ECD (Sino Biological Inc.) and Fc-tagged TNFR2-ECD (Sino Biological Inc.) were diluted in 100 mM sodium acetate buffer, pH 5.0 and immobilized to 1200 RU using amine- coupling chemistry while preligand assembly domain (PLAD) of TNFR1 was expressed and purified as previously reported (C.H. Lo, et al., SLAS Discov. 2017 Sep;22(8):950-961), diluted in 100 mM sodium acetate buffer, pH 4.5 and immobilized using amine coupling to 600 RU. Reference channels underwent the same activation and blocking procedure for the amine coupling without the addition of a ligand. ABYTNFRI-I, non-binder, and TNFa were serially diluted twofold in running buffer. The samples were injected at a rate of 30 uL/min and 25 °C. The complex was allowed to associate and dissociate for 180 s each. The sensor surface was regenerated between analyte sample injections with a 30 s injection of 10 mM glycine, pH 2.1 at 20 uL/min. Data were collected at 10 Hz and then analyzed using the Biacore S200 Evaluation software (vl.1.1). Sensorgrams were double referenced to the reference channel and zero analyte samples. Affinity binding curves were fit to a steady-state binding model. Data was plotted using GraphPad Prism (v9.0.1). NF-KB-Luciferase Reporter Gene Assay

The NF-KB-luciferase reporter assay was performed as previously described.⁵⁶'⁵⁷ Briefly, HEK293 cells were transfected with the NF-KB-luciferase reporter genes in a 10 cm plate with Lipofectamine 3000. The next day, cells were lifted with TrypLE and resuspended in phenol red- free DMEM. Transfected cells (7500 cells/well) were dispensed in 96-well white, solid-bottom plates and incubated with increasing concentrations of ABYTNFRI-I (1 pM -10 pM) or PBS (negative control) in the presence (0.6 nM) and absence of TNF-a (ab9642, Abeam) for 24 hours at 37 °C. After incubation, 70 pL of Dual -Gio Luciferase Reagent (Promega, Madison, WI) was added and incubated at room temperature for 15 min, and firefly luminescence was measured using a Cytation 3 Cell Imaging Multi-Mode Reader luminometer. Next, 70 pL of Dual-Glo Stop & Gio Reagent was added and incubated at room temperature for 15 min, and luminescence was measured using a luminometer.

IKBU Degradation Assay

HEK293 cells were cultured into six-well plates at 0.4 million/ml and incubated overnight. Next day, cells were treated with increasing concentration of ABYTNFRI-I (1 pM - 10 pM) for 2 hours, followed by 30 min of TNF-a (0.6 nM). Cells were lifted with trypsin and lysed with native lysis buffer containing 1% protease inhibitor for 30 min on ice and centrifuged at 13,000 rpm at 4 °C for 15 minutes. The total protein concentration of lysates was determined by bicinchoninic acid assay (BCA), and equal amounts of total protein (80 pg) were mixed with 4x Bio-Rad sample buffer and boiled for 5 minutes. Protein samples were resolved by SDS-PAGE and immunoblotted with anti-FcBa and P-actin.

TNF-TNFR1 pulldown Assay

The effect of ABYTNFRI-I on the TNF-TNFR1 interaction was determined by a pull-down assay with anti-Flag magnetic beads. HEK293 cells with endogenous expression of TNFR1 were lysed with native lysis buffer and protein concentration was determined using BCA. Ten pL of anti -FLAG magnetic beads were incubated with 30 pL of 25 pg/mL FLAG-tagged TNF (ALX- 522-008-C050, Enzo Life Sciences) for two hours at 4 °C. Unbound TNF was then removed using magnet, and the anti-FLAG beads were washed three times with 0.1% PBSA. TNF coated beads were incubated with 250 pl of HEK293 lysate (7.5 mg/ml) with and without ABYTNFRI-I, incubated overnight, and washed three times with PBSA. 10 pL of lx loading dye (1610747, BioRad) was added to the 10 pL of beads and pipetted up and down 5 times to elute the proteins. Using a magnet, the 10 pL of dye was then removed and placed in a separate tube. This elution step was repeated three more times for each sample for a total of 40 pL of loading dye per sample. Pulled-down samples were resolved by SDS-PAGE and immunoblotted with anti-Flag and anti- TNFR1 antibodies.

Effect of ABYTNFRI-I on conformation of TNFR1

For lifetime measurements, HEK293T cells were transfected using Lipofectamine 3000 with TNFR1ACD-GFP and TNFR1ACD-GFP: TNFR1ACD-RFP (1 :6 ratio) for a total of 20 pg in 10 cm plates for 24 hours and transfection was confirmed using EVOS fluorescence microscopy. The cells were harvested using TrypLE (Invitrogen), washed three times with PBS and resuspended in PBS at a concentration of 1 million cells/mL. Cells were dispensed (50 pL/well) into a 384-well plate and treated with soluble ABYTNFRI-I (0.00001-10 pM) and incubated for 1-2 hours. After being incubated, the donor lifetime was measured using a fluorescence lifetime plate reader (Fluorescence Innovations, Inc., Minneapolis, MN). Time- resolved fluorescence waveforms for each well were fitted to single-exponential decays using least-squares minimization global analysis software (Fluorescence Innovations, Inc.) to give donor lifetime (TD) and donor-acceptor lifetime (TDA). FRET efficiency (E) was then calculated based on Eq. (1)

E = 1 - \ T_D / (1)

Effect of ABYTNFRI-I on TNFR2 Function

The effect of ABYTNFRI-I on TNFR2 function was determined by membrane-TNF induced v-rel avian reticuloendotheliosis viral oncogene homolog B (RelB) activity⁵⁸'⁶⁰ using western blotting. HUVEC cells were plated in a 6 well plate at 1 million cells per well and incubated overnight. Next day, cells were transfected with 0.5 pg of pCMV6-TNF vector to express membrane TNF using Lipofectamine 3000. After six hours, transfecting media was replaced with fresh media and cells were then treated with the ABYTNFRI-I and incubated overnight. Next day, cells were lifted and lysed with a native lysis buffer, and protein concentrations were determined using BCA. Proteins samples were resolved by SDS-PAGE and immunoblotted with anti-RelB antibody.

Statistical Analysis

Data are presented as mean ± SD unless stated otherwise. To determine statistical significance for all experiments, data analysis was performed using a two-tailed unpaired t test with P values determined using GraphPad software. Values of P < 0.05 were considered statistically significant. GraphPad style in using asterisks to denote P values in figures was used (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). RESULTS

Discovery of TNFRl-binding ABY using yeast surface display

Affibody molecules with specific binding to the extracellular domain of TNFR1 were isolated via directed evolution from an affibody library comprising 2xl0⁹ unique affibody variants in a yeast surface display format. Magnetic activated cells sorting (MACS) and fluorescence- activated cell sorting (FACS) selection methods (Figure 1) enriched TNFR1 -specific binders using a target protein isolated from the lysate of mammalian cells expressing the TNFR1 with a deleted cytoplasmic domain replaced by green fluorescent protein (TNFR1 ACD-GFP).

The GFP enables immobilization of TNFR1 ACD-GFP on magnetic beads via anti-GFP antibody as well as fluorescent detection via FACS. A similar construct with lymphocyte-activation gene 3 (LAG3)-GFP, a transmembrane immune checkpoint receptor that has a very low sequence homology with TNFR1, served as a negative control (Figure 1).

The affibody library was displayed on the yeast surface and incubated with bare magnetic agarose beads to eliminate any non-specific binders. The population which did not bind to bare beads was then incubated with beads saturated with LAG3-GFP to select against non-desired binding to GFP or general features of transmembrane receptors. The remaining affibody- displaying yeast were then sorted for the desired binding to TNFR1 ACD-GFP. Bound yeast were amplified and the selection scheme was iterated twice more. After the three rounds of MACS selection, FACS was performed with selection for binding to TNFR1 ACD-GFP from cell lysates. DNA from isolated TNFRl-binding affibodies were mutated using error-prone PCR targeting the helical paratope of the genes of enriched ABY variants. The mutagenized affibody population was further enriched with two MACS with TNFR1ACD-GFP coated beads and one FACS with TNFR1 ACD-GFP expressing cell lysates. After these seven sorts for TNFR1 binders and one round of mutagenesis, the enriched affibody population showed significant binding to TNFR1ACD-GFP (Figure 2A) whereas binding was not observed to LAG3-GFP cell lysate (Figure 2B). These results suggests that the population evolved specifically with TNFR1-ABY interactions rather than non-specific interactions in the selection process. Next, six clones in this evolved population were randomly selected and sequenced. Five out of six clones showed high sequence diversity in the functional population with amino acid variations at the initially diversified helical sites (Figure 3 A).

Binding affinity of ABYTNFRI-I to TNFR1 ACD-GFP expressing HEK293 cells

The binding of soluble ABY variants to the extracellular domain of TNFR1 was tested using flow cytometry. The five affibody binders were produced in E. coll using a pET expression system with a C-terminal Hise-tag and purified by metal affinity chromatography (Figure 3B). To determine the binding of soluble ABY variants (including ABYTNFRI) to TNFR1, HEK293 cells with stable expression of TNFR1 ACD-GFP were incubated, separately, with each of the five soluble ABY variants. One clone (ABYTNFRI-I) showed significant binding to TNFR1ACD-GFP expressing cells, and four clones showed weak binding (ABYTNFRI-2 IO 5) (Figure 3C).

Subsequently, the affinity of the TNFR1 binder (ABYTNFRI-I) for TNFR1 was determined using flow cytometry. To evaluate the specificity of ABYTNFRI-I, we examined its affinity for TNFR2, a homologous member of the TNFR superfamily (52% similar and 30% identical to TNFR1, determined using the European Bioinformatics Institute’s LALIGN software). The binding affinity of ABYTNFRI-I for TNFR1 or TNFR2 was measured by titrating it with HEK293T or cells transiently overexpressing TNFR1 ACD-GFP or TNFR2ACD-GFP. First, we checked the surface expression levels of the TNFR1 ACD-GFP and TNFR2ACD-GFP using fluorescence microscopy and measured the median fluorescence intensities of these receptors using flow cytometry. Cells transfected with TNFR1 ACD-GFP (Figure 4A) or TNFR2ACD-GFP (Figure 4B) showed similar transfection efficiencies.

Median fluorescence intensities of TNFR1 ACD-GFP (Figures 10A-10B) and TNFR2ACD-GFP (Figure 10C) were comparable throughout the ABYTNFRI-I or a non-binder titration (Figure 4C). Titration showed an affinity [KD (dissociation constant)] of about 22 ± 15 nM for TNFR1 ACD-GFP (Figure 4D) (also see Figure 13 that showed KD of about 16 ± 4 nM in an earlier study). HEK293T cells have endogenous expression of TNFR1, resulting in moderate binding. No additional binding was observed when cells expressed TNFR2ACD-GFP (Figure 4D) (also see Figure 14 that showed that ABYTNFRI-I does not cross-react with cell membrane-bound TNFR2 ACD-GFP in an earlier study). These results indicate that ABYTNFRI-I binds to TNFR1 but not to TNFR2, and a non-binder didn’t show any binding to TNFR1 (Figure 4D).

Direct binding of ABYTNFRI-I to TNFR1

To determine whether affibody acted directly on TNFR1, we performed measurements with surface plasmon resonance (SPR). To do this, recombinant Fc-tagged TNFR1 ECD, Fc- tagged TNFR2 ECD or purified pre-ligand assembly domain of TNFR1 (PLAD) were immobilized onto the SPR chip, which was followed by flowing ABYTNFRI-I, a non-binder, or TNF through the chip to allow for binding. While ABYTNFRI-I showed dose-dependent binding to TNFR1 ECD (Figures 11 A-l IB), TNFR2 ECD (Figures 11C-1 ID), and PLAD (Figures 5A-5B), with binding affinities (KD) of 73 ± 1 nM, 129 ± 28 nM, and 377 ± 66 nM, respectively, a non- binder showed no dose-dependent binding to TNFR1-ECD (Figures 11E-11F), TNFR2-ECD (Figures 11G-11H), and PLAD (Figures 5C-5D). It is interesting that the ABYTNFRI-I has a 5.2- fold higher binding affinity to the TNFR1-ECD than the PLAD domain. These findings indicate that ABYTNFRI-I may need both cysteine-rich domain one (PLAD) and two for binding. TNF showed dose-dependent binding to TNFR1-ECD (Figures 111-11 J), TNFR2-ECD (Figures 11K- 1 IL), but did not bind to PLAD (Figures 5E-5F). We note the difference between the binding affinities of ABY NFRi-i to soluble TNFR2-ECD (SPR data) and membrane bound TNFR2ACD- GFP (Figure 4D). The fact that ABYTNFRI-I has no effect on membrane-bound TNFR2 in cells suggests that there is either a difference in the structure of the recombinant soluble ECD or in the availability of the binding motif in the plasma membrane.

Effect of ABYTNFRI-I on TNF-induced IK Bo degradation and NF-KB activation

The effect of ABYTNFRI-I on TNF-induced inflammation was assessed using cell-based functional assays. Two metrics were used to quantify the degree of TNF-induced TNFR1 activation: IKBGI degradation and NF-KB activation. The dose-dependent effect of ABYTNFRI-I or a non-binder on TNF-induced IxBa degradation was determined by immunoblotting for IxBa and normalized using P-actin as a loading control (Figure 6A). After TNF treatment, IxBa was degraded to 10% of the basal IxBa levels in HEK293 cells (Figure 6A).

ABYTNFRI-I inhibited IxBa degradation in a dose-dependent manner with an IC50 of 7 ± 6 nM (Figure 6A-6B), while a non-binder had no effect on IxBa degradation (Figure 6A-6B). To test the effect of ABYTNFRI-I or a non-binder on TNF-induced NF-KB activation, a luciferase reporter assay was performed using HEK293 cells. The luciferase reporter gene will be only transcribed when NF-KB signaling pathway is active. TNF triggers NF-KB activation that involves nuclear translocation of the NF-KB transcription factors. Transcription of luciferase reporter gene is directly proportionally to the magnitude of NF-KB activation. While ABYTNFRI-I inhibited TNF- induced NF-KB activation in a dose-dependent manner, with an IC50 of 0.23 ± 0.07 nM (Figure 6C), a non-binder had no effect on TNF-induced NF-KB activation (Figure 6D). Taken together, these results confirm that ABYTNFRI-I is an antagonist of TNF-induced inflammation.

Next, to rule out the possibility that the inhibitory effect of ABYTNFRI-I on TNF-induced biological responses was not caused by its cytotoxic effect, the cytotoxicity of ABYTNFRI-I in HEK293 cells was investigated using the MTT assay. No appreciable cytotoxicity is observed, which suggests that inhibitory effect of ABYTNFRI-I.I is not due to cell death (Figure 6E). We note the variance in IC50 for IxBa and NF-KB, which could be attributed to the differences between the experimental conditions in these two assays: TNF induces complete degradation of IxBa within minutes, so in the IxBa degradation assay HEK293 cells were incubated with TNF for only 20 minutes. The NF-KB luciferase reporter gene activity was assayed 48 hours after transfection of HEK293T cells and 24 hours after TNF treatment.

Next, we sought to determine if the functional effect of ABYTNFRI-I resulted from binding to TNFR1 and modifying that protein's function rather than by blocking proteins in other signaling pathways. So, we investigated the effects of ABYTNFRI-I on CRISPR-generated TNFR1 wild-type Hapl (TNFR1-WT) and TNFR1 knockout Hapl (TNFR1-K0) cells. First, we measured the surface expression of TNFR1 in these two cell lines. These results suggest that there is -75% knockdown of TNFR1 expression following CRISPR treatment (Figure 7A-B). While TNFR1- WT cells showed TNF-induced IKB degradation, TNFR1-K0 cells did not respond to TNF, indicating that they were not functionally active. Next, we examined the impact of ABYTNFRI-I on TNF-induced IxBa degradation. In TNFR1-WT cells, ABYTNFRI-I inhibited IKB degradation in a dose-dependent manner (Figure 7C), but it had no impact on TNFR1-K0 cells (Figure 7D). These results further confirm that ABYTNFRI-I inhibits TNF-induced IKB degradation via TNFR1.

Next, the binding and functionality of ABYTNFRI-I were tested in the mouse cell line L929. Binding studies indicate ABYTNFRI-I binds to mouse TNFR1 with a KD of 11 pM (Figure 12A), which is a 100-fold weaker affinity than that of human TNFR1. We then tested the effect of ABYTNFRI-I.I on mouse-TNF-induced IxBa degradation. These results suggest that ABYTNFRI-I partially inhibits TNF-induced IxBa degradation in mouse L929 cells (Figure 12B).

Mode of action of ABYTNFRI-I

After determining the function of ABYTNFRI-I, we then aimed to investigate if the anti- TNFR1 function of ABYTNFRI-I results from blocking TNF binding. Whether the ABYTNFRI-I acts competitively or non-competitively with TNF is of particular interest since a competitive mechanism would alter the free TNF levels thereby serving as a potential source of side effects; a competitive mechanism would also require higher potency to compete with autocrine signaling. To determine the mode of action of ABYTNFRI-I, we first tested the effect of TNF on ABYTNFRI-I binding to TNFR1 using flow cytometry. These studies showed that TNF does not affect ABYTNFRI-I binding to TNFR1 (Figure 8 A). Next, TNF-TNFR1 interactions were monitored in the absence and presence of the ABYTNFRI-I or with a known TNF blocker⁶¹ (H398 antibody) or a non-binder using coimmunoprecipitation (Figure 8B). Western blot analysis of coimmunoprecipitated samples showed that ABYTNFRI-I does not affect ligand binding: TNF binding to TNFR1 was not statistically significantly different in the presence or absence of ABYTNFRI-I (Figure 8C). This data suggest that ABYTNFRI-I does not compete with TNF for binding to TNFR1.

We then tested whether the anti-TNFRl function of ABYTNFRI-I results from blocking receptor-receptor interactions or altering receptor conformational dynamics. We have previously shown that it is possible to inhibit receptor activation by inhibiting TNF receptor-receptor interactions or conformational dynamics.⁶²'⁶⁴ So, we investigated whether the binding of ABYTNFRI-I causes any conformational changes in TNFR1 or inhibiting TNF receptor-receptor interactions using the time-resolved fluorescence energy transfer (TR-FRET) assay. We have previously used this method to monitor TNF receptor oligomerization (increase in basal FRET), disruption of receptor-receptor interactions (decrease in basal FRET), confirmational changes or structural rearrangements (change in basal FRET).

Experiments were carried out in HEK293T cells transiently expressing TNFR1 ACD-GFP (donor) and co-expressing TNFR1 ACD-GFP and TNFR1ACD-RFP (acceptor). Donor lifetime was measured in the presence and absence of the acceptor and then used to calculate FRET efficiency. Measurements showed a significant decrease in the fluorescence lifetime of the donor in the presence of the acceptor compared with the donor alone, which confirms efficient energy transfer between the FRET pairs (Figure 8D). These results confirm that TNFR1 exists as ligand independent oligomers. We then evaluated the effect of ABYTNFRI-I on ligand independent TNFR1 interactions. Surprisingly, ABYTNFRI-I treated cells showed FRET that was similar to untreated cells (Figure 8E). No change in FRET indicates that the binding of ABYTNFRI-I probably does not inhibit receptor-receptor interactions or cause any confirmational changes or conformational rearrangements are too small to change the distance between the cytoplasmic ends of preassembled TNFR1 receptors in this study. This data further confirms the ABYTNFRI-I is a non-competitive inhibitor of TNFR1.

Effect of ABYTNFRI-I on TNFR2 Function

To study the effect of ABYTNFRI-I on TNFR2 function, its effect on TNFR2 -induced activation of Rel-B was tested. Recently, it has been shown that the non-canonical NF-KB pathway selectively responds to a subset of TNFR superfamily members.⁵⁸'⁶⁰ The non-canonical pathway depends on ligand-induced processing of NF-KB precursor protein, pl 00, which mediates activation of the p52/RelB complex.⁵⁸'⁶⁰ Previous studies showed that the membrane-TNF (m- TNF) induces plOO processing via TNFR2.⁶⁵ So, we tested the effect of ABYTNFRI-I on m-TNF- induced RelB activation in HUVEC cells. Indeed, transfection of HUVEC cells with m-TNF plasmid resulted in a 5 ± 2-fold increase in RelB expression relative to untransfected cells (Figure 9 A). And m-TNF transfected cells treated with ABYTNFRI-I had no statistically significant effect on RelB activation (Figure 9B). Taken together, these results confirm that ABYTNFRI-I specifically inhibits TNFR1 function without affecting TNFR2.

DISCUSSION

Despite the clinical success of anti-TNF biologies,⁶⁶'⁷⁰ the use of these drugs is limited because of high cost, adverse side effects, and inconsistent patient response including a reduced ability of the immune system to fight infections.⁷¹'⁷⁴ These deficiencies led to the development of novel antagonistic anti-TNFRl antibodies and antibody derivatives.¹⁹'²² For example, Atrosab, a humanized IgG derived antagonistic anti-TNFRl -specific antibody, is capable of inhibiting TNF- induced IL-6 and IL-8 release with nanomolar potency (IC50 165 nM for IL-6 and 84 nM for IL- 8 production).¹⁹'^20,22 However, Atrosab failed in phase 1 clinical trial due to dose-dependent side effects, which was subsequently credited to a slight agonistic activity of the therapy.^{20, 22} The agonism was attributed to Atrosab ’s bivalency, which would cross-link TNFR1 receptors and lead to activation by creating a network of TNFRls.^20,22 This led to the development of Atrosimab, a monovalent and improved derivative of Atrosab⁷⁵'⁷⁶ with high affinity and efficacy (IC50 54.5 nM for IL-6 and 24.2 nM for IL-8 production).^21,75'⁷⁶ Atrosimab is in early preclinical stages for the treatment of hepatic fibrosis and non-alcoholic steatohepatitis. Like Atrosimab, ABYTNFRI-I is monovalent and thus should not suffer from unwanted induction of receptor clusters. Another nanobody-based selective inhibitor of TNFR1, TNF receptor-one Silencer, reduced secretion of IL-6, IL-8 and TNF in ex vivo and in vivo models of inflammation and also inhibited TNF -induced NF-KB activity, resulting in an IC50 of 324 nM.⁷⁷

All of these anti-TNFRl antibodies or nanobodies work by blocking TNF binding to TNFR1. Here, in contrast, we have emphasized the specific advantage of a non-competitive inhibitor such as ABYTNFRI-I, and we note again its very high potency (NF-KB activation IC50 of 0.23 ± 0.07 nM). In a recent set of studies, we used small-molecule discovery to prove that non-competitive regulation of TNFR1 is possible by altering receptor conformations.^{57, 62} While the potency of our best small-molecule compound was only in the low micromolar range, herein we have demonstrated higher potency and high-selectivity affibody inhibitors for this novel, noncompetitive approach to efficiently targeting TNFR1.

Non-competitive inhibitors do not having to outcompete picomolar-affinity TNF binding⁷⁸ or micromolar affinity TNFR1-TNFR1 assembly^{25, 79}, providing a clear benefit from a drug dosing perspective. However, there may be other less apparent advantages. For example, it has been reported that soluble TNFR1 binds TNF and thereby reduces the amount of free TNF available for binding to membrane-bound TNF receptors.⁸⁰ Antagonists that act by blocking TNF binding to TNFR1 reduces soluble TNFRl’s capacity to neutralize physiological levels of TNF, thus increasing the concentration of free TNF. In theory, this increase in free TNF could overstimulate membrane-TNFR2. Thus, an inhibitor which minimizes disruption to the physiological levels of free TNF could ameliorate unwanted side effects and therefore might be preferable.

We note with curiosity the discrepancy between the binding of ABYTNFRI-I to soluble TNFR2- ECD (as measured by SPR, Figures 11C-11D) and the lack of binding and activation of membrane-bound TNFR2ACD-GFP as measured in cells (Figure 4D and Figure 9). That ABYTNFRI-I is inactive against membrane-bound TNFR2 in cells suggests a difference in either the structure of the recombinant, immobilized receptor ECD, or in the accessibility of the binding motif in the plasma membrane. Although further study may be needed, without wanting to be bound by theory, we prefer the latter explanation given the consistency across experimental modalities for TNFR1 binding and activation. Different accessibility in the two receptors’ binding domains in the plasma membrane could be due to differences in the extent of oligomeric clustering of the two receptors, though little has been published on this point. Extensive oligomerization could cause molecular crowding and preclude binding of ABYTNFRI-I to TNFR2. While TNF receptor oligomerization has been investigated via biochemical^{25, 81-82} and/or crystallization studies,^{78, 83} only recently have studies using super-resolution techniques begun to provide the kind of critical molecular detail that could eventually help explain our findings.⁸⁴ Indeed, molecules like ABYTNFRI-I may eventually also be useful as probes to understand still elusive details regarding key differences in molecular architecture in the TNF superfamily. Furthermore, the discovery of a potent functional affibody by testing only five representative affibodies after enriching binders without epitope- or mechanism-driven decisions may have been accidental. It is reasonable to assume that affibodies will preferentially bind at hot spots associated with natural binding interfaces. However, many additional clones would need to be sequenced and mechanistically characterized in order to determine the relative frequency of binders that compete with TNF, compete with TNFR1 dimerization, or trigger particular conformations.

There are several potential next steps in advancing ABYTNFRI-I as a therapeutic. First, while we have shown that the ABYTNFRI-I inhibits TNFR1 activation without disrupting TNF binding, further efforts could be geared to understand how ABYTNFRI-I impacts receptor conformational states and organization in the membrane. Additional studies could also use epitope mapping to identify the binding interface of the affibody and TNFR1, providing further insight into a mechanism of action. Numerous non-antibody proteins have been identified in clinical research, which is evidence of the potential of these molecules for use in human therapy.⁸⁵ However, compared to small molecule drugs, protein-based therapeutics have a higher risk of being immunogenic and giving rise to anti-drug antibodies (AD As).⁸⁶ The consequent AD As may cause allergic reaction and may impact the effectiveness, pharmacokinetics, and bioavailability of protein therapeutics.^86-89 Several factors can affect the immunogenicity of protein therapeutics including protein sequence and structure, aggregation, and degradation that can lead to exposure of buried antigenic sites.^90-92 Therefore, immunogenicity mitigation strategies may be considered as part of the development of protein therapeutics. Overall, in this Example, the human TNFR1 specific ABYTNFRI-I was developed to neutralize pro-inflammatory TNFR1 signaling without affecting TNFR2 function, indicating that ABYTNFRI-I can be a viable therapy for inflammatory diseases. It was shown herein that the ABYTNFRI-I inhibits TNFR1 activation without interfering with TNF binding or TNFR2 function. In previous studies, small molecule TNFR1 antagonists were identified that stabilize nonfunctional conformational states of TNFR1 that are independent of ligand binding or receptor dimerization (Lo, C. H. et la., Set Signal 2019, 12 (592)). There is a probability that ABYTNFRI-I also inhibits TNFR1 signaling via stabilizing the nonfunctional conformational states of TNFR1. Epitope mapping may provide further insight into mechanism of action of ABYTNFRI-I. It was demonstrated herein that ABYTNFRI-I selectively inhibits ligand induced IKBOL degradation and NF-KB activity with nanomolar potency, and do not block TNF binding or affect TNFR2 function.

In summary, a synthetic affibody ligand antagonist of tumor necrosis factor receptor 1 was engineered in this Example as potential therapy. In particular, yeast display and directed evolution was used herein to identify a synthetic affibody (ABY) ligand with high binding affinity, potency, and specificity for TNFR1. Functional assays showed that the affibody inhibits TNF-induced IKBOL degradation and NF-KB activation in HEK293 cells. Using biochemical techniques, it was demonstrated that the affibody does not block TNF binding, thereby enhancing inhibitory robustness. The affibody does not block TNFR2 function, providing greater specificity. The potency, mechanism of action, and specificity of the engineered anti-TNFRl affibody make it a potential therapeutic candidate for TNFR1 related diseases, such as inflammatory or autoimmune diseases.

Abbreviations

FACS - fluorescence-activated cell sorting

FRET - Forster resonance energy transfer

GFP - green fluorescent protein

HEK293 - human embryonic kidney cells

HUVEC - human umbilical vein endothelial cells

LAG3 - lymphocyte activation gene 3

MACS - magnetically activated cells sorting

TNF - tumor necrosis factor

TNFR1 - tumor necrosis factor receptor 1 Documents Cited in Example 1

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All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference, including Vunnam N. et al., Mol.

Pharmaceutics 2023, 20, 4, 1884-1897. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMS What is claimed is:

1. An isolated TNFR1 binding polypeptide, comprising one or more helix domains selected from the group consisting of:

(a) a helix 1 domain comprising an amino acid sequence having at least 60% sequence identity to an amino acid sequence of SEQ ID NO: 2; and

2. The isolated polypeptide of claim 1, comprising one or more helix domains selected from the group consisting of:

(a) a helix 1 domain comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence of SEQ ID NO:2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence of SEQ ID NO: 8.

3. The isolated polypeptide of claim 2, comprising one or more helix domains selected from the group consisting of:

(a) a helix 1 domain comprising the amino acid sequence of SEQ ID NO:2; and

(b) a helix 2 domain comprising the amino acid sequence of SEQ ID NO:8.

4. The isolated polypeptide of any one of claims 1-3, comprising: a helix 1 domain comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2.

5. The isolated polypeptide of any one of claims 1-4, comprising: a helix 1 domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.

6. The isolated polypeptide of any one of claims 1-5, comprising: a helix 1 domain comprising SEQ ID NO:2.

7. The isolated polypeptide of any one of claims 1-6, comprising: a helix 2 domain comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8.

8. The isolated polypeptide of any one of claims 1-7, comprising: a helix 2 domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8.

9. The isolated polypeptide of any one of claims 1-8, comprising: a helix 2 domain comprising SEQ ID NO: 8.

10. The isolated polypeptide of claim 1, comprising:

(a) a helix 1 domain comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8.

11. The isolated polypeptide of claim 10, comprising:

(a) a helix 1 domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2; and

(b) a helix 2 domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8.

12. The isolated polypeptide of claim 11, comprising:

(a) a helix 1 domain comprising SEQ ID NO:2; and

(b) a helix 2 domain comprising SEQ ID NO:8.

13. The isolated polypeptide of claims 1-12, comprising an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 19.

14. The isolated polypeptide of claim 13, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 19.

15. The isolated polypeptide of claim 14, comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 19.

16. The isolated polypeptide of claim 15, comprising the amino acid sequence of SEQ ID NO:19.

17. The isolated polypeptide of any one of claims 1-12, further comprising a helix 3 domain having at least 80% sequence identity to SEQ ID NO: 13.

18. A composition comprising the isolated polypeptide of any one of claims 1-17, and a carrier.

19. An isolated nucleic acid comprising a nucleotide sequence encoding the isolated polypeptide of any one of claims 1-17.

20. A vector comprising the nucleic acid of claim 19.

21. A cell comprising the nucleic acid of claim 19 or the vector of claim 20.

22. The cell of claim 21, which is a bacterial cell (e.g., E. coli).

23. A method of inhibiting the activity of TNFR1, comprising contacting TNFR1 with the isolated polypeptide of any one of claims 1-17.

24. The method of claim 23, wherein the TNFR1 is contacted in vitro.

25. The method of claim 23, wherein the TNFR1 is contacted in vivo.

26. The method of any one of claims 23-25, wherein the activity of the TNFR1 is inhibited by at least about 25% as compared to a control.

27. The method of any one of claims 23-25, wherein the activity of the TNFR1 is inhibited by at least about 40% as compared to a control.

28. A method for treating an inflammatory or autoimmune disorder in a mammal, comprising administering an effective amount of the isolated polypeptide of any one of claims 1-17 to the mammal.

29. The method of claim 28, further comprising administering at least one additional therapeutic agent to the mammal.

30. The isolated polypeptide of any one of claims 1-17 for the prophylactic or therapeutic treatment of an inflammatory or autoimmune disorder.

31. The use of the isolated polypeptide of any one of claims 1-17 to prepare a medicament for the treatment of an inflammatory or autoimmune disorder in a mammal.

32. The method, polypeptide or use of any one of claims 28-31, wherein the inflammatory or autoimmune disorder is selected from the group consisting of arthritis, psoriasis, Crohn's disease, ulcerative colitis, and asthma.

33. The isolated polypeptide of any one of claims 1-17 for use in diagnosis or medical therapy.

34. A kit comprising the isolated polypeptide of any one of claims 1-17, packaging material, and instructions for administering the isolated polypeptide to a mammal to treat an inflammatory or autoimmune disorder.