IL-17F-specific capture agents, compositions, and methods of use and manufacture

Abstract

The present application provides stable peptide-based IL-17F capture agents and methods of use as detection agents. The present application also provides methods of making IL-17F capture agents.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 192,899, filed July 15, 2015, U.S. Provisional Patent Application No. 62 / 277,430, filed January 11, 2016, and U.S. Provisional Patent Application No. 62 / 309,756, filed March 17, 2016, each of which is incorporated herein by reference in its entirety. Background Technology

[0003] Human interleukin-17 (IL-17A) is a pro-inflammatory cytokine secreted by activated T cells. IL-17A is an effective target for the treatment of severe plaque psoriasis. The IL-17 cytokine family includes six distinct homodimer cytokines (IL-17A-F) and heterodimers, IL-17A / F. IL-17F is the closest homologue to IL-17A, sharing 50% sequence similarity. Both IL-17A and IL-17F are secreted both as disulfide-linked homodimers (32 to 38 kDa) and as covalently linked heterodimers (40 to 45 kDa) of IL-17A / F. Similar to IL-17A, IL-17F activates both immune and non-immune cells to induce pro-inflammatory regulatory factors. These regulatory factors can induce the recruitment of neutrophils at sites of inflammation, promote local tissue destruction, induce tumor angiogenesis, enhance osteoclastogenesis, and prevent pathogen infection, thereby leading to disease progression and host protection.

[0004] IL-17A is an effective target for treating severe plaque psoriasis. Current approaches block the interaction between IL-17A and its cell surface receptors by neutralizing circulating IL-17A. This is primarily achieved through monoclonal antibodies and their fragments.

[0005] Members of the IL-17 cytokine family regulate their activity by binding to the IL-17 receptor family, which includes five related members (IL-17RA-IL-17RE). Both IL-17A and IL-17F bind as homodimers or heterodimers to heterodimeric receptor complexes formed between IL-17RA and IL-17RC. While the activity of IL-17F appears to correlate with that of IL-17A, their potency differs, consistent with differences in receptor binding affinity.

[0006] Due to the role of IL-17A and IL-17F in immunity and immune-mediated diseases, they have become a hot area for therapeutic drug development. The natural abundance of circulating IL-17A and IL-17A / F is very low, which is a challenge for detecting these biomarkers by traditional sandwich immunoassay. Detecting circulating levels of each homodimer (IL17A, IL-17F) as well as IL-17A / F heterodimer with high sensitivity will help to understand the role of each cytokine in the process of disease and treatment. SUMMARY

[0007] The present disclosure relates to chemically synthesized capture agents designed to bind to detect interleukin 17F (IL-17F) (referred to as protein catalyzed capture agents or PCC agents), methods of making the capture agents utilizing iterative in situ click chemistry, methods of detecting IL-17F using the capture agents and detection utilizing the methods.

[0008] In one aspect, provided herein is a stable synthetic capture agent that specifically binds IL-17F, wherein the capture agent comprises one or more designed anchor ligands. In certain embodiments, the capture agent comprises two anchor ligands connected by a linker. In another aspect, provided herein is a composition comprising one or more synthetic capture agents that specifically bind IL-17F as described herein. According to some embodiments, the capture agent binds IL-17F with greater affinity than IL-17A. According to certain embodiments, the capture agent binds IL-17F with at least 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, or 1000 times greater affinity than IL-17A.

[0009] In another aspect, provided herein is a stable synthetic capture agent that specifically binds IL-17F, wherein the capture agent comprises a first ligand having affinity for a first epitope on IL-17F, a second ligand having affinity for a second epitope on IL-17F, and a linker covalently linking the first ligand to the second ligand. In particular, the first ligand binds the first epitope (or a synthetic form thereof) in isolation, and the second ligand binds the second epitope (or a synthetic form thereof) in isolation. In the capture agent, the first ligand and the second ligand cooperatively bind the first epitope and the second epitope of IL-17F, respectively.

[0010] In another aspect, provided herein is a method for detecting IL-17F in a biological sample, comprising the step of treating the biological sample with one or more capture agents described herein.

[0011] Anchor ligand

[0012] In one embodiment of the capture agent, the capture agent comprises two ligands that specifically bind to two different epitopes of IL-17F. These anchor ligands (sometimes referred to herein simply as "ligands") can then be bound to one another through a linker that increases affinity for IL-17F. In certain embodiments, there is a first ligand and a second ligand that bind to a first epitope and a second epitope, respectively.

[0013] According to certain embodiments, the first epitope comprises an amino acid sequence of FFQKPES (SEQ ID NO: 1) or FFQKPESCPPVPGG (SEQ ID NO: 2). In certain embodiments, the first epitope is between 5 and 20 amino acids in length. In other embodiments, the first epitope is between 7 and 13 amino acids in length. In other embodiments, the first epitope is at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0014] According to certain embodiments, the second epitope comprises an amino acid sequence of NENQRVS or GIINENQRVS. In certain embodiments, the second epitope is between 5 and 20 amino acids in length. In other embodiments, the second epitope is between 7 and 10 amino acids in length. In other embodiments, the second epitope is at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0015] According to certain embodiments, the first ligand comprises an amino acid sequence selected from the group consisting of FYKTH (SEQ ID NO: 5), FYKQH (SEQ ID NO: 6), FYLTH (SEQ ID NO: 7), FYLQH (SEQ ID NO: 8), RRATS (SEQ ID NO: 9), and RRAQS (SEQ ID NO: 10). According to certain embodiments, the first ligand comprises an amino acid sequence selected from the group consisting of RRATS (SEQ ID NO: 9) and RRAQS (SEQ ID NO: 10). In certain embodiments, the first ligand is cyclic. In certain embodiments, the first ligand comprises a 1,4-substituted-1,2,3-triazole residue (Tz4) or a 1,5-substituted-1,2,3-triazole residue (Tz5).

[0016] According to certain embodiments, the second ligand comprises an amino acid sequence selected from the group consisting of KYGEV (SEQ ID NO: 11), LYGEV (SEQ ID NO: 12), VHKSG (SEQ ID NO: 13), VHLSG (SEQ ID NO: 14), QKHGP (SEQ ID NO: 15), TKHGP (SEQ ID NO: 16), QLHGP (SEQ ID NO: 17), TLHGP (SEQ ID NO: 18), YDLQR (SEQ ID NO: 19), YDLTR (SEQ ID NO: 20), YDKQR (SEQ ID NO: 21), YDKTR (SEQ ID NO: 22), KKGWP (SEQ ID NO: 23), KLGWP (SEQ ID NO: 24), LKGWP (SEQ ID NO: 25), LLGWP (SEQ ID NO: 26), RSYNL (SEQ ID NO: 27), and RSYNK (SEQ ID NO: 28). According to certain embodiments, the second ligand comprises an amino acid sequence selected from the group consisting of TKHGP (SEQ ID NO: 16), QKHGP (SEQ ID NO: 15), KKGWP (SEQ ID NO: 23), and RSYNK (SEQ ID NO: 28). In certain embodiments, the second ligand is cyclic. In certain embodiments, the second ligand comprises a 1,4-substituted-1,2,3-triazole residue (Tz4) or a 1,5-substituted-1,2,3-triazole residue (Tz5).

[0017] According to certain embodiments, the first ligand comprises the sequence RRATS (SEQ ID NO: 9) and the second ligand comprises the sequence QKHGP (SEQ ID NO: 15). In other embodiments, the first ligand comprises the sequence RRATS (SEQ ID NO: 9) and the second ligand comprises the sequence RSYNK (SEQ ID NO: 28). In other embodiments, the first and second ligands are cyclic and comprise a Tz4 residue.

[0018] Linker

[0019] According to certain embodiments, the capture agent further comprises a linker that simultaneously binds the first ligand and the second ligand. According to certain embodiments, the length of the linker corresponds to the distance between the first epitope and the second epitope. The length of the linker must be at least the distance between the first epitope and the second epitope. In certain embodiments, the linker is longer than the distance between the first epitope and the second epitope. According to certain embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the distance between the first epitope and the second epitope.

[0020] According to certain embodiments, the length of the linker is about to about about to about or about to about In certain embodiments, the length of the linker is about

[0021] In other embodiments, the linker comprises one or more ethylene glycol repeat units. In some embodiments, the linker is PEG1, PEG2, PEG3, PEG4, or PEG5. In other embodiments, the linker comprises a peptide. In other embodiments, the linker comprises an amino acid. In particular embodiments, the linker is glycine. In other embodiments, the linker comprises an alkylene moiety, wherein the alkylene moiety is optionally substituted with one or more moieties provided herein.

[0022] Triazole linkage

[0023] In one embodiment of the capture agent, the anchor ligand and the second ligand are linked together by a 1,4-substituted-1,2,3-triazole residue (Tz4). In another embodiment, the second ligand and the third ligand are linked together by a 1,4-substituted-1,2,3-triazole residue (Tz4). In yet another embodiment, the third ligand and the fourth ligand are linked together by a 1,4-substituted-1,2,3-triazole residue (Tz4). In yet another embodiment, the anchor ligand and the second ligand are linked together by a 1,4-substituted-1,2,3-triazole residue, the second ligand and the third ligand are linked together by a 1,4-substituted-1,2,3-triazole residue. In yet another embodiment, the anchor ligand and the second ligand are linked together by a 1,4-substituted-1,2,3-triazole residue, the second ligand and the third ligand are linked together by a 1,4-substituted-1,2,3-triazole residue, and the third ligand and the fourth ligand are linked together by a 1,4-substituted-1,2,3-triazole residue.

[0024] Capture agent

[0025] According to certain embodiments, the capture agent has a structure selected from the group consisting of:

[0026]

[0027]

[0028] According to other embodiments, the capture agent has a structure selected from the group consisting of:

[0029]

[0030]

[0031] Property

[0032] In certain embodiments, the IL-17F capture agents provided herein are stable over a wide range of temperatures, pH, storage times, storage conditions, and reaction conditions, and in certain embodiments, the capture agents are more stable than comparable antibodies or biologies. In certain embodiments, the capture agents are stable as lyophilized powder. In certain embodiments, the capture agents are stable when stored at temperatures ranging from about -80 °C to about 60 °C. In certain embodiments, the capture agents are stable at room temperature. In certain embodiments, the capture agents are stable in human serum for at least 24 hours. In certain embodiments, the capture agents are stable at a pH ranging from about 3 to about 12. In certain embodiments, the capture agents are stable as a powder at a temperature of about 60 °C for two months.

[0033] Detectable label

[0034] In some embodiments, the capture agent is labeled with a tag selected from the group consisting of biotin, copper-DOTA, biotin-PEG3, aminooxyacetate, 19 FB, 18 FB, and FITC-PEG3. In other embodiments, the capture agent is labeled with a detectable moiety selected from the group consisting of 64 Cu DOTA, 68 Ga DOTA, 18 F, 64 Cu, 68 Ga, 89 Zr, 124 I, 86 Y, 94m Tc, 110m In, 11 C, and 76 Br. In other embodiments, the tag is a fluorescent tag. In particular embodiments, the detectable tag is18 F.

[0035] Methods and uses

[0036] As used herein, the term "capture agent of the invention" or "capture agents of the invention" refers to a synthetic proteinaceous capture agent that binds IL-17F as described herein.

[0037] Also provided are methods of detecting IL-17F in a subject, comprising the step of contacting a biological sample from the subject with one or more capture agents of the invention. Also provided is the use of one or more capture agents of the invention for detecting IL-17F in a subject.

[0038] Also provided are methods of detecting IL-17F in a biological sample using an immunoassay, wherein the immunoassay utilizes a capture agent as described herein, wherein the capture agent replaces an antibody or equivalent thereof in the immunoassay. In certain embodiments, methods of identifying, detecting, quantifying or isolating IL-17F in a biological sample using a capture agent as described herein are provided. In one embodiment of the method, the immunoassay is selected from the group of Western blot, pull-down assay, dot blot and ELISA.

[0039] Also provided are methods of detecting the presence of IL-17F in a human or mammalian subject, the method comprising the steps of:

[0040] a) administering one or more capture agents of the invention to a biological sample of the subject, wherein each capture agent is linked to a detectable moiety; and

[0041] b) detecting the moiety linked to each capture agent in the subject; wherein detection of the moiety indicates the presence of IL-17F in the subject.

[0042] Also provided herein are methods of detecting IL-17F in a sample, comprising:

[0043] a) exposing the sample to one or more capture agents of the invention, wherein each capture agent is linked to a detectable moiety;

[0044] b) binding IL-17F in the biological sample to the capture agent, and

[0045] c) detecting the moiety linked to each capture agent on a substrate; wherein detection of the moiety on the substrate indicates the presence of IL-17F in the sample.

[0046] Kit

[0047] Provided herein in certain embodiments are kits comprising one or more capture agents of the present application. In certain embodiments, these kits can be used to identify, detect, quantify, and / or isolate IL-17F, and in certain embodiments, the kits can be used for the diagnosis and / or staging of a disorder associated with the presence of IL-17F. In certain embodiments, the kits provided herein comprise: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent is suitable for binding IL-17F, and (b) a wash solution or instructions for preparing a wash solution, wherein the combination of the adsorbent and the wash solution allows for the detection of IL-17F. In other embodiments, the kits provided herein can be used to treat a disorder associated with the presence of IL-17F.

[0048] In certain embodiments, the kits can further comprise instructions for suitable operating parameters, either in the form of a label or in the form of a separate insert. For example, the kits can have standard instructions informing the consumer / kits user how to wash the probe after contacting the plasma sample or other tissue sample with the probe.

[0049] In certain embodiments, the kits comprise (a) one or more capture agents that specifically bind IL-17F; and (b) a detection reagent. Such kits can be prepared from the materials described herein.

[0050] The kits provided herein can optionally comprise standard or control information and / or control amounts of material, such that a test sample can be compared to the control information standard and / or control amounts to determine whether the test amount of IL-17F detected in the sample is an amount that is consistent with the diagnosis of a particular disorder.

[0051] Synthesis of capture agent

[0052] Provided herein are methods of making (i.e., synthesizing) IL-17F-specific capture agents of the present application. In one embodiment, the method comprises the steps of:

[0053] a. selecting a first ligand that binds to a first epitope on a target protein,

[0054] b. selecting a second ligand that binds to a second epitope on the target protein,

[0055] c. selecting a linker, wherein the length of the linker allows for the linker to bind to both the first ligand and the second ligand when the first ligand and the second ligand specifically bind to the first epitope and the second epitope, respectively, and

[0056] d. combining the linker with the first ligand and the second ligand, thereby producing a synthetic capture agent that specifically binds to the target protein.

[0057] In certain embodiments, the following steps are employed to identify the ligand:

[0058] 1) pre-cleanup to eliminate non-specific binders,

[0059] 2) product screening to identify hits generated from the epitope template in situ click chemistry,

[0060] 3) target screening against His-tagged IL-17F protein, and

[0061] 4) another target screening against His-tagged IL-17F protein in 2% (v / v) human serum to identify peptides whose binding to IL-17F is not interfered by serum proteins.

[0062] In certain embodiments, the first epitope and the second epitope are separated from each other by a distance of about to about about to about or about to about or about In some embodiments, the linker is longer than the distance between the first epitope and the second epitope. Optionally, the linker is 10 to 50%, 5 to 25%, or 1 to 10% longer than the distance between the first epitope and the second epitope.

[0063] In certain embodiments, the capture agent has a binding affinity to the target protein that is greater than any of the ligands. In some embodiments, the capture agent has a binding affinity that is at least 50, 75, or 90% of the binding affinity of a full cooperative binder. In other embodiments, the capture agent has a binding affinity that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the binding affinity of a full cooperative binder.

[0064] In certain embodiments, the target protein is a synthetic epitope, wherein the synthetic epitope comprises a sequence of at least 20 amino acids of a full-length protein, wherein at least one amino acid of the synthetic epitope comprises an azido group or an ethynyl group. In some embodiments, the synthetic epitope is a sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 200, 250, or 300 amino acids of a full-length protein. In some embodiments, at least two amino acids of the synthetic epitope comprise an azido group or an ethynyl group. In other embodiments, at least 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the synthetic epitope comprise an azido group or an ethynyl group.

[0065] According to certain embodiments, the full-length protein is a naturally occurring protein. According to other embodiments, the naturally occurring protein is IL-17.

[0066] According to certain embodiments, the capture agent binds the synthetic epitope and the full-length protein with a binding affinity that is at least 50% of the binding affinity of the fully synergistic binder. According to certain embodiments, the capture agent binds the synthetic epitope and the full-length protein with a binding affinity that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the binding affinity of the fully synergistic binder. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1: Epitopes derived from IL-17F protein. Figure 1A The difference in the N-terminal sequence exists in the mature protein that distinguishes IL-17A (SEQ ID NO: 37) from IL-17F (SEQ ID NO: 38). The uniqueness of this region corresponds to Arg-31 to Thr-79 in IL-17F. Figure 1B The sequence of the designed Epitope 1 fragment, which contains a biotin-PEG3 assay handle and a click handle strategically substituted with azide (C48Az4; Az4 = L-azidolysine), (biotin-PEG3-FFQKPES(SEQ ID NO: 1) [Az4]PPVPGGS) (SEQ ID NO: 32). Figure 1C The sequence of the designed Epitope 2 fragment, which contains a biotin-PEG3 assay handle and a click handle strategically substituted with azide (I62Az4), (biotin-PEG3-GI[Az4]NENQRVS) (SEQ ID NO: 33).

[0068] Figure 2: In vitro characterization of macrocycles developed against IL-17F Epitope 1. Figure 2A Sandwich ELISAs against PEG3-biotin modified Cy(RRATS) (SEQ ID NO: 9) and Cy(RRAQS) (SEQ ID NO: 10) human IL-17F proteins yielded EC 50 values of 66 to 52 nM. Similar assays with biotinylated monoclonal antibody (TA319597, Origene) showed similar binding affinities. Figure 2B Dot ELISAs against both macrocycle ligands (PEG3-biotin-modified Cy(RRATS) (SEQ ID NO: 9) and Cy(RRAQS) (SEQ ID NO: 10)) human IL-17F and IL-17A proteins indicated preferential binding to IL-17F. Figure 2CMass spectrometry of Cy(RRATS)(SEQ ID NO: 9)-PEG3-biotin. Figure 2D Mass spectrometry of Cy(RRAQS)(SEQ ID NO: 10)-PEG3-biotin.

[0069] Figure 3: Mapping of macrocycles binding to IL-17F epitope 1. Figure 3A Synthesis of His-tagged IL-17F epitopes to contain strategically scrambled sequences that can be performed at the N- or C-terminus of the click handle position (C48S). 1) SEQ ID NO: 39, 2) SEQ ID NO: 40, 3) SEQ ID NO: 41. Scrambled residues are shown in italics. Figure 3B Dot ELISA of His-tagged IL-17F epitopes against two macrocyclic peptide ligands (PEG3-biotin-modified Cy(RRATS)(SEQ ID NO: 9) and Cy(RRAQS)(SEQ ID NO: 10)) indicated preferential binding to the sequence FFQKPES (SEQ ID NO: 1).

[0070] Figure 4: In vitro characterization of macrocycles developed against IL-17F epitope 2. Figure 4A Sandwich ELISA against human IL-17F protein for PEG3-biotin modified Cy(QKHGP)(SEQ ID NO: 15), Cy(TKHGP)(SEQ ID NO: 16), Cy(KKGWP)(SEQ ID NO: 23), and Cy(RSYNK)(SEQ ID NO: 28) yielded EC 50 values of 72 to 15 nM. Figure 4B Dot ELISA of human IL-17F and IL-17A proteins against four macrocyclic peptide ligands (PEG3-biotin-modified Cy(QKHGP)(SEQ ID NO: 15), Cy(TKHGP)(SEQ ID NO: 16), Cy(KKGWP)(SEQ ID NO: 23), and Cy(RSYNK)(SEQ ID NO: 28)) indicated preferential binding to IL-17F. Figure 4C Dot ELISA of human IL-17F and IL-17A proteins against macrocyclic peptide ligands (RRATS (SEQ ID NO: 9), RSYNK (SEQ ID NO: 28), QKHGP (SEQ ID NO: 15)) indicated preferential binding to IL-17F or IL-17A.

[0071] Figure 5: 3-D structure representation of IL-17F homodimer. The sequences of IL-17F epitopes 1-2 are overlaid with a macrocycle (Tz = triazole) representing a representative targeting epitope. The distance measured between the two epitopes is indicated by a dashed line. Figure 5A , In monomeric IL-17F protein, the distance between the sequences FFQKPES (SEQ ID NO: 1) (in IL-17F epitope 1) and IL-17F epitope 2 (NENQRVS (SEQ ID NO: 3)) is about Using a linker of similar length to the distance between the two binding sites on the protein, a bi-ligand containing a macrocycle targeting each of the two IL-17F epitopes can be synthesized. Figure 5B , In homodimeric IL-17F protein, the distance between the sequence FFQKPES (SEQ ID NO: 1) from one monomer (in IL-17F epitope 1) and IL-17F epitope 2 (NENQRVS (SEQ ID NO: 3)) from the other monomer is about Using a linker of similar length to the distance between the two binding sites on the bridged protein dimer, another bi-ligand containing a macrocycle targeting each of the two IL-17F epitopes can be synthesized. PDB ID: 1JPY.

[0072] Figure 6: Structure of synergistic bi-ligand candidate compounds connecting two macrocycles with linkers ranging from 4.4 to Figure 6A Biotin-PEG3-Cy(RRATS) (SEQ ID NO: 9)-Gly-Cy(QKHGP) (SEQ ID NO: 15) Figure 6B Biotin-PEG3-Cy(RRATS) (SEQ ID NO: 9)-PEG1-Cy(QKHGP) (SEQ ID NO: 15) Figure 6C Biotin-PEG3-Cy(RRATS) (SEQ ID NO: 9)-PEG2-Cy(QKHGP) (SEQ ID NO: 15) Figure 6D Biotin-PEG3-Cy(RRATS) (SEQ ID NO: 9)-PEG3-Cy(QKHGP) (SEQ ID NO: 15) Figure 6E Biotin-PEG3-Cy(RRATS) (SEQ ID NO: 9)-PEG4-Cy(QKHGP) (SEQ ID NO: 15) Figure 6F ​, Biotin-PEG3-Cy(RRATS) (SEQ ID NO: 9)-PEG5-Cy(QKHGP) (SEQ ID NO: 15)

[0073] Figure 7 : Development of a starting point for PCC binders to a protein target. The initial goal is to identify one or more PCCs (12) that bind to an epitope on a protein target, and one or more different PCCs (13) that bind to a second epitope. Other PCCs that bind to a third epitope, a fourth epitope, etc. can also be useful. The PCC approach to targeting epitopes teaches that this can be accomplished by screening a library of peptides against synthetic epitopes (SynEps) (14, 15). SynEps are polypeptides that have the sequence of a naturally occurring target epitope, except that one site contains an artificial amino acid that presents an azido or ethynyl chemical group (16) (referred to as a click handle). The SynEps are further modified to include a detection handle, such as a biotin group, at the N- or C-terminus (17). The screening process has been previously described (Das, S. et al. A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands. Angew. Chem. Int. Ed. Engl. 2015, incorporated by reference in its entirety). Using this process, at least one unique peptide binder to each of at least two epitopes on the target can be identified. These peptide binders are validated by performing binding assays against the intact protein target (11) as well as against the SynEps. For these binding assays, the SynEps are prepared with the naturally occurring residues in place of the click handles (16). Ideally, the different regions of the target protein to which the different ligands bind will be relatively close (a few nanometers or less) in the tertiary protein structure. Even a single SynEp, the screening can yield a PCC that binds to two different sites.

[0074] Figure 8 : PCCs that bind to two different sites. The region representing the epitope of interest (12) is highlighted against the dim background of the full-length protein (11). The amino acid residues that are replaced with click handles in the SynEp structure are indicated with asterisks (22). In the SynEp screening step, PCCs that bind to either the N-terminal side (23) or the C-terminal side (24) of the epitope can be identified.

[0075] Figure 9: Evaluate optimal linker length. Shown is the first PCC (31) binding to the N side of one epitope (12) and the second PCC (32) binding to the C side of the second epitope (13). This binding arrangement is analyzed together with the structure of a protein from, e.g., a protein database to allow estimation of the length of the optimal linker (33). Such an evaluation can reduce the number of selected candidate linkers to very small. In one example, this evaluated length value can be used to select one or two length-matching polyethylene glycol oligomers for testing. The best linker (34) is the one that brings the affinity of the bi-ligand closest to that of a perfectly cooperative binder.

[0076] Figure 10 : IL-17F and IL-17A are close homologs. Alignment of IL-17F (SEQ ID NO: 38) with IL-17A (SEQ ID NO: 37). Residues in dark green are identical, residues in yellow are homologous, and residues not highlighted are unique.

[0077] Figure 11 : Generate ligands for IL-17F and IL-17A. Use two epitopes to generate an anchor ligand for IL-17F and one epitope to generate an anchor ligand for IL-17A.

[0078] Figure 12: Optimized PCC bi-ligand for IL-17F by using cooperativity design. Figure 12A , Map full-length IL-17F. Develop PCCs for epitopes labeled LI and L2. Figure 12B , Show IL-17F unfolding plot for target region. Distance between chemically accessible points on PCCs is evaluated as Figure 12C , Demonstrate that a linker (PEG3) with a length closest to produces the best binder (about 10-fold). The bi-ligand is in the form of biotin-PEG3-Cy(RSYNK)(SEQ ID NO: 28)-PEG x -Cy(RRATS)(SEQ ID NO: 9) (x = 1 to 5). Figure 12D , Show graph that a linker (PEG3) with a length closest to produces the best binder (about 10-fold).

[0079] Figure 13: Structure of a bi-ligand with two macrocycles connected with PEG linkers ranging from 8.8 to Figure 13A, biotin-PEG3-Cy(RSYNK)(SEQ ID NO: 28)-PEG1-Cy(RRATS)(SEQ ID NO: 9) Figure 13BBiotin-PEG3-Cy(RSYNK) (SEQ ID NO: 28)-PEG2-Cy(RRATS) (SEQ ID NO: 9) Figure 13C Biotin-PEG3-Cy(RSYNK) (SEQ ID NO: 28)-PEG3-Cy(RRATS) (SEQ ID NO: 9) Figure 13D Biotin-PEG3-Cy(RSYNK) (SEQ ID NO: 28)-PEG4-Cy(RRATS) (SEQ ID NO: 9) Figure 13E Biotin-PEG3-Cy(RSYNK) (SEQ ID NO: 28)-PEG5-Cy(RRATS) (SEQ ID NO: 9)

[0080] Figure 14 : Plasmodium falciparum histidine-rich protein-2 (Pf.HRP-2). Sequence map of Pf.HRP-2 is shown (SEQ ID NO: 42).

[0081] Figure 15 : Structure and EC of YKYYR (SEQ ID NO: 29) dimer. 50 Data.

[0082] Figure 16 : cy(YKYYR) (SEQ ID NO: 29)-linker-cy(GWNVDL) (SEQ ID NO: 30) bi-ligand developed by library screening with a linker. DETAILED DESCRIPTION

[0083] The following description of the application is merely exemplary in nature and is not intended to limit the scope of the application, as described. Those skilled in the art will be able to make various modifications and variations thereto without departing from the scope of the present application. It should be noted that all statements concerning specific modifications are intended to include equivalents thereof and that all references to a specific compound are intended to include possible racemates, enantiomers, diastereomers, tautomers, stereoisomers, and mixtures thereof, as well as isotopically enriched forms such as deuterium.

[0084] Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations of the word, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0085] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0086] Definitions

[0087] "Amino" refers to the -NH2 group.

[0088] "Cyano" refers to the -CN group.

[0089] "Hydroxy" or "hydroxyl" refers to the -OH group.

[0090] "Imino" refers to the =NH substituent.

[0091] "Nitro" refers to the -NO2 group.

[0092] "Oxo" refers to the =O group.

[0093] "Sulfo" refers to the =S group.

[0094] "Alkyl" refers to a straight or branched chain hydrocarbon group, which is saturated or unsaturated (i.e., containing one or more double and / or triple bonds), which consists only of carbon and hydrogen atoms, having from one to twelve carbon atoms (C1-C12alkyl), preferably one to eight carbon atoms (C1-C8alkyl), or one to six carbon atoms (C1-C6alkyl), and which is attached to the rest of the molecule by a single bond, such as for example methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, ethylene, prop-1- enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, acetylene, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted. 12 Alkyl), preferably one to eight carbon atoms (C1-C8alkyl), or one to six carbon atoms (C1-C6alkyl), and which is attached to the rest of the molecule by a single bond, such as for example methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, ethylene, prop-1- enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, acetylene, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0095] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain of carbon and hydrogen atoms only, linking the rest of the molecule to a radical group, which is saturated or unsaturated (i.e., contains one or more double and / or triple bonds), and has from one to twelve carbon atoms, such as methylene, ethylene, propylene, n-butylene, ethlenyl, propenyl, n-butenyl, propynyl, n-butynyl, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise indicated herein, an alkylene chain can be optionally substituted.

[0096] "Alkoxy" refers to a radical of the formula -OR a where R a is an alkyl group containing one to twelve carbon atoms as defined above. Unless stated otherwise in the specification, an alkoxy group can be optionally substituted.

[0097] "Aminocarbonyl" refers to a radical of the formula -C(=O)NR a R a where each R a is independently H, alkyl, or a linker moiety.

[0098] "Alpha-aminocarbonyl" refers to a radical of the formula -C(=O)CR b (NR a R a ), where each R a is independently H, alkyl, or a linker moiety, and R b is H or alkyl. In some embodiments, the alpha-aminocarbonyl is part of a cyclic moiety (e.g., a peptide), where the carbonyl is within the ring and the amino group (NR a R a ) is outside the ring. For example, in certain embodiments, the alpha-aminocarbonyl can be used for Edman degradation of a cyclic peptide.

[0099] "Alpha-amidocarbonyl" refers to a radical of the formula -C(=O)CR b (N(C=O)R a R a ), where each R a is independently H, alkyl, or a linker moiety, and R b is H or alkyl. In some embodiments, the alpha-amidocarbonyl is part of a cyclic moiety (e.g., a peptide), where the carbonyl is within the ring and the amid group (N(C=O)R a R a ) is outside the ring.

[0100] "Alkylamino" refers to a radical of the formula -NHR a or -NR a R a where each R a is independently an alkyl radical containing from 1 to 12 carbon atoms as defined above. Unless otherwise stated in the specification, an alkylamino group can be optionally substituted.

[0101] "Thioalkyl" refers to a radical of the formula -SR a where R a is an alkyl radical containing from 1 to 12 carbon atoms as defined above. Unless otherwise stated in the specification, a thioalkyl group can be optionally substituted.

[0102] "Aryl" refers to a hydrocarbyl ring system group comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl groups can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which can include fused ring systems or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from acanthrylene, acenaphthylene, acenaphthene, azulene, azoline, benzene, fluoranthene, fluorene, indacene, indacene, indene, indene, naphthalene, peryleno, phenanthrene, pyrene, and triphenylene. Unless otherwise stated in the specification, the term "aryl" or the prefix "ar-" (e.g., in "aralkyl") refers to aryl groups including optionally substituted aryl groups.

[0103] "Arylalkyl" refers to a radical of the formula -R b -R c where R b is an alkylene chain as defined above, and R c is one or more aryl groups as defined above, e.g., benzyl, diphenylmethyl, and the like. Unless otherwise stated in the specification, an aralkyl group can be optionally substituted.

[0104] "Cycloalkyl" or "carbocyclic" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, which can include a fused or bridged ring system having from 3 to 15 carbon atoms, preferably having from 3 to 10 carbon atoms, which is saturated or unsaturated, and which is attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, and the like. Unless otherwise stated in the specification, a cycloalkyl group can be optionally substituted.

[0105] "Cycloalkylalkyl" refers to a radical of the formula -R b R d where R bis an alkylene chain as defined above, R d is a cycloalkyl group as defined above. Unless stated otherwise in the specification, the cycloalkylalkyl group can be optionally substituted.

[0106] “Fused” refers to any ring structure described herein that is fused to an existing ring structure in a compound of the invention. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure that becomes part of the fused heterocyclyl ring or the fused heteroaryl ring can be replaced with a nitrogen atom.

[0107] “Halo” or “halogen” means bromo, chloro, fluoro, or iodo.

[0108] “Haloalkyl” means an alkyl group as defined above substituted by one or more halogen groups as defined above, for example trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated otherwise in the specification, the haloalkyl group can be optionally substituted.

[0109] “Heterocyclyl” or “heterocycle” means a stable 3- to 18-membered non-aromatic ring radical consisting of two to twelve carbon atoms and one to six heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, the heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or spiro ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl group can optionally be oxidized; the nitrogen atoms can optionally be quaternized; and the heterocyclyl group can be partially or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolizinyl, octahydroisoindolizinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4- piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, and 1,1-dioxothiomorpholinyl. Unless stated otherwise in the specification, the heterocyclyl group can be optionally substituted.

[0110] “N-heterocyclyl” means a heterocyclyl group as defined above containing at least one nitrogen, wherein the point of attachment of the heterocyclyl group to the rest of the molecule is through a nitrogen atom in the heterocyclyl group. Unless stated otherwise in the specification, the N-heterocyclyl group can be optionally substituted.

[0111] “Heterocyclylalkyl” means a radical of the formula -R b R e wherein R bIt is an alkylene chain as defined above, R e It is a heterocyclic group as defined above, and if the heterocyclic group is a nitrogen-containing heterocyclic group, it may be attached to an alkyl group on the nitrogen atom. Unless otherwise specified in the specification, the heterocyclic alkyl group may be optionally substituted.

[0112] "Heteroaromatic group" refers to a 5- to 14-membered ring system group comprising a hydrogen atom, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For the purposes of this invention, the heteroaromatic group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused ring or bridged ring systems; and the nitrogen, carbon, or sulfur atom in the heteroaromatic group may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples include, but are not limited to, nitrogen-containing heteroaromatic groups. Benzyl, acridine, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxacyclopentenyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxacycloheptenyl, 1,4-benzodioxylalkyl, benzonaphthofuranyl, benzooxazolyl, benzodioxacyclopentenyl, benzodioxinyl, benzopyranyl, benzopyranoneyl, benzofuranyl, benzofuranyl Benzyl ketone, benzothiophene (benzobenzyl thio), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazole, cenolinyl, dibenzofuranyl, dibenzobenzyl thio, furanyl, furanone, isothiazolyl, imidazoyl, indazole, indolyl, indazole, isoindolyl, dihydroindolyl, isoindololinyl, isoquinolinyl, indazinyl, isoxazolyl, naphthidyl, oxadiazolyl, 2-oxazaza The aromatic groups include alkyl, oxazolyl, ethylene oxide, 1-pyridyl oxide, 1-pyrimidinyl oxide, 1-pyrazinyl oxide, 1-pyridazinyl oxide, 1-phenyl-1H-pyrroloyl, phenazinyl, phenothiazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purine, pyrroloyl, pyrazolyl, pyridinyl, pyridinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quininecycloyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and phenylthioyl (i.e., thiophene). Unless otherwise specified in the specification, heteroaryl groups may be optionally substituted.

[0113] "N-Heteroaryl" refers to a heteroaryl group as defined above containing at least one nitrogen atom, wherein the connection point between the heteroaryl group and the remainder of the molecule is through a nitrogen atom in the heteroaryl group. Unless otherwise specified in the specification, the N-heteroaryl group may be optionally substituted.

[0114] "Heteroarylalkyl" refers to the formula -R b R f The group, wherein R bis an alkylene chain as defined above, R f is a heteroaromatic group as defined above. Unless otherwise specified in the specification, a heteroaromaticalkyl group can be optionally substituted.

[0115] The term "substituted" as used herein means any of the above groups (e.g., alkyl, alkylene, alkoxy, alkylamino, aminocarbonyl, α-aminocarbonyl, α-amido, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl) in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines groups; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also denotes where one or more hydrogen atoms in any of the above groups are replaced by a higher-order bond (e.g., a double or triple bond) to a heteroatom such as, but not limited to: an oxygen in oxo, carbonyl, carboxyl, and ester groups; and a nitrogen in imine, oxime, hydrazone, and nitrile groups. For example, "substituted" includes one or more hydrogen atoms in any of the above groups replaced by -NR g R h , -NR g C(=O)R h , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g CNR g SO2R h , -OC(=O)NR g R h , -OR g , -SR g , -SOR g , -SO2R g , -OSO2R g , -SO2OR g , =NSO2R g , and -SO2NR g R hsubstituted. "Substituted" further means that one or more hydrogen atoms in any of the above groups is replaced by a bond to an amino, cyano, hydroxy, imino, nitro, oxo, thioxo, halogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. Additionally, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents. g g g h g g h substituted. In the foregoing, R g and R h are the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further means that one or more hydrogen atoms in any of the above groups is replaced by a bond to an amino, cyano, hydroxy, imino, nitro, oxo, thioxo, halogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. Additionally, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents.

[0116] "Prodrug" means a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound of the application (i.e., a disclosed trapping agent). Thus, the term "prodrug" refers to a metabolic precursor of a compound of the application that is pharmaceutically acceptable. A prodrug can be inactive until converted to its active form in vivo. Prodrugs are typically rapidly transformed in vivo to yield the parent compound, for example, by hydrolysis in the blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7 9, 21 24 (Elsevier, Amsterdam) for a discussion of prodrugs). A review of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0117] ​​​​​​The term "prodrug" is also meant to include any covalently bonded carriers, which when administered to a mammalian subject, release the active compound of the application in vivo. Prodrugs of the compounds of the application can be prepared by modifying functional groups present on the compounds of the application in such a way that their activity is not adversely affected but their solubility or other properties are improved. Prodrugs include compounds of the application wherein a hydroxy, amino, or mercapto group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or free mercapto group, respectively, of the parent compound of the application. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols or amide derivatives of amine functional groups, among others, in the compounds of the application.

[0118] The application disclosed herein is also intended to cover all pharmaceutically acceptable disclosed capture agents that are isotopically-labeled by having one or more atoms replaced by an atom having the same atomic number, but an atomic mass or mass number that is different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be found in the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I. These radiolabeled compounds are useful in determining or measuring the effectiveness of a compound by characterizing, for example, the site or mode of action or binding affinity to a pharmacologically important site. Certain isotopically-labeled capture agents disclosed, for example, those into which radioactive isotopes are 3 H) and carbon-14 (i.e. 14 C) are particularly preferred for this purpose in view of their ease of incorporation and facile detection by standard means.

[0119] Substitution with heavier isotopes such as deuterium (i.e. 2 H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence can be preferred in some circumstances.

[0120] Substitution with positron emitting isotopes such as 11 C,18 F、 15 O and 13 N substitutions, can be used for positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically-labeled capture agents generally can be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0121] The application disclosed herein is also intended to cover in vivo metabolic products of the disclosed capture agents. Such products can result, for example, from oxidative, reductive, hydrolytic, amidation, esterification, etc. of the administered compound, primarily due to enzymatic processes. The application, therefore, includes compounds produced by a process comprising administering a compound of this application to a mammal for a time and sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound of this application to an animal, such as rat, mouse, guinea pig, monkey, or human, allowing an adequate time for metabolism to occur, and determining the presence of radioactivity in urine, blood, or other biological media isolated from the subject.

[0122] "Mammal" includes humans and domestic animals such as laboratory test animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife.

[0123] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted aryl" means that the aryl group can or can not be substituted and that the description includes aryl groups that are substituted and aryl groups that do not have a substituent.

[0124] "Pharmaceutically acceptable carrier, diluent or excipient" includes any and all solvents, dispersion media, diluents, or other fluid vehicles, adjuvants, excipients, flow regulators, sweeteners, diluting agents, preservatives, dyes / colorants, flavor enhancers, surface active or wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, or emulsifying agents, which are nontoxic to the subjects under treatment and are acceptable for use in humans or household animals.

[0125] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0126] "Pharmaceutically acceptable acid addition salt" refers to those salts of the free base which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1 -hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0127] "Pharmaceutically acceptable base addition salt" refers to those salts of the free acids which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of inorganic or organic bases to free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenylglycine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0128] The compounds of the present application (trapping agents) or pharmaceutically acceptable salts thereof can contain one or more asymmetric centers and thus can exist in enantiomeric or diastereomeric forms. The present application is meant to include all such possible isomers, as well as, their racemic and optically pure forms. Optical active (+) and (-), (R) and (S), or (D) and (L) isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques (e.g., chromatography and fractional crystallization). Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor (e.g., a chiral high pressure liquid chromatography (HPLC) resolved form), and chiral chromatography of mixtures. When the compounds described herein contain olefinic double bonds, other geometric isomers are possible. Unless otherwise specified, all geometric isomers are intended to be encompassed by the description and claims of this application. Similarly, all tautomers are also intended to be encompassed by the description and claims of this application. (D)-amino acids (also referred to as D-amino acids) are referred to herein in lower case (e.g., D-valine is referred to as "v"), while (L)-amino acids (also referred to herein as L-amino acids) are referred to in upper case (e.g., L-valine or valine is referred to as "V"). Glycine is achiral and is referred to as "G".

[0129] "stereoisomers" are compounds which have the same molecular formula but different spatial arrangements of atoms. Accordingly, stereoisomers have the same connective bond sequence but differ in stereochemical configuration. The term "stereoisomers" includes enantiomeric and diastereomeric forms of compounds. The term "enantiomers" refers to two stereoisomers of a compound, which are non- superimposable mirror images of each other. The term "diastereomers" refers to stereoisomers having different spatial arrangements of atoms but which are not mirror images of each other.

[0130] "tautomers" refers to a proton shift from one atom to another atom of the same molecule. The present application includes any tautomers of the compounds described.

[0131] Crystallization typically produces solvates of the compounds of the present application. As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a compound of the present application with one or more molecules of solvent. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present application can exist in a hydrate form, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the like, as well as corresponding solvated forms. The compounds of the present application can be true solvates, while in other cases, the compounds of the present application can merely retain adventitious solvent or be a mixture of water and some adventitious solvent.

[0132] The term "capture agent" as used herein refers to a composition comprising two or more target binding moieties, the composition specifically binds to a target protein through those target binding moieties. Each target binding moiety, alone or in combination with other target binding moieties, displays a binding affinity for a target protein. In certain embodiments, each target binding moiety binds to a target protein via one or more non-covalent interactions, including, for example, hydrogen bonds, hydrophobic interactions, and van der Waals interactions. A capture agent can comprise one or more organic molecules, including, for example, polypeptides, peptides, polynucleotides, and other non-polymeric molecules. In some aspects, the capture agent is a protein catalyzed capture agent (PCC).

[0133] The term "epitope" as used herein refers to a unique molecular surface of a protein, e.g., IL-17F. Typically, an epitope is a polypeptide that can function on its own as a limited sequence of 10 to 40 amino acids.

[0134] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to an amino acid sequence comprising a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0135] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, such as serine, glycine, proline, etc., as well as those modified after synthesis, such as hydroxyproline, gamma-carboxyglutamate, O-phosphoserine, etc., and their isomers. The term "amino acid analog" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl, an amino, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The term "amino acid mimetic" refers to chemical compounds that have a structure different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0136] The term "non-natural amino acid" as used herein refers to an amino acid that differs in its side chain functionality from the 20 naturally occurring amino acids (alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan, phenylalanine). The non-natural amino acid can be a close structural mimic of one of the twenty natural amino acids, or it can introduce entirely new functionality and chemical reactivity, or it can be as hydrophobic as a natural amino acid. The non-natural amino acid can replace an existing amino acid in a protein (substitution), or it can be an addition to the wild type sequence (insertion). Incorporation of non-natural amino acids can be achieved by known chemical methods, including solid phase peptide synthesis or native chemical ligation, or by biological methods.

[0137] The terms "specifically binds," "selectively binds," "selectively binds," or "binds specifically" as used herein means that the capture agent binds to an epitope on a predetermined antigen. Typically, the capture agent binds with an affinity of about less than 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or even lower affinity (K D ) to the predetermined antigen.

[0138] The term "K D " as used herein refers to the dissociation equilibrium constant of a particular capture agent-antigen interaction. Typically, the capture agents of the present application bind to the predetermined antigen with a dissociation equilibrium constant of less than about 10 -6 M, 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, or 10 -10 M or even lower (K D ). For example, the dissociation equilibrium constant is determined using surface plasmon resonance (SPR) technology in a Biacore instrument using the antigen as the ligand and the capture agent as the analyte, the capture agent binding to the predetermined antigen with an affinity corresponding to KD that is at least 10-fold lower, for example, at least 100-fold lower, for example, at least 1000-fold lower, for example, at least 10,000-fold lower, for example, at least 100,000-fold lower than the affinity with which the capture agent binds to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount by which the affinity is lower depends on the K D of the capture agent, such that when the K D of the capture agent is very low (i.e., the capture agent is highly specific), the amount by which the affinity of the antigen is lower than the affinity of a non-specific antigen can be at least 10,000-fold.

[0139] As used in this article, the term "k" d (sec) -1 () refers to the dissociation rate constant of a specific trapping agent-antigen interaction. This value is also known as k. off value.

[0140] As used in this article, the term "k" a (M) -1 ×sec -1 () refers to the binding rate constant of a specific capture agent-antigen interaction.

[0141] As used in this article, the term "K" D "(M) refers to the dissociation equilibrium constant of a specific capture agent-antigen interaction."

[0142] As used in this article, the term "K" A (M) -1 ) refers to the binding equilibrium constant of a specific capture agent-antigen interaction, and is expressed by k a Divide by k d And thus obtained.

[0143] "Pharmaceutical composition" refers to the compounds of this invention and formulations commonly accepted in the art for delivering bioactive compounds to mammals, such as humans. Such media include all pharmaceutically acceptable carriers, diluents, or excipients.

[0144] As used in this article, the term "symptom" generally refers to a disease, event, or change in health status. A change in health status may be associated with a specific disease or event, in which case the change may occur concurrently with or before the disease or event. When a change in health status precedes a disease or event, it can serve as a predictor of that disease or event. For example, a change in health status might be a change in the expression level of a specific gene associated with a disease or event. Alternatively, a change in health status may be unrelated to a specific disease or event.

[0145] As used in this article, the terms “treat,” “treating,” or “treatment” generally refer to preventing a condition or event, slowing the onset or progression of a condition or delaying the occurrence of an event, reducing the risk of developing a condition or experiencing an event, preventing or delaying the development of symptoms associated with a condition or event, reducing or terminating symptoms associated with a condition or event, causing a condition to completely or partially subside, mitigating the severity of a condition or event, or some combination thereof.

[0146] An "effective amount" or "therapeutically effective amount" as used herein refers to the amount and period of time required to effect the desired treatment. The therapeutically effective amount of the disclosed capture agent can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the capture agent to elicit a desired response in the individual.

[0147] The term "stable" as used herein with respect to a capture agent or a pharmaceutical formulation of a capture agent protein catalyzed, refers to an agent or formulation that retains structural and functional integrity for a sufficient period of time to be utilized in the methods described herein.

[0148] The term "synthetic" as used herein with respect to a protein catalyzed capture agent or a capture agent refers to a capture agent produced by chemical means rather than biological means.

[0149] Unless otherwise indicated, sequence identity / similarity values provided herein were obtained using the BLAST 2.0 suite of programs employing default parameters (Altschul et al., (1997) Nucleic Acids Res. 25:3389-402).

[0150] As understood by one of ordinary skill in the art, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequence, which can be homopolymer tracts, short-period repetitive sequences, or regions rich in one or more amino acids. Such low complexity regions can align between unrelated proteins even if other regions of the proteins are completely dissimilar. A number of low complexity filters can be employed to reduce such low complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17: 149-63) and XNU (Claverie and States, (1993) Comput. Chem. 17: 191-201) low complexity filters can be used, alone or in combination.

[0151] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When referring to a percentage of sequence identity, 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 having 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 can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making this adjustment are well known in the art. Typically, this involves scoring a conservative substitution as a partial rather than complete 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 one. For example, according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4: 11-17, conservative substitutions are calculated as follows: Scores for non-conservative substitutions are set to zero.

[0152] As used herein, "percent sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can 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 percent sequence identity 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 comparison window and multiplying the result by 100 to yield the percent sequence identity.

[0153] The term "substantial identity" or "substantially identical" in the context of polynucleotide sequences, refers to a sequence having 50 to 100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%, when compared and aligned for maximum correspondence against a reference sequence using one of the comparison programs described, employing standard parameters. The skilled artisan will recognize that these values can be adjusted as appropriate by consideration of codon degeneracy, amino acid similarity, reading frame positioning, and the like, to determine corresponding identity of proteins encoded by two nucleotide sequences. Substantial identity of amino acid sequences for these purposes normally means 55 to 100%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95% sequence identity.

[0154] In certain embodiments, the term "IL-17F" as used herein refers to human IL-17F. In some embodiments, IL-17 comprises the following amino acid sequence or an amino acid sequence substantially identical thereto.

[0155] 1 MTVKTLHGPA MVKYLLLSIL GLAFLSEAAA RKIPKVGHTF FQKPESCPPV PGGSMKLDIG

[0156] 61 IINENQRVSM SRNIESRSTS PWNYTVTWDP NRYPSEVVQA QCRNLGCINA QGKEDISMNS

[0157] 121 VPIQQETLVV RRKHQGCSVS FQLEKVLVTV GCTCVTPVIH RVQ (SEQ ID NO: 31)

[0158] In other embodiments, IL-17F is the protein encoded by the gene denoted by Entrez Gene ID No. 112744.

[0159] Development of IL-17F capture agents

[0160] Antibodies are currently the default detection agents for diagnostic platforms. However, antibodies have several drawbacks, including high cost, poor stability, and in many cases lack of proper characterization and high specificity. An ideal replacement for diagnostic assays should be synthetic, stable to a range of thermal and chemical conditions, and display high affinity and specificity for the target of interest.

[0161] High quality monoclonal antibodies have low nanomolar affinities and high target specificities. Interestingly, structural and genetic analyses of the antigen recognition surface suggest that most of the molecular diversity of the variable loops is contained in a single highly variable loop (CDR-H3). In humans, this loop ranges in size from 1 to 35 residues (average 15), can adopt a wide range of structural conformations, and is the primary cause of interaction with antigen. The other five loops are significantly less diverse and adopt only a few conformations. This suggests that a carefully selected "anchor" peptide can dominate the pattern and strength of the interaction between a capture agent and its target protein. It also suggests that the other polypeptide components, while providing only modest contributions to the total interaction energy, can provide important scaffolding features and specificity elements.

[0162] In situ click chemistry is a technique that separates a small molecule enzyme inhibitor into two parts and then expands each part into a small library containing an ethyne functional group in one part and an azido group in the other. The enzyme itself then assembles the "best fit" inhibitor from these library components by selectively promoting the 1,3-dipolar cycloaddition between the ethyne and azido groups to form a triazole linkage (the "click" reaction). The protein effectively acts as a very selective variant of the Cu(I) catalyst that is normally used for this coupling. The enzyme only promotes the click reaction between these library components that bind to the protein component in the correct orientation. The resulting inhibitor can exhibit far superior affinity characteristics relative to the starting inhibitor that was based on the two libraries.

[0163] Sequential in situ click chemistry extends the in situ click chemistry concept to enable the discovery of multi-ligand capture agents (see: USSN 20100009896, incorporated herein by reference). This process was previously used to generate a tri-ligand capture agent for the model protein carbonic anhydrase II (CAII). Sequential in situ click chemistry has several advantages. First, the structural information about the protein target is replaced with the ability to sample very large chemical space to identify the ligand components of the capture agent. For example, the starting ligands can be identified by screening a protein against a large (> 106 elements) one-bead-one-compound (OBOC) peptide library, where the peptides themselves can be composed of natural, unnatural, and / or artificial amino acids. The resulting anchor ligands are then used in an in situ click screen, again utilizing a large OBOC library to identify the bi-ligand binders. A second advantage is that the process can be repeated such that the bi-ligands are used as anchors to identify tri-ligands, and so on. The final capture agent can then be scaled up using relatively simple and mostly automated chemical reactions, and can be developed with a tag such as a biotin group as an inherent part of its structure. This approach allows the exploration of branched, cyclic, and linear capture agent architectures. Although many strategies for the directed assembly of multi-ligands to proteins have been described, most require detailed structural information about the target to guide the screening strategy, and most methods (e.g., the original in situ click method) are optimized for low diversity small molecule libraries.

[0164] Embodiments of the present invention further generalize the in situ click application to easily find anchor ligands with in situ click. In previous methods, a known binder was required to start the ligands. The present method provides a mechanism to find anchor ligands from scratch.

[0165] As described herein, a bi-ligand capture agent that specifically binds IL-17F was synthesized using an iterative in situ click chemistry approach. This in situ click chemistry approach includes two steps. First, two "anchor" ligands that bind IL-17F at different but relatively close sites were discovered. Second, an appropriate size linker that binds both of these ligands was discovered, resulting in a capture agent with higher affinity for IL-17F.

[0166] Capture agents produced by the methods disclosed herein were found to exhibit binding affinity for IL-17F. The capture agents were shown to function as both a capture and detection agent in an ELISA assay, and to effectively immunoprecipitate IL-17F.

[0167] IL-17F capture agents

[0168] In one aspect, provided herein are stable synthetic capture agents that specifically bind IL-17F, wherein the capture agent comprises two or more "anchor" ligands (also referred to herein simply as "ligands") and linkers, wherein the ligands selectively bind IL-17F.

[0169] In certain embodiments, the ligands comprise one or more polypeptides or peptides. In some of these embodiments, the target binding moiety comprises one or more peptides comprising D-amino acids, L-amino acids, and / or amino acids substituted with a functional group selected from the group consisting of substituted and unsubstituted alkyl groups, substituted and unsubstituted azido groups, substituted and unsubstituted alkynyl groups, substituted and unsubstituted biotinyl groups, substituted and unsubstituted azallyl groups, substituted and unsubstituted polyethylene glycol groups, and substituted and unsubstituted 1,2,3-triazole groups.

[0170] In certain embodiments, the ligands are interconnected via covalent bonds by the linkers. In some of these embodiments, the ligands and linkers are interconnected via amide bonds or 1,4-disubstituted-1,2,3-triazole bonds as shown below:

[0171]

[0172] In those embodiments in which the ligands and linkers are interconnected via 1,4-disubstituted-1,2,3-triazole bonds, the 1,4-disubstituted-1,2,3-triazole bonds can be formed by Cu-catalyzed azide / alkyne cycloaddition (CuAAC).

[0173] In certain embodiments, the ligands and linkers are interconnected by Tz4 bonds having the following structure:

[0174]

[0175] In certain embodiments, the ligands and linkers are interconnected by Tz5 bonds having the following structure:

[0176]

[0177] In those embodiments in which one or more ligands and linkers are interconnected via amide bonds, the amide bonds can be formed by coupling a carboxylic acid group and an amine group in the presence of a coupling agent (e.g., O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N-hydroxy-7-azabenzotriazole (HOAt), or diisopropylethylamine (DIEA) in DMF).

[0178] In certain embodiments, the capture agents provided herein are stable over a range of reaction conditions and / or storage times. A "stable" capture agent, as used herein, retains the ability to specifically bind to a target protein. In certain embodiments, the capture agents provided herein are more stable than an antibody that binds to the same target protein under one or more reaction and / or storage conditions. For example, in certain embodiments, the capture agents provided herein are more resistant to proteolytic degradation than an antibody that binds to the same target protein.

[0179] In certain embodiments, the capture agents provided herein have a shelf life of greater than six months, meaning that they are stable over a period of more than six months of storage. In some of these embodiments, the shelf life of the capture agent is one or more years, two or more years, or more than three years. In some of these embodiments, the capture agent is stored as a lyophilized powder. In certain embodiments, the shelf life of the capture agents provided herein is longer than the shelf life of an antibody that binds to the same target protein.

[0180] In certain embodiments, the capture agents provided herein are stable over a temperature range of about -80 °C to about 120 °C. In certain of these embodiments, the capture agents are stable over a temperature range of -80 °C to -40 °C, -40 to -20 °C, -20 to 0 °C, 0 to 20 °C, 20 to 40 °C, 40 to 60 °C, 60 to 80 °C, and / or 80 to 120 °C. In certain embodiments, the capture agents provided herein are more stable than an antibody that binds to the same target protein over a wider temperature range, and / or remain stable for a longer period of time at a particular temperature than an antibody that binds to the same target protein.

[0181] In certain embodiments, the capture agents provided herein are stable over a pH range of about 3.0 to about 8.0. In certain embodiments, the range is about 4.0 to about 7.0. In certain embodiments, the range is about 7.0 to about 8.0.

[0182] In certain embodiments, the capture agents provided herein are stable in human serum for more than 12 hours. In certain of these embodiments, the capture agents are stable in human serum for more than 18 hours, more than 24 hours, more than 36 hours, or more than 48 hours. In certain embodiments, the capture agents provided herein are stable in human serum for a longer period of time than an antibody that binds to the same target protein. In certain embodiments, the capture agents are stable in powder form for two months at a temperature of about 60 °C.

[0183] In certain embodiments, the capture agents provided herein can comprise one or more detection tags, including, for example, biotin, copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Cu-DOTA), 64 Cu DOTA,68 Ga DOTA, 18 F, 64 Cu, 68 Ga, 89 Zr, 124 I, 86 Y, 94m Tc, 110m In, 11 C, 76 Br, 123 I, 131 I, 67 Ga, 111 In and 99m Tc or other radiolabeling products, which can include gamma emitters, proton emitters, positron emitters, tritium, or other detectable cover tags (i.e., gadolinium), etc. In particular embodiments, the detection tag is 18 F. In certain embodiments, the capture agents provided herein can be modified for use as imaging agents. The imaging agents can be used as diagnostic agents.

[0184] In certain embodiments, the capture agents provided herein can be modified to obtain a desired chemical or biological activity. Examples of desired chemical or biological activities include, but are not limited to, improved solubility, stability, bioavailability, detectability, or reactivity. Examples of particular modifications that can be introduced to the capture agents include, but are not limited to, cyclization of the capture agents by formation of disulfide bonds; modification of the capture agents with other functional groups or molecules. Similarly, the capture agents can be synthesized to bind to non-canonical or non-biological epitopes on proteins, thereby increasing their versatility. In certain embodiments, the capture agents can be modified by modifying the synthetic blocks of the target binding moieties prior to the coupling reaction.

[0185] Methods of making / screening capture agents

[0186] Methods for screening target binding moieties and / or preparing capture agents comprising these target binding moieties are also found in International Publication Nos. WO2012 / 106671, WO2013 / 033561, WO2013 / 009869, and WO2014 / 074907, each incorporated by reference herein in its entirety.

[0187] In some implementations, two separately recognized ligands that bind to two different regions of the same protein (target) are chemically linked together to form a biligand. By optimizing the linker between the two ligands, the biligand formed by the ligand and linker can exhibit a binding affinity far superior to that of either ligand alone. This enhanced binding effect is called binding cooperativity. For an ideal cooperative conjugate, the thermodynamic binding energies of the individual ligands to the target will sum to produce the binding energy of the linked biligand. This means that the binding affinity constant (K0) of the linked biligand is... D ) will be the product of the binding affinity of each ligand (i.e., K) D =K D1 ×K D2 (where subscripts 1 and 2 refer to the two ligands). In practice, fully concerted binding is rarely achieved, if any. Therefore, a comparison between the properties of linked biligands and those of fully concerted binders can provide a measure of how optimally the two ligands are linked.

[0188] If the protein target has a known and well-defined tertiary (folded) structure, a key aspect of this targeting approach involves strategies for identifying ligands that bind to preferred regions of the protein, followed by methods for identifying optimized linkers. If the protein does not have a well-defined tertiary structure, this disclosure describes strategies aimed at still achieving a significant measure of co-binding from both ligands.

[0189] Figure 7 The starting point for developing a set of PCC conjugates targeting protein targets (11) is described. Initial target identification identifies one or more PCCs (12) that bind to one epitope on the protein target, and one or more different PCCs (13) that bind to a second epitope. Other PCCs that bind to epitopes, such as third and fourth epitopes, may also be useful. The epitope-targeted PCC approach teaches that this can be achieved by screening peptide libraries targeting synthetic epitopes (SynEps), such as… Figure 7 As shown in (14, 15). SynEp is a polypeptide with a naturally occurring target epitope sequence, differing in that one site contains an artificial amino acid presenting an azide or acetylene chemical group (16) (referred to as a click handle). SynEp is further modified to include a detection handle, such as a biotin group, at the N-terminus or C-terminus (17). The screening process can be performed using any process disclosed herein or known in the art. By screening, at least one unique peptide conjugate for each of at least two epitopes on the target can be identified. These peptide conjugates are validated by binding assays against the intact protein target (11) and against SynEps. For these binding assays, SynEps are prepared using naturally occurring residues instead of the click handle (16).

[0190] Ideally, the different regions of the target protein to which the different ligands bind will be relatively close (a few nanometers or less) in the tertiary protein structure. Even a single SynEp, screen can yield PCCs that bind to two different sites. In Figure 8 In the middle, the region representing the epitope of interest (12) is highlighted against the dim background of the full-length protein (11). The amino acid residues that are replaced with click handles in the SynEp structure are indicated with asterisks (22). In the SynEp screen step, PCCs that bind to the N-terminal side (23) or the C-terminal side (24) of the epitope can be identified.

[0191] Once the PCCs that target the epitope are identified, there are several ways to select a linker.

[0192] In a first embodiment, if the folded structure of the protein is known, and if the PCCs bind to the folded structure, then this information, together with the knowledge of which PCCs bind to which epitopes, can be used to estimate the optimal linker length. This is shown in Figure 9 The figure shows one PCC (31) that binds to the N side of one epitope (12) and a second PCC (32) that binds to the C side of a second epitope (13). Analysis of this binding arrangement together with the structure of the protein from, for example, the Protein Data Bank, allows an estimate of the optimal linker (33) length. Such an estimate can reduce the number of candidate linkers to be selected to a very small number. In one example, this estimated length can be used to select one or two polyethylene glycol oligomers of matching length for testing. The best linker (34) is the one that brings the affinity of the bi-ligand closest to that of a perfectly cooperative binder.

[0193] In a second embodiment, if the folded structure of the protein is not known, or if the protein simply does not have a well-defined folded structure, then as much information as possible is used to determine the composition of a library of candidate linker molecules. This library is then screened to identify the optimal linker.

[0194] In a third embodiment, if the folded structure of the protein is not known, or if the protein simply does not have a well-defined folded structure, then the existing knowledge about the protein is used to simply select a linker to add the two PCCs. Even if this does not identify an optimized, perfectly cooperative binder, the linked bi-ligand will almost certainly outperform either of the two single ligands due to the cooperative effect.

[0195] In vitro

[0196] To detect IL-17F in a solution, the capture agent of the application is detectably labeled, then contacted with the solution, and then the formation of a complex between the capture agent and the IL-17F target can be detected. As an example, a fluorescently labeled capture agent can be used in an in vitro IL-17F detection assay, in which the capture agent is added to a solution of IL-17F to be tested under conditions that allow binding to occur. The complex between the fluorescently labeled capture agent and the IL-17F target can be detected and quantified by, for example, measuring the increase in fluorescence polarization of the complexed peptide relative to the fluorescence polarization of the free peptide.

[0197] Alternatively, a sandwich-type "ELISA" assay can be used, in which the capture agent is immobilized on a solid support such as a plastic tube or well, then a solution suspected of containing IL-17F is contacted with the immobilized binding moiety, unbound material is washed away, and the complexed polypeptide is detected using a suitable detection reagent for recognizing IL-17F.

[0198] To detect or purify soluble IL-17F in a solution, the capture agent of the application can be immobilized on a solid substrate such as a chromatography support or other matrix material, and then the immobilized binding moiety can be loaded or contacted with the solution under conditions suitable for the formation of a capture agent / IL-17F complex. The unbound portion of the solution can be removed, and the complex can be detected, for example, using an anti-IL-17F antibody or an anti-binding polypeptide antibody, or the IL-17F can be released from the binding moiety under appropriate elution conditions.

[0199] In vivo diagnostic imaging

[0200] A particularly preferred use of the capture agents of the application is for producing a visually readable image of IL-17F or IL-17F-expressing cells in a biological fluid, for example such as in human serum. The IL-17F capture agents disclosed herein can be converted into imaging reagents by conjugating the capture agent to a label suitable for diagnostic detection. Preferably, the capture agent, which exhibits much greater specificity for IL-17F than for other serum proteins, is conjugated or linked to a tag suitable for the detection method used. For example, the capture agent can be conjugated with or without a linker to a paramagnetic chelate suitable for magnetic resonance imaging (MRI), the capture agent can be conjugated to a radioactive label suitable for x-ray, positron emission tomography (PET), single photon emission computed tomography (SPECT), or scintigraphic imaging (including chelators for radioactive metals), the capture agent can be conjugated to an ultrasound contrast agent (e.g., stabilized microbubbles, microballoons, microspheres, or what have been called gas-filled "liposomes") suitable for ultrasound detection, or the capture agent can be conjugated to an optical imaging dye.

[0201] In another embodiment, rather than directly labeling the capture agent with a detectable label or radiotherapeutic construct, one or more peptides or constructs of the application can be conjugated to, for example, avidin, biotin, or an antibody or antibody fragment that binds to a detectable label or radiotherapeutic agent.

[0202] A, magnetic resonance imaging

[0203] The IL-17F capture agents described herein can advantageously be conjugated to paramagnetic metal chelates to form contrast agents for MRI.

[0204] Preferred paramagnetic metal ions have atomic numbers of 21 to 29, 42, 44, or 57 to 83. This includes ions of transition metals or lanthanides that have one, more preferably five or more, unpaired electrons and a magnetic moment of at least 1.7 Bohr magnetons. Preferred paramagnetic metals include, but are not limited to, chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), europium (III), and ytterbium (III), chromium (III), iron (III), and gadolinium (III). The trivalent cation Gd 3+ Gd(III) is particularly preferred for MRI contrast agents due to its high relaxivity and low toxicity, with the further advantage that it exists in only one biologically accessible oxidation state, which minimizes unwanted metallo-metabolic breakdown in the patient. Another useful metal is Cr 3+ , which is less expensive. Gd(III) chelates have been used in clinical and radiological MR applications since 1988, and about 30% of MRI examinations now employ gadolinium-based contrast agents.

[0205] Paramagnetic metal chelators are molecules with one or more polar groups that act as ligands for paramagnetic metals and complex with paramagnetic metals. Suitable chelators are known in the art and include acids with methylenephosphonic acid groups, carbohydroxamine acid groups, carboxyethylidene groups, or carboxymethylene groups. Examples of chelators include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA), 1-substituted-1,4,7- tricarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Additional chelating ligands are ethylenylbis-(2-hydroxy-phenylglycine) (EHPG) and its derivatives, including 5-CI-EHPG, 5-Br-EHPG, 5-methyl-EHPG, 5-t-butyl-EHPG, and 5-sec-butyl-EHPG; benzo-diethylenetriaminepentaacetic acid (benzo-DTPA) and its derivatives, including bis-benzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA; bis-2(hydroxybenzyl)-ethylenedi-amine diacetic acid (HBED) and its derivatives; the class of macrocyclic compounds comprising at least 3, more preferably at least 6, carbon atoms, and at least two heteroatoms (O and / or N), which macrocyclic compounds can consist of one ring, or of two or three rings connected together at heterocyclic elements, such as benzo-DOTA, bis-benzo-DOTA, and benzo-NOTA (where NOTA is 1,4,7-triazacyclononane-N,N',N"-triacetic acid), benzo-TETA, benzo-DOTMA (where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid)), and benzo-TETMA (where TETMA is 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyltetraacetic acid)); derivatives of 1,3- propylene-diamine tetraacetic acid (PDTA) and triethylenetetraamine hexaacetic acid (TTNA); derivatives of 1,5,10-N,N',N"-tris(2,3-dihydroxybenzoyl)-tricresol (LICAM); and 1,3,5-N,N',N"-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). The preferred chelator for use in the present application is DTPA, and the use of DO3A is particularly preferred.Representative chelators and chelating groups contemplated by the present application are exemplified in WO 98 / 18496, WO 86 / 06605, WO 91 / 03200, WO 95 / 28179, WO 96 / 23526, WO 97 / 36619, PCT / US98 / 01473, PCT / US98 / 20182, U.S. Patent No. 4,899,755, U.S. Patent No. 5,474,756, U.S. Patent No. 5,846,519, and U.S. Patent No. 6,143,274, all of which are incorporated herein by reference.

[0206] According to the present application, the chelator of the MRI contrast agent is coupled to the IL-17F capture agent. The position of the chelator should be chosen so as not to interfere with the binding affinity or specificity of the IL-17F capture agent. The chelator can also be attached anywhere on the capture agent.

[0207] Generally, the IL-17F capture agent can be directly or covalently bound to the metal chelator (or other detectable label), or it can be coupled or conjugated to the metal chelator using a linker, which can be, but is not limited to, an amide, urea, acetal, ketal, diester, carbonyl, carbamate, thiourea, sulfone, thioester, ester, ether, disulfide, lactone, imine, phosphoryl, or phosphodiester linkage; a substituted or unsubstituted saturated or unsaturated alkyl chain; a straight, branched, or cyclic chain of single amino acids or different amino acids (e.g., an extension of the N- or C-terminus of the IL-17F binding moiety); a derivatized or non-derivatized polyethylene glycol (PEG), polyoxyethylene, or polyvinylpyrrolidine chain; a substituted or unsubstituted polyamide chain; a derivatized or non-derivatized polyamine, polyester, polyethyleneimine, polyacrylate, polyvinyl alcohol, polyglycerol, or oligosaccharide (e.g., dextran) chain; an alternating block copolymer; malonic acid, succinic acid, glutaric acid, adipic acid, and pimelic acid; caproic acid; simple diamines and diols; any other linker disclosed herein; or any other simple polymeric linker known in the art (see, e.g., WO 98 / 18497 and WO 98 / 18496). Preferably, the molecular weight of the linker can be tightly controlled. The molecular weight can range from less than 100 to greater than 1000. Preferably, the molecular weight of the linker is less than 100. Additionally, it can be desirable to utilize a biodegradable linker in vivo to provide an efficient excretion pathway for the imaging reagent of the present application. Such biodegradable functional groups can include ester, diester, amide, phosphate, ether, acetal, and ketal functional groups, depending on their position within the linker.

[0208] Typically, these linkers can be employed to conjugate the metal chelate and the IL-17F capture agent using known methods (WO 95 / 28967, WO 98 / 18496, WO 98 / 18497 and the discussion therein). The IL-17F binding moiety can be attached via an amide bond at the N-terminus or C-terminus to, for example, the metal coordinating backbone nitrogen of the metal chelate or to the acetate arm of the metal chelate itself. The present disclosure contemplates attachment of the chelate at any position, provided that the metal chelate retains the ability to tightly bind the metal to minimize toxicity.

[0209] MRI contrast agents prepared according to the disclosure herein can be used in the same manner as conventional MRI contrast agents. Certain MR techniques and pulse sequences can be preferred to enhance the contrast of the site from background blood and tissue. These techniques include, but are not limited to, black-blood angiographic sequences such as the fast spin echo sequence (Alexander, A. et al., 1998. Magn. Reson. Med., 40:298-310) and the flow-spoiled gradient echo sequence (Edelman, R. et al., 1990. Radiology, 177:45-50) which attempt to darken the blood. These methods also include flow-independent techniques that enhance the contrast difference such as inversion recovery prepared sequences or saturation recovery prepared sequences which will increase the contrast between IL-17F expressing tissue and background tissue. Finally, magnetization transfer preparations can also improve the contrast with these agents (Goodrich, K. et al., 1996. Invest. Radiol, 31 :323-32).

[0210] The labeled reagent is administered to the patient in the form of an injectable composition. The method of administering the MRI contrast agent is preferably parenteral, meaning intravenous, intraarterial, intrathecal, interstitial, or intracranial administration. For imaging of IL-17F expressing tissue (e.g., a tumor), intravenous administration or intraarterial administration is preferred. For MRI, the subject is expected to receive a dose of the contrast agent sufficient to enhance the MR signal of the IL-17F expressing site by at least 10%. After injection of the IL-17F capture agent containing the MRI reagent, the patient is scanned in an MRI machine to determine the location of any IL-17F expressing site. In a therapeutic setting, once the IL-17F expressing site (e.g., fluid or tissue) is identified, an anti-cancer agent (e.g., an inhibitor of IL-17F) can be administered immediately, if desired, and the patient can be subsequently scanned to visualize the viral load.

[0211] B, Nuclear Imaging (Radionuclide Imaging) and Radiotherapy

[0212] The IL-17F capture agents of the present application can be conjugated to a radionuclide reporter molecule suitable for scintigraphic, SPECT or PET imaging and / or to a radionuclide suitable for radiotherapy. Constructs in which the IL-17F capture agent is conjugated to both a radionuclide chelator for diagnostic imaging and a chelator for radiotherapy are within the scope of the present application.

[0213] For use as a PET agent, the disclosed capture agents can be complexed with one of a variety of positron emitting metal ions, such as 51 Mn, 52 Fe, 60 Cu, 68 Ga, 72 As, 94 mTc or 110 In. The binding moieties of the present application can also be labeled by halogenation with a radionuclide such as 18 F, 124 I, 125 I, 131 I, 123 I, 77 Br and 76 Br. Preferred metal radionuclides for scintigraphic imaging or radiotherapy include 99m Tc, 51 Cr, 67 Ga, 68 Ga, 47 Sc, 51 Cr, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 90 Y, 88 Y, 153 Sm, 166 Ho, 165 Dy, 166 Dy, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 117 mSn, 149Pm, 161 Tb, 177 Lu, 198 Au and 199 Au. The metal selected will be determined according to the desired therapeutic or diagnostic application. For diagnostic purposes, preferred radionuclides include 64 Cu, 67 Ga, 68 Ga, 99m Tc and 111 In. For therapeutic purposes, preferred radionuclides include 64 Cu, 90 Y, 105 Rh, 111 In, 117 mSn, 149 Pm, 153 Sm, 161 Tb, 166 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Ln, 186 / 188 Re and 199 Au. 99m Tc is very useful for diagnostic applications due to its low cost, availability, imaging properties, and high specific activity. 99m The nuclear and radioactive properties of Tc make this isotope an ideal scintigraphic imaging agent. The isotope has a single photon energy of 140 keV and a radioactive half-life of about 6 hours, and is readily available from 99 Mo- 99m Tc generators. 18 F, 4-[ 18 F]fluorobenzaldehyde ( 18 FB), Al[ 18 F]-NOTA, 68 Ga-DOTA and 68 Ga-NOTA are typical radionuclides for conjugation to IL-17F capture agents for diagnostic imaging.

[0214] Metal radionuclides can be chelated by, for example, straight chain, macrocyclic, terpyridine and N3S, N2S2or N4chelators (see also U.S. Patent No. 5,367,080, U.S. Patent No. 5,364,613, U.S. Patent No. 5,021,556, U.S. Patent No. 5,075,099, U.S. Patent No. 5,886,142), and other chelators known in the art, including but not limited to HYNIC, DTPA, EDTA, DOTA, DO3A, TETA, NOTA and diaminobis thiols (BAT) chelators (see also U.S. Patent No. 5,720,934). For example, N4chelators are described in U.S. Patent No. 6,143,274; U.S. Patent No. 6,093,382; U.S. Patent No. 5,608,110; U.S. Patent No. 5,665,329; U.S. Patent No. 5,656,254; and U.S. Patent No. 5,688,487. Certain N3S chelators are described in PCT / CA94 / 00395, PCT / CA94 / 00479, PCT / CA95 / 00249, and U.S. Patent No. 5,662,885; U.S. Patent No. 5,976,495; and U.S. Patent No. 5,780,006. Chelators can also include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3) containing N3S, N2S2systems such as MAMA (monoamide monoamino dithiol), DADS (N2S diamine dithiol), CODADS, and the like. These ligand systems and a variety of other systems are described in, for example, Liu, S and Edwards, D., 1999. Chem. Rev., 99:2235-2268 and references therein.

[0215] Chelators can also include complexes containing ligand atoms that do not present themselves in a tetradentate array to the metal. These include the boronic acid adducts of technetium and rhenium dioximes, described for example in U.S. Patent No. 5,183,653; U.S. Patent No. 5,387,409; and U.S. Patent No. 5,118,797, the disclosures of which are incorporated by reference herein in their entireties.

[0216] As previously described, the chelator can be covalently linked directly to the IL-17F capture agent via a linker, and then directly labeled with a selected radiometal (see WO 98 / 52618, U.S. Patent No. 5,879,658 and U.S. Patent No. 5,849,261).

[0217] Comprising 18 F, 4-[ 18 F]fluorobenzaldehyde 18 FB), Al[ 18 F]-NOTA, 68 Ga-DOTA and68 IL-17F capture agents of Ga-NOTA are preferred for diagnostic imaging. Radio-technetium complexes can also be used for diagnostic imaging, and radio-rhenium complexes are particularly useful for radiotherapy. In forming a radio-technetium complex with a reagent of the present application, the technetium complex, preferably 99m Tc pertechnetate is reacted with the reagent in the presence of a reducing agent. Preferred reducing agents are dithionite, stannous ion, and ferrous ion; the most preferred reducing agent is stannous chloride. The method for preparing such a complex is conveniently provided in kit form, which includes a sealed vial containing a predetermined amount of a reagent of the present application to be labeled and a sufficient quantity of a reducing agent for labeling with 99m Tc to label the reagent. Alternatively, the complex can be formed by reacting a peptide of the present application conjugated to an appropriate chelating agent with a preformed unstable technetium complex and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art. For example, a transfer ligand such as tartrate, citrate, gluconate, or mannitol can be employed to form the unstable complex. Useful 99m Tc pertechnetate includes alkali metal salts such as sodium salts, or ammonium or lower alkylammonium salts.

[0218] The preparation of a complex of the present application wherein the metal is radio-rhenium can be accomplished using a rhenium starting material in either the +5 or +7 oxidation state. An example of a compound wherein the rhenium is in the Re(VII) state is NH4ReO4or KReO4. Re(V) can be obtained, for example, as [ReOCl4](NBu4), [ReOCl4](AsPh4), ReOCl3(PPh3)2, and as ReO2(pyridine) 4+ wherein Ph is phenyl and Bu is n-butyl. Other rhenium reagents capable of forming rhenium complexes can also be used.

[0219] The radio-labeled PET, SPECT, or scintigraphic imaging agent provided by the present application contains a suitable amount of radioactivity. Typically, the unit dose to be administered has from about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi of radioactivity. The solution injected in a unit dose is from about 0.01 mL to about 10 mL. It is generally preferred that the radio-complex be formed in a solution containing a concentration of from about 0.01 mCi to 100 mCi / mL of radioactivity.

[0220] A typical dose of the radionuclide-labeled IL-17F trap according to the present application is provided as 10 to 20 mCi. After injection of the radionuclide-labeled IL-17F trap into the patient, the area of uptake of the agent is imaged and the amount of radioactivity present at the site is quantified using a gamma camera calibrated for the gamma ray energy of the nuclide incorporated into the imaging agent. Imaging of the site in vivo can be accomplished in a few minutes. However, if desired, imaging can be performed several hours or even longer after injection of the radiolabeled peptide into the patient. In most cases, a sufficient amount of the administered dose will accumulate in the area to be imaged in about 0.1 hour to allow scintiphotos to be taken.

[0221] Suitable dosage regimens for the radiotherapeutic compounds of the present application are known to those skilled in the art. The compounds can be administered using a number of methods, including but not limited to single or multiple intravenous or intraperitoneal injections, with an amount of radioactivity sufficient to cause damage or ablation of the targeted IL-17F-expressing tissue, but not so much as to cause substantial damage to non-target (normal tissue). The amount and dose required will vary depending on the energy and half-life of the isotope used, the extent of uptake and clearance of the agent from the body, and the mass of the IL-17F-expressing tissue, and will vary for different constructs. Generally, the dose can range from a single dose of about 30 to 50 mCi to a cumulative dose of up to about 3 Ci.

[0222] The radiotherapeutic compositions of the present application can include physiologically acceptable buffers, and can require a radioprotectant to prevent radiation damage to the compound prior to injection. Radioprotectants are known to those skilled in the art, and can include, for example, p-aminobenzoic acid, ascorbic acid, gelatin acid, and the like.

[0223] In addition to the radionuclide, a single or multiple vial kit containing all components necessary to prepare the complex of the present application is a component of the present application.

[0224] A single vial kit preferably contains the chelating ligand, the stannous salt source or other pharmaceutically acceptable reducing agent, and is appropriately buffered with a pharmaceutically acceptable acid or base to adjust the pH to a value of about 3 to about 9. The type and amount of reducing agent used depends on the nature of the exchange complex to be formed. Appropriate conditions are well known to those skilled in the art. The contents of the kit are preferably in lyophilized form. Such single vial kits can optionally contain labile or exchange ligands such as gluceptate, gluconate, mannitol, malate, citrate or tartarate, and can also contain reaction modifiers such as diethylenetriaminepentaacetic acid (DPTA), ethylenediaminetetraacetic acid (EDTA) or alpha, beta or gamma-cyclodextrin for improving the radiochemical purity and stability of the final product. The kit can also contain stabilizers, bulking agents such as mannitol designed to aid in the freeze-drying process, and other additives known to those skilled in the art.

[0225] A multiple vial kit preferably contains the same general components, but uses more than one vial in reconstituting the radiopharmaceutical. For example, one vial can contain all the ingredients necessary to form the labile Tc(V) complex upon addition of a pertechnetate salt (e.g., a stannous source or other reducing agent). The pertechnetate salt is added to this vial, and after an appropriate time, the contents of this vial are added to a second vial containing the ligand and a buffer appropriate to adjust the pH to its optimum value. After a reaction time of about 5 to 60 minutes, the complex of the present application is formed. Advantageously, the contents of both vials of this multiple vial kit are lyophilized. Reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. can be present in either or both vials, as described above.

[0226] Also provided herein are methods of incorporating a 18 F radiolabeled auxiliary group onto an IL-17F capture agent. In one embodiment, 4-[ 18 F]fluorobenzaldehyde ( 18 FB) is conjugated to a capture agent bearing an aminooxy moiety, resulting in the formation of an oxime. In another embodiment, 4-[ 18 F]fluorobenzaldehyde is conjugated to a capture agent bearing an acylhydrazine moiety, resulting in the formation of a hydrazone adduct. 4-Fluorobenzaldehyde can be prepared in 18 F form using 18 F ion by displacement of a leaving group using known methods.

[0227] 18 F-labeled capture agents can also be prepared from a capture agent bearing a thiosemicarbazide moiety under conditions that promote the formation of a thiosemicarbazino acid or by utilizing a 18 F-labeled bisulfite adduct complex.

[0228] The above method is particularly suitable for labelling a capture agent, such as those described herein, which can be modified during synthesis to contain a nucleophilic hydroxylamine, aminothiourea or hydrazine (or hydrazide) moiety which can be used to react with the labelled aldehyde. The method can be used for any capture agent which can accommodate a suitable nucleophilic moiety. Typically the nucleophilic moiety is attached to the N-terminus of the peptide, but the skilled person will appreciate that the nucleophile can also be attached to an amino acid side chain or the C-terminus of the peptide. Methods are provided for the synthesis of radiolabelled peptide sequences in which 4- 18 F]fluorobenzaldehyde is reacted with a peptide sequence containing a hydroxylamine, aminothiourea or hydrazine (or hydrazide) group, thereby forming the corresponding oxime, thiosemicarbazone or hydrazone, respectively.4- 18 F]fluorobenzaldehyde is typically generated in situ by acid catalysed decomposition of an addition complex of 4- 18 F]fluorobenzaldehyde and sodium bisulphite. The use of the bisulphite addition complex improves the speed of purification as, unlike the aldehyde, the complex can be concentrated to dryness. The formation of the complex is also reversible under both acidic and basic conditions. In particular, when the complex is contacted with a peptide containing a hydroxylamine, aminothiourea or hydrazine (or hydrazide) group in an acidic medium, the active free 4- 18 F]fluorobenzaldehyde is consumed as it is formed in situ, producing the corresponding 18 F]radiolabelled peptide sequence.

[0229] In the case where the oxime, thiosemicarbazone or hydrazone linkage is unstable in vivo, an additional reduction step can be employed to reduce the double bond linking the peptide to the 18 F]substrate. The corresponding reduced peptide bond will enhance stability. The skilled person is aware of various methods which can be used to perform such a reduction step. The reductive amination procedure described in Wilson et al., Journal of Labeled Compounds and Radiopharmaceuticals, XXVIII(10), 1189-1199, 1990 can also be used to form a Schiff base comprising a peptide and 4- 18 F]fluorobenzaldehyde, and this Schiff base is reduced directly using a reducing agent such as sodium cyanoborohydride.

[0230] 4-[ 18F]Fluorobenzaldehyde can be prepared as described in Wilson et al., Journal of Labeled Compounds and Radiopharmaceuticals, XXVIII(10), 1189-1199, 1990; Iwata et al., Applied radiation and isotopes, 52, 87-92, 2000; Poethko et al. The Journal of Nuclear Medicine, 45, 892-902, 2004; and Schottelius et al. Clinical Cancer Research, 10, 3593-3606, 2004. Na 18 F is added to a mixture of kryptofix and K2CO3. Anhydrous acetonitrile can be added and the solution evaporated in a heating block under a stream of argon. Additional portions of acetonitrile can be added and evaporated to completely dry the sample. 4-Trimethylammonium benzaldehyde triflate can be dissolved in DMSO and added to the dry F-18. The solution can then be heated in a heating block. The solution can be briefly cooled, diluted with water and passed through a Oasis HLB LP extraction cartridges are filtered. The cartridges can be washed with 9: 1 water: acetonitrile and water to remove unbound 18 F and unreacted 4-trimethylammonium benzaldehyde triflate. 4-[ 18 F]Fluorobenzaldehyde can then be eluted from the cartridge with methanol in the eluent.

[0231] Therapeutic applications

[0232] Provided herein in certain embodiments are methods of identifying, detecting, quantifying, and / or isolating IL-17F in a biological sample using the IL-17F capture agents disclosed herein. In certain embodiments, these methods utilize immunoassays, in which the capture agent replaces an antibody or its equivalent. In certain embodiments, the immunoassay can be a Western blot, a pull-down experiment, a dot blot, or an ELISA.

[0233] A biological sample for use in the methods provided herein can be selected from the group consisting of organs, tissues, body fluids, and cells. Where the biological sample is a body fluid, the fluid can be selected from the group consisting of blood, serum, plasma, urine, sputum, saliva, fecal matter, spinal fluid, cerebrospinal fluid, lymphatic fluid, skin secretions, respiratory secretions, intestinal secretions, urogenital tract secretions, tears, and milk. Organs include, for example, adrenal glands, bladder, bone, brain, breast, cervix, esophagus, eye, gall bladder, genitalia, heart, kidney, large intestine, liver, lung, lymph node, ovary, pancreas, pituitary, prostate, salivary gland, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, thymus, trachea, thyroid, testicle, ureter, and urethra. Tissues include, for example, epithelial tissue, connective tissue, nervous tissue, and muscle tissue.

[0234] Provided herein in certain embodiments are methods of diagnosing and / or classifying (e.g., staging) a disorder associated with IL-17F expression using the IL-17F capture agents disclosed herein. In certain embodiments of these embodiments, the method comprises (a) obtaining a biological sample from a subject; (b) assaying the sample for the presence or absence of IL-17F using an IL-17F capture agent; (c) comparing the level of IL-17F to a predetermined control range for IL-17F; and (d) diagnosing a disorder associated with IL-17F expression based on the difference in the level of IL-17F in the biological sample compared to the predetermined control.

[0235] In other embodiments, the IL-17F capture agents disclosed herein are used as mutation-specific targeted therapeutics. In certain aspects of this embodiment, the IL-17F capture agents are administered alone without delivery of DNA, radiopharmaceuticals, or another active agent.

[0236] The IL-17F capture agents of the present application can also be used to target genetic material to cells expressing IL-17F. Genetic material can include nucleic acids of natural or synthetic origin, such as RNA or DNA, including recombinant RNA and DNA as well as antisense RNA and DNA. Types of genetic material that can be used include, for example, genes carried on expression vectors such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), and defective or "helper" viruses, antisense gene nucleic acids, single- and double-stranded RNA, and DNA therefor, and analogs thereof (e.g., phosphorothioate and phosphorodithioate oligodeoxynucleotides). Additionally, the genetic material can be combined with, for example, lipids, proteins, or other polymers. Delivery vehicles for genetic material can include, for example, viral particles, retroviruses or other gene therapy vectors, liposomes, complexes of lipids (particularly cationic lipids) and genetic material, complexes of dextran derivatives and genetic material, and the like.

[0237] In one embodiment, the capture agent of the present application is used for gene therapy. In this embodiment, genetic material or one or more delivery vehicles containing genetic material can be conjugated to one or more IL-17F capture agents of the present disclosure and administered to a patient.

[0238] The therapeutic agents and IL-17F capture agents disclosed herein can be linked or fused in known ways, optionally utilizing the same types of linkers discussed elsewhere in this application. Preferred linkers will be substituted or unsubstituted alkyl chains, amino acid chains, polyethylene glycol chains, and other simple polymeric linkers known in the art. More preferably, if the therapeutic agent is itself a protein known to be encoded by a DNA sequence, the therapeutic protein and IL-17F binding polypeptide can be co-expressed from the same synthetic gene, produced using recombinant DNA technology as described above. The coding sequence for the IL-17F binding polypeptide can be fused in frame with the coding sequence for the therapeutic protein, such that the peptide is expressed at the amino terminal or carboxy terminal end of the therapeutic protein or at a position between the ends, if it is determined that this placement will not destroy the desired biological function of the therapeutic protein or IL-17F binding polypeptide. A particular advantage of this general approach is that concatamerization of multiple IL-17F capture agents in tandem is possible, thereby increasing the number and concentration of IL-17F binding sites associated with each therapeutic protein. In this way, IL-17F binding avidity is increased, which would be expected to improve the efficacy of the recombinant therapeutic fusion protein.

[0239] The following examples are provided to better illustrate the claimed invention and are not to be construed as limiting the scope of the application. To the extent that specific materials are mentioned, this is only by way of exemplification, and is not intended to limit the scope of the application. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the application.

[0240] Examples

[0241] Example 1, IL-17F Epitope Design

[0242] The primary sequences of IL-17F and IL-17A were examined to understand where regions of identity or similarity exist, and where differences exist. In order to generate a macrocyclic peptide ligand that can effectively discriminate between IL-17F and IL-17A, attention was focused on sequences unique to the two proteins. Sequence differences that distinguish IL-17F from IL-17A were found to occur in the N-terminal region of the mature protein. In IL-17F, the uniqueness of this region corresponds to Arg-31 to Thr-79 Figure 1A ).

[0243] Two polypeptide epitopes were chemically synthesized and used as targets for the generation of specific macrocyclic peptide ligands against IL-17F. The epitopes were designed with a biotin-PEG3 assay handle and a strategically substituted azido click handle (Az4 = L-azidolysine). The site of click handle substitution in each epitope was determined by examining the protein structure and identifying amino acids (a) that are surface exposed and do not interact with other atoms in the protein and (b) that are chemically similar to Az4. IL-17F Epitope 1 (Phe-40 to Ser-54) is located very close to the N-terminus of IL-17F and substituted with Az4 at the central position (Cys-48). IL-17F Epitope 2 (Gly-60 to Ser-69) is located after Epitope 1 in the primary sequence and substituted with Az4 at Ile-62. The sequences of Epitope 1 and Epitope 2 are shown in Figure 1B and C.

[0244] Example 2, Screening for Macrocyclic Anchors against IL-17F Epitope 1

[0245] Screening was performed with an OBOC library of triazole cyclized in the form of H2N-Pra-Cy(XXXXX)-Met-TG (SEQ ID NO: 34), where S NH2 resin (S 30 902, Rapp Polymere), X = one of 17 L-amino acids (lacking Cys, Met and Ile), Pra = L- propargylglycine, Cy() = triazole cyclization through flanking Pra and Az4 residues. A four-step screening approach was employed to identify macrocycles against IL-17F Epitope 1 fragment: 1) pre-cleanup to eliminate non-specific binders, 2) product screening to identify hits generated by epitope template in situ click chemistry, 3) target screening against His-tagged IL-17F protein, and 4) additional target screening against His-tagged IL-17F protein in 2% (v / v) human serum to identify those peptides whose binding to IL-17F is not affected by serum proteins.

[0246] Pre-cleanup. Swollen library beads (500 mg) were blocked with blocking buffer (25 mM Tris-HCl, 150 mM NaCl, 1% (w / v) BSA and 0.05% (v / v) Tween-20, pH 7.6) overnight at 4°C, then washed three times with blocking buffer. Streptavidin-alkaline phosphatase (V559C, Promega) diluted 1 : 10,000 in 5 mL blocking buffer was added to the beads and incubated for 1 hour at room temperature with gentle shaking. Subsequently, the beads were washed with 3 x 3 mL TBS (25 mM Tris-HCl, 150 mM NaCl, pH 7.6) (1 minute each), 3 x 3 mL 0.1 M glycine pH 2.8 wash buffer, 3 x 3 mL TBS, then 3 x 3 mL alkaline phosphatase buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, pH 9) (5 minutes each). Binding was visualized by incubating the beads in the presence of 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT) substrate (S3771, Promega) for 25 minutes. Purple beads indicated background binders and were removed by pipette and discarded. Remaining clear beads were collected and stripped with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes, washed 10 times with water, and incubated overnight in 1-methyl-2-pyrrolidinone (NMP) to destain.

[0247] Product screening was performed using IL-17F epitope 1. Pre-cleaned beads were washed 10 times with water and 3 times with TBS. The beads were then incubated at room temperature for 1.5 hours with 3 mL of 100 μM TBS solution of the IL-17F epitope 1 fragment (Biotin-PEG3-FFQKPES (SEQ ID NO:1)[Az4]PPVPGGS) (SEQ ID NO:32) to allow for in-situ click reaction. The beads were washed 10 times with TBS and then incubated with 7.5 M guanidine hydrochloride at pH 2.0 for 1 hour to remove all IL-17F epitopes not covalently linked to the beads. These beads were washed 10 times with TBS and then blocked with blocking buffer for 2 hours. Streptavidin-alkaline phosphatase (V559C, Promega) diluted 1:10,000 in 5 mL of blocking buffer was added and the reaction was continued for 1 hour to detect the presence of IL-17F epitopes clicked onto the beads. The beads were then washed with 3×3 mL TBS (1 min each), 3×3 mL 0.1 M glycine pH 2.8 wash buffer, 3×3 mL TBS, and then 3×3 mL alkaline phosphatase (pH 9) buffer (5 min each). Following this, the beads were developed with BCIP / NBT for 25 min as outlined in the pre-cleaning section. Vibrating beads with purple epitope conjugation were selected by pipette and stored. These vibrating beads (25 in total: 5 dark purple and 20 medium to light purple) were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 min to remove attached streptavidin, washed 10 times with water, and incubated overnight in NMP for destaining.

[0248] Target screening was performed using His-tagged IL-17F protein. The hit products were washed 10 times with water and stored in TBS at 4°C. These 25 beads were then transferred... 8162 The beads were filtered through centrifuge tubes (cellulose acetate membrane) and incubated with blocking buffer for 3 hours at room temperature. The beads were washed three times with blocking buffer and then incubated with 150 nM full-length His-labeled IL-17F protein (ab167911, Abcam) in blocking buffer (prepared by adding 0.5 μL of His-labeled IL-17F protein to 200 μL of blocking buffer) for 1 hour at room temperature. The beads were washed three times with blocking buffer and then incubated with 500 μL of 1:10,000 anti-6×His... Antibody [HIS-1] (alkaline phosphatase conjugated) (ab49746, Abeam) was incubated in blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΙ_ blocking buffer, 3 x 500 μΙ_ TBS, then 3 x 500 μΙ_ alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads that bound to the IL-17F protein were selected by pipette and saved. 20 beads were purple, indicating binding to both the IL-17 epitope and protein, while 5 beads were clear, indicating no binding to the IL-17F protein. The 20 target hit beads were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove the bound protein, washed 10 times with water, and incubated overnight in NMP to destain.

[0249] Target screening was performed with His-tagged IL-17F protein in 2% (v / v) human serum. The target hit beads were washed 10 times with water. These 20 beads were incubated with blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΙ_ blocking buffer, 3 x 500 μΙ_ TBS, then 3 x 500 μΙ_ alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads that bound to the IL-17F protein were selected by pipette and saved. 20 beads were purple, indicating binding to both the IL-17 epitope and protein, while 5 beads were clear, indicating no binding to the IL-17F protein. The 20 target hit beads were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove the bound protein, washed 10 times with water, and incubated overnight in NMP to destain. 8162 centrifuge tube filters (cellulose acetate membrane) for 7 hours. The beads were rinsed three times with blocking buffer, then incubated with 150 nM full-length His-tagged IL-17F protein (ab167911, Abeam) in blocking buffer containing 2% (v / v) human serum (HS-30, Omega) for 1 hour at room temperature (preparation: 1.25 μΙ_ His-tagged IL-17F protein + 10 μΙ_ filtered serum + 490 μΙ_ blocking buffer). Note: Prior to screening, the beads were blocked with 2% (v / v) human serum (HS-30, Omega) for 1 hour at room temperature. 8162 The tube filters removed particulate matter from the serum by centrifugation (7000 rpm, 30 seconds). The beads were washed three times with blocking buffer, then incubated with 500 μΙ_ 1 : 10,000 anti-6xHis antibody (ab49999, Abeam) in blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΙ_ blocking buffer, 3 x 500 μΙ_ TBS, then 3 x 500 μΙ_ alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads that bound to the IL-17F protein were selected by pipette and saved. 20 beads were purple, indicating binding to both the IL-17 epitope and protein, while 5 beads were clear, indicating no binding to the IL-17F protein. The 20 target hit beads were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove the bound protein, washed 10 times with water, and incubated overnight in NMP to destain. Antibody [HIS-1] (alkaline phosphatase conjugated) (ab49746, Abeam) was incubated in blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΙ_ blocking buffer, 3 x 500 μΙ_ TBS, then 3 x 500 μΙ_ alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads that bound to the IL-17F protein were selected by pipette and saved. 20 beads were purple, indicating binding to both the IL-17 epitope and protein, while 5 beads were clear, indicating no binding to the IL-17F protein. The 20 target hit beads were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove the bound protein, washed 10 times with water, and incubated overnight in NMP to destain.

[0250] Edman degradation on an Applied Biosystems 494 Protein Sequencer at the Caltech Protein / Peptide Micro Analytical Laboratory. The Edman sequencer cannot distinguish between 1) residues K (lysine) and L (leucine), and 2) residues Q (glutamine) and T (threonine). The sequencing results including K / L and Q / T variants are shown in Table 1.

[0251] Table 1: Sequences of macrocyclic peptide hits identified against IL-17F epitope 1

[0252]

[0253] These candidate peptides were resynthesized on cleavable resin, purified by reverse phase HPLC using a C18 column (Phenomenex Luna, 5 μm, 250 x 10 mm), and tested for affinity and specificity against IL-17F protein by ELISA.

[0254] Synthetic data for IL-17F epitope 1 hits

[0255] Cy(FYKQH) (SEQ ID NO: 6)-PEG3-biotin. MALDI-MS (m / z): C 65 H 97 N 17 O 14 S (M+H) calculated 1399.72; found 1401.36.

[0256]

[0257] Cy(FYKQH) (SEQ ID NO: 6)-PEG3-biotin. MALDI-MS (m / z): C 66 H 98 N 18 O 14 S (M+H) calculated 1399.72; found 1401.36.

[0258]

[0259] Cy(FYKQH) (SEQ ID NO: 6)-PEG3-biotin. MALDI-MS (m / z): C 65 H 96 N 16 O 14 ​S(M+H) calculated 1357.70; found 1360.15.

[0260]

[0261] Cy(FYLQH) (SEQ ID NO: 8) - PEG3-biotin. MALDI-MS (m / z): C 66 H 97 N 17 O 14 S(M+H) calculated 1384.71 ; found 1386.24.

[0262]

[0263] Cy(RRATS) (SEQ ID NO: 9) - PEG3-biotin. MALDI-MS (m / z): C 53 H 94 N 20 O 14 S(M+H) calculated 1269.70; found 1269.91 Figure 2C ).

[0264]

[0265] Cy(RRAQS) (SEQ ID NO: 10) - PEG3-biotin. MALDI-MS (m / z): C 54 H 95 N 21 O 14 S(M+H) calculated 1295.71 ; found 1296.19 Figure 2D ).

[0266]

[0267] Example 3, in vitro testing with IL-17F epitope 1 targeted ligands

[0268] Sandwich ELISA. Black 96-well NeutrAvidin-coated high-binding-capacity plates (15510, Pierce) were coated with 2 μM macrocyclic peptide ligand in TBS (25 mM Tris-HCl, 150 mM NaCl, pH 7.6) at room temperature for 2 hours. A control was prepared by coating with 4 μg / mL biotinylated monoclonal antibody-IL17F (TA319597, Origene) in TBS. The plates were aspirated and washed with TBS (5×) and washing buffer (0.05% (v / v) Tween-20 in PBS, 1×). Full-length His-labeled IL-17F protein (ab167911, Abcam) was serially diluted in washing buffer (800 to 0 nM) and incubated in designated microwells at room temperature for 90 minutes. The microwells were aspirated and then washed with washing buffer (10×). To detect the bound IL-17F protein, alkaline phosphatase (AP)-conjugated anti-6×His protein was prepared at a dilution of 1:10,000. Antibody [HIS-1] (ab49746, Abcam) was added to the microwells and incubated at room temperature for 1 hour. The plate was then aspirated and washed with washing buffer (5×). The microwells were developed using the AP fluorescent substrate system (S1000, Promega). Fluorescence emission at 535 nm was recorded using a Beckman Coulter DTX880 photometer with an excitation wavelength of 430 nm. Titration curves were fitted using a four-parameter regression curve fitting program (Origin 8.5) to determine EC. 50 value.

[0269] The results are as follows Figure 2A As shown. The binding affinity of PEG3-biotin-modified Cy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ ID NO:10) was tested in ELISA format. For these assays, serially diluted full-length His-labeled IL-17F protein was captured using macrocyclic peptide ligands immobilized on NeutrAvidin-coated plates. Cy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ ID NO:10) of human IL-17F protein showed EC50 values ​​of 66 ± 9 nM and 52 ± 5 nM, respectively. 50 Value. Biotinylated monoclonal antibody-IL17F, as determined in a similar assay, showed similar binding affinity.

[0270] Spot ELISA (selectivity assay of IL-17F versus IL-17A). Black 96-well NeutrAvidin-coated high-binding-capacity plates (15510, Pierce) were coated with 2 μM macrocyclic peptide ligand in TBS (pH 7.6) solution at room temperature for 2 hours. The plates were aspirated and washed with TBS (5×) and wash buffer (0.05% (v / v) Tween-20 in PBS solution, 1×). 200 and 50 nM of full-length His-labeled IL-17F (ab167911, Abcam) and IL-17A (ab166882, Abcam) proteins were prepared in wash buffer and incubated in designated microwells at room temperature for 90 minutes. The microwells were aspirated and then washed with wash buffer (10×). To detect the binding of IL-17F and IL-17A proteins, alkaline phosphatase (AP)-conjugated anti-6×His antibodies were prepared at a dilution of 1:10,000. Antibody [HIS-1] (ab49746, Abcam) was added to the microwells and incubated at room temperature for 1 hour. The plate was then aspirated and washed with washing buffer (5×). The micropores were developed using the AP fluorescent substrate system (S1000, Promega). Fluorescence emission at 535 nm was recorded using a Beckman Coulter DTX880 photometer with an excitation wavelength of 430 nm.

[0271] The results are as follows Figure 2B As shown. The selectivity of PEG3-biotin-modified Cy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ ID NO:10) was tested using ELISA. For these assays, full-length His-labeled IL-17F and IL-17A proteins were captured using macrocyclic peptide ligands immobilized on NeutrAvidin-coated plates. Both Cy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ ID NO:10) showed a 2:1 selectivity for IL-17F in the concentration range of 200 to 50 nM. These results confirm the selectivity of the epitope targeting strategy.

[0272] A directional assay was performed to determine the orientation of the macrocycle binding to IL-17F epitope 1. Black 96-well NeutrAvidin-coated high binding capacity plates (15510, Pierce) were coated with a 2 mM solution of macrocyclic peptide ligand in TBS (pH 7.6) for 2 hours at room temperature. A control was performed by coating with a 4 pg / mL solution of biotinylated monoclonal anti-IL17F (TA319597, Origene) in TBS. The plates were aspirated and then washed with TBS (5x) and wash buffer (0.05% (v / v) Tween-20 in PBS, lx). Chemically synthesized His-tagged IL-17F epitope was prepared at 2 mM in wash buffer and incubated in the designated microwells for 90 minutes at room temperature. Wash buffer without epitope was added as a control. The microwells were aspirated and then washed with wash buffer (10x). To detect bound IL-17F epitope, alkaline phosphatase (AP) conjugated anti-6xHis antibody [HIS-1] (ab49746, Abeam) was prepared at a 1 : 10,000 dilution and added to the microwells for 1 hour at room temperature. The plates were aspirated and washed with wash buffer (5x). The microwells were developed with the AP fluorometric substrate system (S1000, Promega). Fluorescence emission at 535 nm was recorded by a Beckman Coulter DTX880 spectrophotometer using an excitation wavelength of 430 nm. Data are shown after subtraction of the no epitope background.

[0273] Results are shown in Figure 3. For this experiment, IL-17F epitope 1 was resynthesized with a His6determination handle and a C48S substituent in place of the click handle. Two additional His-tagged IL-17F epitopes were synthesized to include a strategically scrambled sequence, either N-terminal or C-terminal to the C48S. A dot ELISA was performed for the three His-tagged IL-17F epitopes against the immobilized macrocyclic peptide ligand. ELISA positive signals were obtained for His-tagged IL-17F epitope 1 (C48S) and the click handle C-terminal scrambled epitope (black and red bars) for PEG3-biotin modified Cy(RRATS) (SEQ ID NO: 9) and Cy(RRAQS) (SEQ ID NO: 10). Binding was disrupted when the N-terminal of the click handle epitope was scrambled (blue bar). Thus, the macrocyclic peptide ligand shows a preference for binding to the sequence FFQKPES (SEQ ID NO: 1) within IL-17F epitope 1 (C48S). On the other hand, biotinylated monoclonal anti-IL17F shows no appreciable binding to the His-tagged IL-17F epitopes, as it was raised against the protein immunogen.

[0274] ​In summary, the results of Figures 2-3 demonstrate that the epitope targeting strategy yields macrocyclic peptide ligands that selectively recognize the IL-17F epitope and have affinities below 100 nM for the full-length protein.

[0275] Example 4, Screening for Macrocycles against IL-17F Epitope 2

[0276] Screening against IL-17F Epitope 2 fragment (Biotin-PEG3-GI[Az4]NENQRVS (SEQ ID NO: 33)) was similarly performed using a triazole ring- ligation OBOC library in the form of H2N-Pra-Cy(XXXXX)-Met-TG (SEQ ID NO: 34). The four-step screening method described above was employed to identify macrocycles against IL-17F Epitope 2 fragment: 1) pre-purification to eliminate non-specific binders, 2) product screening to identify hits generated from the epitope template in situ click chemistry, 3) target screening against His-tagged IL-17F protein, and 4) additional target screening against His-tagged IL-17F protein in 1-5% (v / v) human serum to identify those peptides whose binding to IL-17F is not affected by serum proteins.

[0277] Product screening was performed with IL-17F Epitope 2. Product screening was performed by incubating 3 mL of 100 μΜ IL-17F Epitope 2 fragment (Biotin- PEG3-GI[Az4]NENQRVS (SEQ ID NO: 33)) in TBS at room temperature for 1.5 hours to allow the in situ click reaction to proceed. The beads were washed 10 times with TBS and then incubated with 7.5 M guanidine hydrochloride pH 2.0 for 1 hour to remove all IL-17F epitope not covalently attached to the beads. These beads were washed 10 times with TBS and re-blocked with blocking buffer for 2 hours. Streptavidin-alkaline phosphatase (V559C, Promega) diluted 1 : 10,000 in 5 mL blocking buffer was added for 1 hour to detect the presence of IL-17F epitope clicked to the beads. The beads were then washed with 3 x 3 mL TBS (1 minute each), 3 x 3 mL 0.1 M glycine pH 2.8 wash buffer, 3 x 3 mL TBS, and then 3 x 3 mL alkaline phosphatase (pH 9) buffer (5 minutes each). After this, the beads were developed with BCIP / NBT as outlined for pre-purification for 25 minutes. The purple epitope-conjugated hit beads were selected by pipette and saved. These hit beads (a total of 98: 50 dark purple, 48 medium to light purple) were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove the attached streptavidin, washed 10 times with water, and incubated overnight in NMP to destain.

[0278] His-tagged IL-17F protein. Hit products were washed 10 times with water and stored in TBS at 4°C. These 98 beads were transferred to 8162 centrifuge filters (cellulose acetate membrane) and incubated with blocking buffer for 3 hours at room temperature. Beads were rinsed three times with blocking buffer and then incubated with 150 nM of full-length His-tagged IL-17F protein (ab167911, Abeam) in blocking buffer (preparation: 0.5 μL His-tagged IL-17F protein in 200 μL blocking buffer) for 1 hour at room temperature. Beads were washed three times with blocking buffer and then incubated with 500 μL of 1 : 10,000 anti-6xHis antibody [HIS-1] (alkaline phosphatase conjugated) (ab49746, Abeam) in blocking buffer for 1 hour at room temperature. Beads were then washed with 3 x 500 μL blocking buffer, 3 x 500 μL TBS, then 3 x 500 μL alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, beads were developed with BCIP / NBT for 10 minutes. The purple hit beads that bound to the IL-17F protein were selected by pipette and saved. 53 beads were purple, indicating binding to both the IL-17 epitope and the protein, while 40 beads were clear, indicating no binding to the IL-17F protein. The 53 target hit beads were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove the bound protein, washed 10 times with water, and incubated overnight in NMP to destain.

[0279] His-tagged IL-17F protein in 1% (v / v) human serum. Target hit beads were washed 10 times with water. These 53 beads were incubated with blocking buffer for 7 hours at room temperature in centrifuge filters (cellulose acetate membrane). 8162 centrifuge filters (cellulose acetate membrane) and incubated with blocking buffer for 3 hours at room temperature. Beads were rinsed three times with blocking buffer and then incubated with 150 nM of full-length His-tagged IL-17F protein (ab167911, Abeam) in blocking buffer (preparation: 0.5 μL His-tagged IL-17F protein in 200 μL blocking buffer) for 1 hour at room temperature. Beads were washed three times with blocking buffer and then incubated with 500 μL of 1 : 10,000 anti-6xHis 8162 centrifuge filters (cellulose acetate membrane) and incubated with blocking buffer for 3 hours at room temperature. Beads were rinsed three times with blocking buffer and then incubated with 150 nM of full-length His-tagged IL-17F protein (ab167911, Abeam) in blocking buffer (preparation: 0.5 μL His-tagged IL-17F protein in 200 μL blocking buffer) for 1 hour at room temperature. Beads were washed three times with blocking buffer and then incubated with 500 μL of 1 : 10,000 anti-6xHis Antibody [HIS-1] (alkaline phosphatase conjugated) (ab49746, Abeam) was incubated in blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΐ, blocking buffer, 3 x 500 μΐ, TBS, then 3 x 500 μΐ, alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads were selected by pipette and saved. The 23 hit beads that were not interfered with serum proteins binding to IL-17F protein were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove bound proteins, washed 10 times with water, and incubated overnight in NMP to destain.

[0280] Target screening was performed with His-tagged IL-17F protein in 5% (v / v) human serum to optimize the number of hits. The 23 beads were washed 10 times with water, then incubated with blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΐ, blocking buffer, 3 x 500 μΐ, TBS, then 3 x 500 μΐ, alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads were selected by pipette and saved. The 23 hit beads that were not interfered with serum proteins binding to IL-17F protein were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove bound proteins, washed 10 times with water, and incubated overnight in NMP to destain. 8162 centrifuge tube filters (cellulose acetate membrane) for 7 hours. The beads were rinsed three times with blocking buffer, then incubated with 150 nM full-length His-tagged IL-17F protein (ab 167911, Abeam) in blocking buffer containing 5% (v / v) human serum (HS-30, Omega) for 1 hour at room temperature (preparation: 1.25 μΐ, His-tagged IL-17F protein + 25 μΐ, filtered serum + 475 μΐ, blocking buffer). Note: Prior to screening, the beads were blocked with 5% (v / v) human serum (HS-30, Omega) for 1 hour at room temperature. 8162 The beads were washed three times with blocking buffer, then incubated with 500 μΐ, 1 : 10,000 anti-6xHis antibody (ab 100134, Abeam) in blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΐ, blocking buffer, 3 x 500 μΐ, TBS, then 3 x 500 μΐ, alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads were selected by pipette and saved. The 6 hit beads that were not interfered with an increase in serum protein background binding to IL-17F protein were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove bound proteins, washed 10 times with water, and incubated overnight in NMP to destain. The 6 hit beads were finally washed 10 times with water in preparation for sequencing analysis. Antibody [HIS-1] (alkaline phosphatase conjugated) (ab49746, Abeam) was incubated in blocking buffer for 1 hour at room temperature. The beads were then washed with 3 x 500 μΐ, blocking buffer, 3 x 500 μΐ, TBS, then 3 x 500 μΐ, alkaline phosphatase (pH 9) buffer (centrifuged at 7000 rpm for 30 seconds after each wash). After this, the beads were developed with BCIP / NBT for 10 minutes. The purple hit beads were selected by pipette and saved. The 23 hit beads that were not interfered with serum proteins binding to IL-17F protein were treated with 7.5 M guanidine hydrochloride pH 2.0 for 30 minutes to remove bound proteins, washed 10 times with water, and incubated overnight in NMP to destain.

[0281] Sequencing was performed by Edman degradation. The Edman sequencer cannot distinguish between 1) residues K (lysine) and L (leucine), and 2) residues Q (glutamine) and T (threonine). The sequencing results, including K / L and Q / T variants, are shown in Table 2.

[0282] Table 2: Sequences of macrocyclic peptide hits identified against IL-17F epitope 2

[0283]

[0284]

[0285] These candidate peptides were resynthesized on cleavable resin, purified by reverse phase HPLC using C18 columns, and tested against IL-17F protein by ELISA.

[0286] Synthetic data for IL-17F epitope 2 hits

[0287] Cy(KYGEV) (SEQ ID NO: 11)-PEG3-biotin. MALDI-MS (m / z): C 58 H 93 N 15 O 15 S (M+H) calculated 1272.67; found 1274.02.

[0288]

[0289] Cy(LYGEV) (SEQ ID NO: 12)-PEG3-biotin. MALDI-MS (m / z): C 58 H 92 N 14 O 15 S (M+H) calculated 1257.66; found 1259.13.

[0290]

[0291] Cy(VHKSG) (SEQ ID NO: 13)-PEG3-biotin. MALDI-MS (m / z): C 53 H 89 N 17 O 13 S (M+H) calculated 1204.65; found 1206.20.

[0292]

[0293] Cy(VHLSG) (SEQ ID NO: 14)-PEG3-biotin. MALDI-MS (m / z): C 53 H 88 N 16 O 13 S (M+H) calculated 1189.64; found 1191.11.

[0294]

[0295] Cy(QKHGP) (SEQ ID NO: 15)-PEG3-biotin. MALDI-MS (m / z): C 55 H 90 N 18 O 13 S (M+H) calculated 1243.67; found 1245.18.

[0296]

[0297] Cy(TKHGP) (SEQ ID NO: 16)-PEG3-biotin. MALDI-MS (m / z): C 54 H 89 N 17 O 13 S (M+H) calculated 1216.65; found 1218.25.

[0298]

[0299] Cy(QLHGP) (SEQ ID NO: 17)-PEG3-biotin. MALDI-MS (m / z): C 55 H 89 N 17 O 13 S (M+H) calculated 1228.65; found 1228.84.

[0300]

[0301] Cy(TLHGP) (SEQ ID NO: 18)-PEG3-biotin. MALDI-MS (m / z): C 54 H 88 N 16 O 13 S (M+H) calculated 1201.64; found 1201.73.

[0302]

[0303] Cy(YDLQR) (SEQ ID NO: 19)-PEG3-biotin. MALDI-MS (m / z): C 61 H 98 N 18 O 16 S (M+H) calculated 1371.71 ; found 1372.16.

[0304]

[0305] Cy(YDKQR) (SEQ ID NO: 21)-PEG3-biotin. MALDI-MS (m / z): C 60 H 97 N 17 O 16 S (M+H) calculated 1344.70 ; found 1345.13.

[0306]

[0307] Cy(YDKQR) (SEQ ID NO: 21)-PEG3-biotin. MALDI-MS (m / z): C 61 H 99 N 19 O 16 S (M+H) calculated 1386.72 ; found 1387.00.

[0308]

[0309] Cy(YDKQR) (SEQ ID NO: 21)-PEG3-biotin. MALDI-MS (m / z): C 60 H 98 N 18 O 16 S (M+H) calculated 1359.71 ; found 1359.94.

[0310]

[0311] Biotin-PEG3-Cy(KKGWP) (SEQ ID NO: 23). MALDI-MS (m / z): C 59 H 91 N 17 O 13 S (M+H) calculated 1278.67 ; found 1278.83.

[0312]

[0313] Biotin-PEG3-Cy(KLGWP) (SEQ ID NO: 24). MALDI-MS (m / z): C 59 H 90 N 16 O 13 S (M+H) calculated 1263.66; found 1263.86.

[0314]

[0315] Biotin-PEG3-Cy(LKGWP) (SEQ ID NO: 25). MALDI-MS (m / z): C 59 H 90 N 16 O 13 S (M+H) calculated 1263.66; found 1263.86.

[0316]

[0317] Biotin-PEG3-Cy(LLGWP) (SEQ ID NO: 26). MALDI-MS (m / z): C 59 H 89 N 15 O 13 S (M+H) calculated 1248.65; found 1248.89.

[0318]

[0319] Biotin-PEG3-Cy(RSYNL) (SEQ ID NO: 27). MALDI-MS (m / z): C 57 H 90 N 18 O 16 S (M+H) calculated 1315.65; found 1315.95.

[0320]

[0321] Biotin-PEG3-Cy(RSYNK) (SEQ ID NO: 28). MALDI-MS (m / z): C 57 H 91 N 19 O 16 S (M+H) calculated 1330.66; found 1331.06.

[0322]

[0323] Example 5: In vitro assays using ligands targeting IL-17F epitope 2.

[0324] Sandwich ELISA. These assays were performed using the same protocol as those used to evaluate ligands targeting IL-17F epitope 1.

[0325] The results are as follows Figure 4A As shown. PEG3-biotin-modified Cy(QKHGP) (SEQ ID NO:15), Cy(TKHGP) (SEQ ID NO:16), Cy(KKGWP) (SEQ ID NO:23), and Cy(RSYNK) (SEQ ID NO:28) were tested using ELISA. For these assays, a dilution series of full-length His-labeled IL-17F protein were captured using macrocyclic peptide ligands immobilized on NeutrAvidin-coated plates. Cy(QKHGP) (SEQ ID NO:15) and Cy(TKHGP) (SEQ ID NO:16) of human IL-17F protein showed EC50 values ​​of 64 ± 10 nM and 72 ± 16 nM, respectively. 50 Values. Cy(KKGWP) (SEQ ID NO:23) and Cy(RSYNK) (SEQ ID NO:28) of human IL-17F protein exhibited EC50 values ​​of 24±3 nM and 15±5 nM, respectively. 50 Interestingly, Cy(RSYNK) (SEQ ID NO:28) exhibited a higher binding affinity than similarly determined biotinylated monoclonal antibodies against IL17F.

[0326] Spot ELISA (selectivity assay of IL-17F versus IL-17A). Black 96-well NeutrAvidin-coated high-binding-capacity plates (15510, Pierce) were coated with 2 μM macrocyclic peptide ligand in TBS (pH 7.6) for 2 hours at room temperature. A control was prepared by coating the plates in TBS with 4 μg / mL biotinylated monoclonal antibody-IL17F (TA319597, Origene). The plates were aspirated and washed with TBS (5×) and wash buffer (0.05% (v / v) Tween-20 in PBS, 1×). 100 nM of full-length His-labeled IL-17F (ab167911, Abcam) and IL-17A (ab166882, Abcam) proteins were prepared in wash buffer and incubated in designated microwells at room temperature for 90 minutes. The microwells were aspirated and then washed with wash buffer (10×). To detect the binding of IL-17F and IL-17A proteins, alkaline phosphatase (AP)-conjugated anti-6×His antibodies were prepared at a dilution of 1:10,000. Antibody [HIS-1] (ab49746, Abcam) was added to the microwells and incubated at room temperature for 1 hour. The plate was then aspirated and washed with washing buffer (5×). The micropores were developed using the AP fluorescent substrate system (S1000, Promega). Fluorescence emission at 535 nm was recorded using a Beckman Coulter DTX880 photometer with an excitation wavelength of 430 nm.

[0327] The results are as follows Figure 4B As shown. The selectivity of PEG3-biotin-modified Cy(QKHGP)(SEQ ID NO:15), Cy(TKHGP)(SEQ ID NO:16), Cy(KKGWP)(SEQ ID NO:23), and Cy(RSYNK)(SEQ ID NO:28) was tested using ELISA. For these assays, full-length His-labeled IL-17F and IL-17A proteins were captured using macrocyclic peptide ligands immobilized on NeutrAvidin-coated plates. Cy(KKGWP)(SEQ ID NO:23) and Cy(RSYNK)(SEQ ID NO:28) showed a 4:1 selectivity for 100 nM IL-17F. Other ligands, including Cy(QKHGP)(SEQ ID NO:15) and Cy(TKHGP)(SEQ ID NO:16), and biotinylated monoclonal antibodies against IL-17F showed even higher (almost absolute) selectivity for IL-17F. Furthermore, these results confirm the selectivity of the epitope targeting strategy. Example 6: Designing a connector to covalently link two macrocyclic ligands.

[0328] The tertiary structure of the IL-17F protein was then used as a scaffold to develop a protein exhibiting true co-binding (K) D Biligand PCC agents with a range of <1 nM. Two macrocycles are covalently linked together to produce biligand PCC agents exhibiting high affinity for both epitopes.

[0329] The important aspect of this process is to design an appropriate linker to bridge the distance between the two epitopes of the protein. The 3-D crystal structure of IL-17F (PDB ID: 1JPY) was analyzed in PyMOL (DeLano Scientific) to determine the distance between IL-17F Epitope 1 and IL-17F Epitope 2 (Figure 5). In IL-17F Epitope 1, the focus was placed on the sequence FFQKPES (SEQ ID NO: 1) since the macrocycles Cy(RRATS) (SEQ ID NO: 9) and Cy(RRAQS) (SEQ ID NO: 10) were identified as preferentially interacting with this region (Figure 3). The sequence NENQRVS (SEQ ID NO: 3) within IL-17F Epitope 2 was the putative binding region for the macrocycles Cy(QKHGP) (SEQ ID NO: 15), Cy(TKHGP) (SEQ ID NO: 16), Cy(KKGWP) (SEQ ID NO: 23), and Cy(RSYNK) (SEQ ID NO: 28) due to the location of the N-terminus of the click handle. Since IL-17F naturally exists as a homodimer, the distance between the two epitopes was measured within one monomer and between two monomers. In the monomeric IL-17F protein, the sequence FFQKPES (SEQ ID NO: 1) (in IL-17F Epitope 1) and IL-17F Epitope 2 were separated by approximately 10.5 A. In the homodimeric IL-17F protein, the sequence FFQKPES (SEQ ID NO: 1) (in IL-17F Epitope 1) from one monomer and IL-17F Epitope 2 from the other monomer were separated by approximately 21 A. Thus, a chemical linker of about was used to covalently link one macrocycle targeting IL-17F Epitope 1 with one macrocycle targeting IL-17F Epitope 2. The resulting synergistic bioligand was used to detect or treat IL-17F and IL-17A / F heterodimers. In the homodimeric IL-17F protein, the sequence FFQKPES (SEQ ID NO: 1) (in IL-17F Epitope 1) from one monomer and IL-17F Epitope 2 from the other monomer were separated by approximately 21 A. Thus, a chemical linker of about was used to covalently link one macrocycle targeting IL-17F Epitope 1 with one macrocycle targeting IL-17F Epitope 2. The resulting synergistic bioligand was used to detect or treat IL-17F.

[0330] Example 7, Synthesis of Synergistic Bioligand Candidates

[0331] Synergistic bioligand candidates were synthesized with variable length linkers that covalently linked one macrocycle from IL-17F Epitope 1 with one macrocycle from IL-17F Epitope 2. The linkers that connected the two macrocycles were single poly-ethyleneglycolated amino acids (Fmoc-NH-PEG x- propionic acid; x = 1 to 5) or glycine (Gly). PEG linkers are chosen because they come in a variety of lengths that can bridge the 7 to 10 A distance between two epitopes of a protein. PEG is also expected to show anti-biofouling properties.

[0332] Synergistic bi-ligand candidates were first generated from Cy(RRATS) (SEQ ID NO: 9) (targeting IL-17F epitope 1) and Cy(QKHGP) (SEQ ID NO: 15) (targeting IL-17F epitope 2). Cy(QKHGP) (SEQ ID NO: 15) was prioritized based on its high selectivity for IL-17F. Figure 6 shows the structure of the synergistic bi-ligand candidate Biotin-PEG3-Cy(RRATS) (SEQ ID NO: 9)-PEG3-Cy(QKHGP) (SEQ ID NO: 15). Synergistic bi-ligand candidates can also be generated from Cy(RRATS) (SEQ ID NO: 9) (targeting IL-17F epitope 1) and Cy(RSYNK) (SEQ ID NO: 28) (targeting IL-17F epitope 2). Cy(RSYNK) (SEQ ID NO: 28) was prioritized based on its high affinity for IL-17F. x D

[0333] Example 8, Interleukin 17F (IL17F): Estimation of distance between two ligands using crystal structure and PCC assay data, and selection of optimal linker from this information.

[0334] Figure 10 Sequence similarity of IL-17F and IL-17A is shown. Figure 11 Generation of ligands for IL-17F and IL-17A is shown. Bi-ligand PCCs were prepared for IL-17F that bind to two different epitopes, while mono-ligand PC was prepared for IL-17A. Figure 12A The complete structure of IL-17F (PDB: 1JPY) is provided. The left side of the figure shows the two epitopes L1 and L2 that the PCCs target. These are the two epitopes that distinguish IL-17F from IL-17A. Note that these epitopes are not sequence-adjacent, but are proximal within the folded protein structure. Figure 12B is a depiction of the target region of the protein (from crystal structure) with the binding positions and sequences of the two epitope-targeting PCC macrocycle ligands plotted. Each PCC shows a K D value in the range of 15 to 70 nM.

[0335] ​The chemical handles PCC Cy(RRATS) (SEQ ID NO:9) (targeting IL-17F epitope 1) and Cy(RSYNK) (SEQ ID NO:28) (targeting IL-17F epitope 2) were constructed for further elaboration. This is based on their high affinity for IL-17F (K... D =15nM)Cy(RSYNK) (SEQ ID NO:28) is preferred. When PCCs bind to IL-17F, the distance between these chemical handles is expected to be... This predicts that the optimized connector for connecting two PCCs will have a similar performance to... The length. In Figure 12C In this study, the prediction was validated by testing joints of different lengths based on polyethylene glycol (PEG) oligomers. When the joints were of the closest length... When two PCCs are covalently linked together by PEG oligomers, the resulting dual-ligand Cy(RSYNK)(SEQ ID NO:28)-PEG3-Cy(RRATS)(SEQ ID NO:9) exhibits a 250 pM K-value for IL-17F. D This indicates that the affinity relative to each PCC macrocyclic ligand was improved by >100-fold. Shorter or longer linkers produced biligand affinity that was improved compared to any single ligand, but about 10-fold lower than the optimal linker.

[0336] Figure 13 shows the dual-ligand candidate biotin-PEG3-C(RSYNK)(SEQ ID NO:28)-PEG x The structure of -Cy(RRATS)(SEQ ID NO:9)(x=1 to 5).

[0337] Example 9: Connecting two ligands using the best available knowledge results in a dual-ligand pair with improved affinity compared to individual ligands.

[0338] Having more Dual-ligand conjugates of IL-17F with shorter (PEG1, PEG2) or longer (PEG4, PEG5) linkages showed Kc values ​​of 1 to 4 nM against IL-17F. D Values. These values ​​represent an improvement of 5 to 25 times in affinity relative to each PCC macrocyclic ligand.

[0339] Plasmodium falciparum histidine-rich protein-2 (Pf.HRP-2) is a non-structural protein, but it has many epitopes that are repeated throughout its structure. A series of PCCs targeting various Pf.HRP-2 epitopes have been developed. Figure 14 The sequence map of Pf.HRP-2 is provided.

[0340] A cyclic peptide Cy(YKYYR)(SEQ ID NO:29) was developed targeting the AHHAHHAAD (SEQ ID NO:35) epitope. Cy(YKYYR)(SEQ ID NO:29) exhibits an EC50 of 220 nM. 50 Due to the number of epitope repeats, a synergistic conjugate was sought by simply linking two Cy(YKYYR)(SEQ ID NO:29)PCCs together with a connector of approximately 1.5 nm in length. The resulting linked dual-ligand exhibited an EC50 of 20 nM. 50 ( Figure 15 ).

[0341] Develop a circular PCC (EC) with a variable sequence GWNVDL (SEQ ID NO:30) targeting the C-terminal sequence of Pf.HRP-2 (AHHATDAHHAAAHHEAATHCL (SEQ ID NO:36)). 50 =50 nM). This PCC and cyclic YKYYR (SEQ ID NO:29) were developed by screening a library of 10,000 elements of variable-length connector molecules (see above; EC). 50 The junction between 220 nM and 220 nM. The resulting dual ligands exhibited an EC50 of 540 pM. 50 This resulted in a 100-fold improvement relative to the better of the two ligand components. Figure 16 ). sequence list <110> California Institute of Technology Indi Molecular Inc. <120> IL-17F specific trapping agent, composition, and methods of use and manufacture <130> PIIC6180003P-1 <150> 62 / 309,756 <151> 2016-03-17 <150> 62 / 277,430 <151> 2016-01-11 <150> 62 / 192,899 <151> 2015-07-15 <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 1 Phe Tyr Lys Thr His 1 5 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 2 Phe Phe Gln Lys Pro Glu Ser Cys Pro Pro Val Pro Gly Gly 1 5 10 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 3 Asn Glu Asn Gln Arg Val Ser 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 4 Gly Ile Ile Asn Glu Asn Gln Arg Val Ser 1 5 10 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 5 Phe Tyr Lys Thr His 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 6 Phe Tyr Lys Gln His 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 7 Phe Tyr Leu Thr His 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 8 Phe Tyr Leu Gln His 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 9 Arg Arg Ala Thr Ser 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 Arg Arg Ala Gln Ser 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 11 Lys Tyr Gly Glu Val 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 12 Leu Tyr Gly Glu Val 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 13 Val His Lys Ser Gly 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 14 Val His Leu Ser Gly 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 15 Gln Lys His Gly Pro 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 16 Thr Lys His Gly Pro 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 17 Gln Leu His Gly Pro 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 18 Thr Leu His Gly Pro 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 19 Tyr Asp Leu Gln Arg 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 20 Tyr Asp Leu Thr Arg 1 5 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 21 Tyr Asp Lys Gln Arg 1 5 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 22 Tyr Asp Lys Thr Arg 1 5 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 23 Lys Lys Gly Trp Pro 1 5 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 24 Lys Leu Gly Trp Pro 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 25 Leu Lys Gly Trp Pro 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 26 Leu Leu Gly Trp Pro 1 5 <210> 27 <211> 5 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 27 Arg Ser Tyr Asn Leu 1 5 <210> 28 <211> 5 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 28 Arg Ser Tyr Asn Lys 1 5 <210> 29 <211> 5 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 29 Tyr Lys Tyr Tyr Arg 1 5 <210> 30 <211> 6 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 30 Gly Trp Asn Val Asp Leu 1 5 <210> 31 <211> 163 <212> PRT <213> Homo sapiens <400> 31 Met Thr Val Lys Thr Leu His Gly Pro Ala Met Val Lys Tyr Leu Leu 1 5 10 15 Leu Ser Ile Leu Gly Leu Ala Phe Leu Ser Glu Ala Ala Ala Arg Lys 20 25 30 Ile Pro Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu Ser Cys Pro 35 40 45 Pro Val Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu 50 55 60 Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser 65 70 75 80 Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu 85 90 95 Val Val Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn Ala Gln Gly 100 105 110 Lys Glu Asp Ile Ser Met Asn Ser Val Pro Ile Gln Gln Glu Thr Leu 115 120 125 Val Val Arg Arg Lys His Gln Gly Cys Ser Val Ser Phe Gln Leu Glu 130 135 140 Lys Val Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro Val Ile His 145 150 155 160 Arg Val Gin <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 32 Pro Pro Val Pro Gly Gly Ser 1 5 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> misc_feature <222> (1)..(1) <223> Biotin <220> <221> misc_feature <222> (2)..(2) <223> PEG3 <220> <221> misc_feature <222> (5)..(5) <223> L-Azidohomoalanine <400> 33 Xaa Xaa Gly lie Xaa Asn Glu Asn Gin Arg Val Ser 1 5 10 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> misc_feature <222> (1)..(1) <223> Amide <220> <221> misc_feature <222> (2)..(2) <223> L-propargylglycine <220> <221> misc_feature <222> (3)..(7) <223> any one of 17 L-amino acids (excluding Cys, Met and He) <220> <221> misc_feature <222> (3)..(7) <223> triazole cyclization <220> <221> misc_feature <222> (9)..(9) <223> TentaGel S NH2 resin <400> 34 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Xaa 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 35 Ala His His Ala His His Ala Ala Asp 1 5 <210> 36 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 36 Ala His His Ala Thr Asp Ala His His Ala Ala Ala His His Glu Ala 1 5 10 15 Ala Thr His Cys Leu 20 <210> 37 <211> 155 <212> PRT <213> Homo sapiens <400> 37 Met Thr Pro Gly Lys Thr Ser Leu Val Ser Leu Leu Leu Leu Leu Ser 1 5 10 15 Leu Glu Ala Ile Val Lys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly 20 25 30 Cys Pro Asn Ser Glu Asp Lys Asn Phe Pro Arg Thr Val Met Val Asn 35 40 45 Leu Asn Ile His Asn Arg Asn Thr Asn Thr Asn Pro Lys Arg Ser Ser 50 55 60 Asp Tyr Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg Asn Glu 65 70 75 80 Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg His 85 90 95 Leu Gly Cys Ile Asn Ala Asp Gly Asn Val Asp Tyr His Met Asn Ser 100 105 110 Val Pro Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Pro His 115 120 125 Cys Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser Val Gly Cys 130 135 140 Thr Cys Val Thr Pro Ile Val His His Val Ala 145 150 155 <210> 38 <211> 163 <212> PRT <213> Homo sapiens <400> 38 Met Thr Val Lys Thr Leu His Gly Pro Ala Met Val Lys Tyr Leu Leu 1 5 10 15 Leu Ser Ile Leu Gly Leu Ala Phe Leu Ser Glu Ala Ala Ala Arg Lys 20 25 30 Ile Pro Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu Ser Cys Pro 35 40 45 Pro Val Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu 50 55 60 Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser 65 70 75 80 Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu 85 90 95 Val Val Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn Ala Gln Gly 100 105 110 Lys Glu Asp Ile Ser Met Asn Ser Val Pro Ile Gln Gln Glu Thr Leu 115 120 125 Val Val Arg Arg Lys His Gin Gly Cys Ser Val Ser Phe Gin Leu Glu 130 135 140 Lys Val Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro Val He His 145 150 155 160 His Val Gin <210> 39 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 39 Phe Phe Gin Lys Pro Glu Ser Ser Pro Pro Val Pro Gly Gly Ser 1 5 10 15 <210> 40 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 40 Phe Phe Gin Lys Pro Glu Ser Ser Pro Val Ser Pro Gly Pro Gly 1 5 10 15 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 41 Ser Gin Phe Glu Lys Phe Pro Ser Pro Pro Val Pro Gly Gly Ser 1 5 10 15 <210> 42 <211> 309 <212> PRT <213> Plasmodium falciparum <400> 42 Met Val Ser Phe Ser Lys Asn Lys Val Leu Ser Ala Ala Val Phe Ala 1 5 10 15 Ser Val Leu Leu Leu Asp Asn Asn Ser Ala Phe Asn Asn Leu 20 25 30 Cys Ser Lys Asn Ala Lys Gly Leu Asn Leu Asn Lys Arg Leu Leu His 35 40 45 Glu Thr Gin Ala His Val Asp Asp Ala His His Ala His His Val Ala 50 55 60 Asp Ala His His Ala His His Ala Ala Asp Ala His His Ala His His 65 70 75 80 Ala Ala Asp Ala His His Ala His His Ala Ala Asp Ala His His Ala 85 90 95 His His Ala Ala Asp Ala His His Ala His His Ala Ala Tyr Ala His 100 105 110 His Ala His His Ala Ala Asp Ala His His Ala His His Ala Ser Asp 115 120 125 Ala His His Ala Ala Asp Ala His His Ala Ala Tyr Ala His His Ala 130 135 140 His His Ala Ala Asp Ala His His Ala His His Ala Ser Asp Ala His 145 150 155 160 His Ala Ala Asp Ala His His Ala Ala Tyr Ala His His Ala His His 165 170 175 Ala Ala Asp Ala His His Ala Ala Asp Ala His His Ala Thr Asp Ala 180 185 190 His His Ala His His Ala Ala Asp Ala Arg His Ala Thr Asp Ala His 195 200 205 His Ala Ala Asp Ala His His Ala Thr Asp Ala His His Ala Ala Asp 210 215 220 Ala His His Ala Ala Asp Ala His His Ala Thr Asp Ala His His Ala 225 230 235 240 Ala Asp Ala His His Ala Thr Asp Ala His His Ala Ala Asp Ala His 245 250 255 His Ala Ala Asp Ala His His Ala Thr Asp Ala His His Ala His His 260 265 270 Ala Ala Asp Ala His His Ala Ala Ala His His Ala Thr Asp Ala His 275 280 285 His Ala Thr Asp Ala His His Ala Ala Ala His His Glu Ala Ala Thr 290 295 300 His Cys Leu Arg His 305

Claims

1. A method for generating a synthetic trapping agent that specifically binds to IL-17F, the method comprising: (a) Selecting a first ligand that binds to a first epitope on the IL-17F, wherein the amino acid sequence of the first ligand comprises RRATS (SEQ ID NO:9) or RRAQS (SEQ ID NO:10), wherein the first ligand is cyclic; (b) Selecting a second ligand that binds to the second epitope on IL-17F, wherein the amino acid sequence of the second ligand comprises TKHGP (SEQ ID NO:16), QKHGP (SEQ ID NO:15), KKGWP (SEQ ID NO:23), or RSYNK (SEQ ID NO:28), wherein the second ligand is cyclic; and (c) The connector is covalently bound to the first ligand and the second ligand, thereby producing the synthetic capture agent that specifically binds to the IL-17F.

2. The method according to claim 1, further comprising: Select a connector whose length allows the connector to simultaneously bind the first ligand and the second ligand when the first ligand and the second ligand bind to the first epitope and the second epitope on the IL-17F.

3. The method of claim 2, wherein the connector is selected by evaluating the length of the connector, wherein the length of the connector is evaluated by determining which side of the first ligand binds to the first epitope and which side of the second ligand binds to the second epitope, and by measuring the distance between the binding sides of the first epitope and the second epitope on the folded structure of the IL-17F.

4. The method of claim 2, wherein the adapter is selected by screening a library of candidate adapter molecules, the candidate adapter molecules connecting the first ligand and the second ligand for binding the IL-17F.

5. The method of claim 2, wherein the connector is selected by testing candidate trapping agents, the candidate trapping agents being connected to the candidate connector for binding the IL-17F.

6. The method according to claim 2, wherein the distance between the first tabletop and the second tabletop is to 7. The method of claim 2, wherein the distance between the first tabletop and the second tabletop is to 8. The method of claim 2, wherein the distance between the first tabletop and the second tabletop is to 9. The method of claim 2, wherein the distance between the first tabletop and the second tabletop is 10. The method of claim 1, wherein the connector is 10 to 50% longer than the distance between the first and second positions.

11. The method of claim 1, wherein the connector is 5 to 25% longer than the distance between the first and second positions.

12. The method of claim 1, wherein the connector is 1 to 10% longer than the distance between the first and second positions.

13. The method of claim 1, wherein the synthesized trapping agent has a greater binding affinity for the IL-17F than any of the ligands.

14. The method of claim 13, wherein the synthesized trapping agent has a binding affinity of at least 50% for a fully co-conjugated conjugate.

15. The method of claim 13, wherein the synthesized trapping agent has a binding affinity of at least 75% for a fully co-conjugated conjugate.

16. The method of claim 13, wherein the synthesized trapping agent has a binding affinity of at least 90% for a fully co-conjugated conjugate.

17. The method of claim 1, wherein the first epitope and the second epitope on IL-17F are synthetic epitopes, wherein each of the synthetic epitopes comprises a sequence of at least 20 amino acids of full-length IL-17F, wherein at least one amino acid of each of the synthetic epitopes comprises an azide group or an acetylene group.

18. The method of claim 17, wherein the full-length IL-17F is a naturally occurring protein.

19. The method of claim 17, wherein the synthesized epitope comprises a sequence of at least 50 amino acids of full-length IL-17F.

20. The method of claim 17, wherein the synthesized trap binds the synthesized epitope and the IL-17F with a binding affinity of at least 50% of the binding affinity of a fully co-conjugated conjugate.

21. The method of claim 17, wherein the synthesized trap binds the synthesized epitope and the IL-17F with a binding affinity of at least 75% of the binding affinity of a fully co-conjugated conjugate.

22. The method of claim 17, wherein the synthesized trap binds the synthesized epitope and the IL-17F with a binding affinity of at least 90% of the binding affinity of a fully co-conjugated conjugate.

23. The method of claim 1, wherein the connector is PEG1, PEG2, PEG3, PEG4 or PEG5.

24. The method of claim 1, wherein the connector comprises a peptide.

25. The method of claim 1, wherein the connector comprises an amino acid.

26. The method of claim 25, wherein the connector is glycine.

27. The method of claim 1, wherein the connector comprises an alkylene portion.

28. The method of claim 1, wherein the selection of the first ligand is achieved by screening a first peptide library for a first synthetic epitope and identifying a first library peptide coupled to the first synthetic epitope in the screening, wherein the first synthetic epitope is a peptide comprising a sequence of the first epitope and a click handle, wherein each peptide in the first peptide library comprises a click handle, wherein the click handle of the first synthetic epitope and the click handle of the first library peptide are (1) an azide click handle and an acetylene click handle, or (2) an acetylene handle and an azide click handle, respectively.

29. The method of claim 28, wherein the selection of the second ligand is achieved by screening a second peptide library for a second synthetic epitope and identifying a second library peptide coupled to the second synthetic epitope in the screening, wherein the second synthetic epitope is a peptide comprising a sequence of the second epitope and a click handle, wherein each peptide in the second peptide library comprises a click handle, wherein the click handle of the second synthetic epitope and the click handle of the second library peptide are (1) an azide click handle and an acetylene click handle, or (2) an acetylene click handle and an azide click handle, respectively.

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