Peptides binding LRRC15 and uses thereof

Abstract

The present disclosure relates to peptides that bind to LRRC15. In some cases, the peptides are disulfide-constrained peptides (DCP). In some cases, the peptides bind to murine LRRC15 (muLRRC15).

Description

PEPTIDES BINDING LRRC15 AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims the benefit of U.S. Provisional Application No. 63 / 565,302, filed March 14, 2024 and U.S. Provisional Application No. 63 / 686,649, filed August 23, 2024. The entire disclosures of these prior applications are hereby incorporated by reference each in their entirety.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on March 12, 2025, is named “01164-0035-00PCT. xml” and is 63,149 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD

[0003] The present disclosure relates to peptides that bind to LRRC15. In some cases, the peptides are disulfide-constrained peptides (DCP). In some cases, the peptides bind to murine LRRC15 (muLRRC15).BACKGROUND

[0004] The cell-surface receptor leucine-rich repeat-containing 15 (LRRC15; also known as Lib) is a significant marker expressed on cancer-associated fibroblasts within various solid tumors. For example, overexpression of LRRC15 on cancer-associated fibroblasts has been found to be positively correlated with cancer grade and outcome. Thus, reagents that bind LRRC15 may be useful in applications targeting such tumor-associated cell populations. For instance, such reagents can be used as a marker for such tumor-associated cells in vitro and in vivo, such as in animal model studies. There is a need to develop small, stable molecules that selectively bind muLRRC15. However, obtaining small, stable molecules that bind specifically and with high affinity to protein targets such as murine LRRC15 (muLRRC15) has been challenging.SUMMARY

[0005] The present disclosure relates to particular DCPs binding to muLRRC15, and methods of making and using such DCPs. Disulfide-constrained peptides (DCPs) represent an attractive modality for diagnostic reagents to their improved tissue penetration, high chemical and thermal stability, and tolerance to molecular engineering (Wang et al. Nat Chem Biol 2018). For example, peptides such as DCPs may be particularly useful in diagnostic assays due to their small size, for example compared to larger molecules such as antibodies or antibody binding domains, such as VHH domains (camelid antibodies or nanobodies), Fab, or single chain Fv (scFv) domains. In particular, DCPs have previously been engineered using combinatorial libraries to identify binders against biological relevant protein targets (Thakur et al. ACS Chem Biol 2023). However, in contrast to other binding molecules such as antibodies, identifying suitable target binding reagents from DCP libraries has been relatively difficult.

[0006] Exemplary embodiments include the following:1. A disulfide-constrained peptide (DCP) that binds to murine leucine-rich repeat containing 15 (muLRRC15), comprising the amino acid sequence of any one of SEQ ID NOs: 26, 18, 30, 3-17, 19-25, 27-29, or 31-34, or comprising an amino acid sequence of any one of SEQ ID NOs: 35-41.2. The DCP of embodiment 1, wherein the DCP binds to muLRRC15 with a Kd of 500 nM or less, 250 nM or less, 150 nM or less, 100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less.3. The DCP of embodiment 1 or 2, wherein the DCP binds to a portion of muLRRC15 comprising SEQ ID NO: 2.4. The DCP of any one of embodiments 1-3, wherein the DCP binds to muLRRC15 on the surface of fibroblast cells.5. The DCP of any one of embodiments 1-4, wherein the DCP is 30-32 amino acids in length.6. The DCP of any one of embodiments 1-5, wherein the DCP comprises an amidated C-terminus.7. The DCP of any one of embodiments 1-6, wherein the DCP comprises cysteine residues at positions 2, 11, 17, 21, 23, and 29, optionally wherein the DC comprises 30 amino acids in length.8. The DCP of any one of embodiments 1-7, wherein the DCP further comprisesor is attached to a label.9. The DCP of embodiment 8, wherein the label is or comprises a fluorophore, dye, prosthetic group, and / or radiolabel.10. The DCP of embodiment 9, wherein the radiolabel is a radionuclide, optionally wherein the radionuclide is18F.11. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 26, 18, 30, 3-17, 19-25, 27-29, or 31-34.12. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 26-31, 9, 18, or 34.13. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 26.14. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 18.15. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 30.16. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 31.17. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 28.18. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 27.19. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 34.20. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 9.21. The DCP of any one of embodiments 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 29.22. The DCP of any one of embodiments 1-21, wherein the DCP comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13-34, and wherein the DCP comprises six cysteine residues and wherein disulfide bonds occur between the first and fourth, second and third, and fifth and sixth cysteine residues of the sequence.23. A disulfide-constrained peptide (DCP) that binds to murine leucine-richrepeat-containing 15 (muLRRC15) with a Kd of 500 nM or less, 250 nM or less, 150 nM or less, 100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less.24. The DCP of embodiment 23, wherein the DCP binds to a portion of muLRRC15 comprising SEQ ID NO: 2.25. The DCP of embodiment 23 or 24, wherein the DCP binds to muLRRC15 on the surface of fibroblast cells.26. The DCP of any one of embodiments 23-25, wherein the DCP is 30-32 amino acids in length.27. The DCP of any one of embodiments 23-26, wherein the DCP comprises an amidated C-terminus.28. The DCP of any one of embodiments 23-27, wherein the DCP comprises six cysteine residues, wherein disulfide bonds occur between the first and fourth, second and third, and fifth and sixth cysteine residues of the sequence.29. The DCP of any one of embodiments 23-28, wherein the DCP comprises cysteine residues at positions 2, 11, 17, 21, 23, and 29, optionally wherein the DC comprises 30 amino acids in length.30. The DCP of any one of embodiments 23-29, wherein the DCP further comprises or is attached to a label.31. The DCP of embodiment 30, wherein the label is or comprises a fluorophore, dye, prosthetic group, and / or radiolabel.32. The DCP of embodiment 32, wherein the radiolabel is a radionuclide, optionally wherein the radionuclide is18F.33. A complex comprising the DCP of any one of embodiments 1-32 and muLRRC15 or the extracellular domain of muLRRC15.34. A method of detecting muLRRC15 in vitro, comprising contacting the muLRRC15 with the DCP of any one of embodiments 1-32.35. The method of embodiment 34, wherein the muLRRC15 is expressed on the surface of a cell.36. A method of detecting muLRRC15 in vivo, comprising administering the DCP of any one of embodiments 1-32 to an animal in an amount sufficient to detect muLRRC15 in the animal or in tissues obtained from the animal.37. A method of detecting muLRRC15 in vivo, comprising: a. Administering the DCP of any one of embodiments 8-10 or 31-34 to ananimal; and b. Detecting binding of the DCP to muLRRC15 in the animal or in tissues obtained from the animal, wherein the detection of the binding indicates the presence of muLRRC15 in the animal or in tissues obtained from the animal.38. The method of embodiment 37 or 38, wherein the detecting comprises performing a positron emission tomography (PET) scan or positron emission tomography / computed tomography (PET / CT) scan of the animal.39. The method of any one of embodiments 35-38, wherein the animal is a mouse.40. A polynucleotide encoding the DCP of any one of embodiments 1-32.41. A vector comprising the polynucleotide of embodiment 40.42. A host cell comprising the polynucleotide or vector of embodiment 40 or 41.43. A method of making a DCP of any one of embodiments 1-32, comprising incubating the host cell of embodiment 42 such that the cell expresses the DCP, and optionally isolating the DCP from the host cell.44. A method of making a DCP of any one of embodiments 1-32, comprising chemically synthesizing the DCP.45. A kit comprising the DCP of any one of embodiments 1-32, and optionally further comprising a label, detection assay reagents, and / or instructions for use.

[0007] Additional aspects and embodiments of the disclosure are described elsewhere herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 shows a cartoon overview of phage display for screening the EETI phage libraries against mouse LRRC15. The EETI library designs are shown in Table 1 and SEQ ID NOs: 35-41. Biotinylated mouse LRRC15 was used as the target to capture DCP binders.Mouse LRRC15 amino acid (AA) numbering is based on Uniprot (Q80X72; SEQ ID NO: 1).

[0009] FIG. 2 shows an overview of the high-throughput screening and machine learning platform used in this study for the discovery of DCP binders to mouse LRRC15. 1st generation DCPs are identified from naive phage libraries and binding is validated using in vitro biophysical techniques. Using structure activity relationship analysis, 2nd generation phage libraries are designed and screened to identify enriched sequence motifs. A machine learning model is trained using NGS data from the 2nd generation phage display libraries topredict binding potential of DCP sequences and validated using the 1st generation binding data. The model then generates a large number of novel DCP sequences that were not present in the training or test data set, and the sequences with the highest predicted binding potential are used to build a target-focused library that can be screened against mouse LRRC15.

[0010] FIGS. 3A-3E: FIGS. 3A-3D show single cycle SPR binding sensorgrams showing 1st generation DCPs binding to immobilized mouse LRRC15. All peptides were tested from 10 pM. Hits were identified from the EETI phage library. Binding affinities were calculated using the steady-state affinity model. Data for four different DCPs are shown: EE-E12 (SEQ ID NO: 4; FIG. 3 A), EE-E3 (SEQ ID NO: 7; FIG. 3B), EE-D1 (SEQ ID NO: 3; FIG. 3C), and EE-E1 (SEQ ID NO: 5; FIG. 3D). FIG. 3E shows a competition-based ELISA assay depicting binding of EE-D1 -phage to immobilized muLRRC15 in the presence of synthesized EETI-based 1stgeneration DCPs (data are representative of one independent experiment).

[0011] FIG. 4 shows binding isotherms of EE-D1 (SEQ ID NO: 3) binding to mouse LRRC15. Thermodynamic parameters enthalpy (AH) and entropy (AS) are shown.

[0012] FIGS. 5A-5B: FIG. 5 A shows combinatorial Ala-scanning of EE-D1 (SEQ ID NO: 3) displayed on g8. Values represent the wild type / Ala ratio score for each residue based on the NGS data after two rounds of panning against mouse LRRC15. Residues where Ala was enriched compared to the parent residue have a negative number (light). Residues where the parent residue was enriched compared to Ala have a positive number (dark). FIG. 5B shows library designs of second generation DCP phage libraries.

[0013] FIGS. 6A-6H: FIGS. 6A-6C show evaluations of performance of the modeling. FIG. 6A shows an evaluation of the ML model performance by comparing the ML-derived predicted binding score (x-axis) to the experimentally determined SPR-affinity data (y-axis) for the four 1st generation DCP sequences. FIG. 6B provides a summary of the predicted binding scores, with respect to peptide length, of the computationally generated sequences that passed the criteria of mouse LRRC15 predicted binding > 2.1 and non-specific binding < -0.1. FIG. 6C shows predicted binding scores, with respect to peptide length, of the top 50,000 peptides identified from the computationally generation sequences. FIGS. 6D-6G show the analysis of the accuracy of the ML model and chemical features of the sequences of the ML-library. FIG. 6D shows the predicted non-specific binding score (binding to streptavidin control) of the 50,000 sequences in the ML-library (comprising peptides of 30, 31 and 32 residues in length) all have a non-specific binding score < -0.1. FIGS. 6E-6F shows 2D UMAP plot reflecting the chemical properties of the DCP sequences enriched during 2ndgeneration phage panning compared to the 50,000 member ML-library. Each dot represents one sequence and the distance between any two points approximates their embedding-based similarity in a high-dimensional space. Sequences are colored based on library design for the 2ndgeneration phage libraries (FIG. 6E), or sequence length (FIG. 6F) for the ML library. For reference, the top 1st generation hit EE-D1 is highlighted. FIG. 6G shows sequences of the ML-library, separated by DCP sequence lengths. Residues that contained mutations in the ML-library are designated “X”. FIG. 6H shows the same data from FIG. 6 A including error bars. The r value is the average plus standard deviation.

[0014] FIGS. 7A-7F show single cycle SPR binding sensorgrams showing 2nd generation DCPs binding to immobilized mouse LRRC15. DCPs EE-D1 (SEQ ID NO: 3; FIG. 7 A), EE- D 1-8- 17 (SEQ ID NO: 11; FIG. 7B), EE-D 1-8-9 (SEQ ID NO: 10; FIG. 7D), and EE-D 1-7- 17 (SEQ ID NO: 12; FIG. 7E), and EE-D 1 -7-6 [D8E] (SEQ ID NO: 9; FIG. 7F) were tested from 10 pM. DCPs EE-DI-7-6 (SEQ ID NO: 8; FIG. 7C) and EE-Dl-7-6[D8E] (SEQ ID NO: 9; FIG. 7F) were tested from 100 nM. Binding affinities were calculated using both kinetic and steady state affinity models.

[0015] FIGS. 8A-8L show single cycle SPR binding sensorgrams showing ML-YSD DCPs binding to immobilized mouse LRRC15. DCPs were tested from 100 nM. Binding affinities were calculated using both kinetic and steady state affinity models. The following DCPs are shown: ML-YSD-1 (FIG. 8A; SEQ ID NO: 13), ML-YSD-2 (FIG. 8B; SEQ ID NO: 14), ML-YSD-3 (FIG. 8C; SEQ ID NO: 15), ML-YSD-5 (FIG. 8D; SEQ ID NO: 16), ML-YSD-6 (FIG. 8E; SEQ ID NO: 17), ML-YSD-7 (FIG. 8F; SEQ ID NO: 18), ML-YSD-8 (FIG. 8G; SEQ ID NO: 19), ML-YSD- 10 (FIG. 8H; SEQ ID NO: 21), ML-YSD- 12 (FIG. 81; SEQ ID NO: 23), ML-YSD-11 (FIG. 8J; SEQ ID NO: 22), ML-YSD-13 (FIG. 8K; SEQ ID NO: 24), ML-YSD-9 (FIG. 8L; SEQ ID NO: 20).

[0016] FIGS. 9A-9J show single cycle SPR binding sensorgrams showing ML-PD DCPs binding to immobilized mouse LRRC15. DCPs were tested from 30 nM. Binding affinities were calculated using kinetic affinity models. The following DCPs are shown: ML-PD-02 (FIG. 9A; SEQ ID NO: 25), ML-PD-03 (FIG. 9B; SEQ ID NO: 26), ML-PD-04 (FIG. 9C; SEQ ID NO: 27), ML-PD-05 (FIG. 9D; SEQ ID NO: 28), ML-PD-07 (FIG. 9E; SEQ ID NO: 29), ML-PD-08 (FIG. 9F; SEQ ID NO: 30), ML-PD-09 (FIG. 9G; SEQ ID NO: 31), ML-PD- 12 (FIG. 9H; SEQ ID NO: 32), ML-PD-13 (FIG. 91; SEQ ID NO: 33), ML-PD-14 (FIG. 9J; SEQ ID NO: 34).

[0017] FIGS. 10A-10C show mouse plasma stability of the peptides DI -7-6[D8E] (SEQ ID NO: 9; FIG. 10A), ML-YSD-07 (SEQ ID NO: 18; FIG. 10B), and ML-PD-03 (SEQ IDNO: 26; FIG. IOC). The calculated half-lives (tl / 2) are shown. Data points are shown as the average ± SD of three replicates.

[0018] FIGS. 11 A-l ID: FIGS. 11 A-l IB show representative histograms of flow cytometry plots showing binding of DCP Dl-7-6[D8E]-A488 to NIH / 3T3 cell lines with (FIG. 11 A) and without (FIG. 1 IB) muLRRC15. FIG. 11C shows a representative histogram of a flow cytometry plot showing binding of ML-PD-03-N488 to muLRRC15+ / - NH4 / 3T3 cell lines and KPR3070. FIG. 1 ID shows fluorescence micrographs showing location of ML-PD-03-N488 (DCP) compared to cell nuclei (NucBlue) and outer cell membranes (CellBrite) or both (Overlay). Top row shows staining of NIH / 3T3 cells with NucBlue, CellBrite, the DCP, and an overlay of the stains, respectively left to right. Bottom row shows the same staining of 3T3 cells expressing muLRRC15 (3T3-muLRRC15). The cells were monitored 1 h after adding 500 nM ML-PD-03-N488. As the figure shows, the DCP only stained cells expressing muLRRC15 and co-localized with the cell membrane staining in the 3T3-muLRRC15 cells.

[0019] FIG. 12 provides a schematic showing an SDS-PAGE analysis of mouse LRRC15:EE-D1 DSS cross-linking, on the left, with the detected peptide fragmentation, following protease digestion, showing EE-D1 K12 cross-linking to mouse LRRC15 K86 (residues 4-18 of EE-D1; SEQ ID NO: 43; residues 81-89 of muLRRC15 (SEQ ID NO: 44). The cross-linking site was determined by LC-MS / MS, and a cartoon representation at right highlighted the mouse LRRC15 cross-linking site K86. The mouse LRRC15 structure was generated using (AlphaFold2).

[0020] FIGS. 13A-13C are based on the HDX-MS data. FIG. 13A shows HDX-MS data analysis. The change in the calculated loglO HDX protection factors for mouse LRRC15 residues in the presence of DCP Dl-7-6[D8E] (loglOAPF). Positive loglOAPF values correspond to residues that more slowly exchange backbone amide hydrogen atoms with solvent deuterons in the presence of Dl-7-6[D8E], therefore, highlight the shielded binding site. Magenta - manually set to limit value. Grey shaded region - uncertainty. Black trace - empirical quantification from data. Mouse LRRC15 Y22 residue (Uniprot numbering; SEQ ID NO: 1) in the HDX-MS data is numbered as residue 1. In FIG. 13B, loglOAPF values are mapped onto the mouse LRRC15 structure (AlphaFold2). The mouse LRRC15 cross-linking K86 residue is highlighted in green. In FIG. 13C, mouse LRRC15 residues that are shielded (positive loglOAPF) after the addition of Dl-7-6[D8E] are colored red and underlined. Residues are numbered based on Uniprot (see SEQ ID NO: 1) and the sequence shown in FIG. 13C is provided in SEQ ID NO: 2.

[0021] FIGS. 14A-14N show structural and HDX-MS data on DCP-muLRRC15 binding. FIG. 14A shows the X-ray crystal structure of the ML-YSD-07:muLRRC15-Fab-El complex. Fab-El was used as a structural chaperone to aid with crystallography efforts. Surface representation of muLRRC15 with ML-YSD-07 (ribbon cartoon above the surface) and Fab-El (ribbon cartoon at bottom left). The ML-YSD-07 binding interface with muLRRC15 is further shown in an expanded view. FIG. 14B shows the X-ray crystal structure of the ML-YSD-07:muLRRC15-Fab-El complex. Cartoon representation of muLRRC15 with ML-YSD-07 bound to the concave face and Fab-El bound to the muLRR15 C-terminal region of the extracellular domain. Fab-El was used as a structural chaperone to aid with crystallography efforts. FIG. 14C shows ML-YSD-07 binding interface with muLRRC15, with muLRRC15 contact residues within 4 A of ML-YSD-07 labeled in black italics. The ML-YSD-07 contact residues are represented as sticks and labeled in light gray. FIG. 14D shows an overlay of HDX-MS analysis of the complex of DCP Dl-7-6[D8E] and muLRRC15 against the X-ray crystal binding contact information for the ML-YSD- 07:muLRRC15-Fab-El complex. The shaded regions marked “no sequence coverage” show uncertainty; the black trace marked “slower H-D exchange residues” denotes areas of protection from hydrogen-deuterium exchange, while residues that are within 5 A of ML- YSD-07 interface from the crystal structure are identified with a cross on the x-axis. FIG. 14E shows a comparison of the tertiary structure and disulfide-bond connectivity of the parent EETI and ML-YSD-07 peptides, based on the ML-YSD-07:muLRRC15-Fab-El complex structural data. Disulfide bonds are represented as yellow (light) sticks. ML-YSD-07 cysteines are numbered in order from the N-terminus. 2-D representation of the ML-YSD-07 sequence and cysteine connectivity shows a cysteine knot fold is no longer present, instead forming a ladder-like arrangement. FIG. 14F shows ML-YSD-07 residues that form binding contacts with muLRRC15 as indicated with green circles (SEQ ID NO: 18). FIGS. 14G-14M show the key binding interactions between ML-YSD-07 (top) and muLRRC15 (bottom), with electrostatic (dotted lines), H-bonds (dotted line in FIG. 14L between Y13 and Y157 of muLRRC15), and 7t-stacking (dotted line in FIG. 14L from Y13 to Y205 of muLRRC15) interactions highlighted. FIG. 14G shows residue E10 of M-YSD-07 interacts with K177, N179, and R203 of muLRRC15. FIG. 14H shows W7, W9, W25 account for the majority of the binding interface and shallow pockets on the muLRRC15 surface. Surface patches with the imidazoles of W7 and W9 interact with the side-chains of E153 and Q155, respectively. muLRRC15 residues within 4 A of the ML-YSD-07 Trp’s are represented as yellow sticks. FIG. 141 shows R4 of ML-YSD-07 packed net to R249 of muLRRC15. E3 is solventexposed. FIG. 14J shows Y5 and W6 of ML-YSD-07 pack in the hydrophobic core of the DCP and stabilize the DCP fold and binding conformation. The C-terminus of ML-YSD-07 is near the muLRRC15 surface, whereas the N-terminus is solvent exposed and ideally suited for functionalization. FIG. 14K shows T24, V27 and T28 of ML-YSD-07 pack together and potentially stabilize the turn in loop 5 that enables W25 to contact muLRRC15. FIG. 14L shows R12 of ML-YSD-07 interacts with E206 and E230 of muLRRC15, and Y13 of ML- YSD-07 interacts with Y157 (H-bond) and Y205 (it -stacking) of muLRRC15. FIG. 14M shows DI 4, T15 and D16 of ML-YSD-07 form electrostatic interactions with K273 of muLRRC15. FIG. 14N shows ML-YSD-07 having mutated residues W9A and W25A (ML- YSD-07[W9A][W25A]) does not bind to muLRRC15. Representative SPR sensorgram showing complete lack of binding of ML-YSD-07[W9A][W25A] to immobilized muLRRC15.

[0022] FIGS. 15A-15C show solution-based disulfide mapping experiments that identify a distinct connectivity from the parent EETI-II scaffold. FIG. 15A provides a diagram of the partial reduction method used to determine the disulfide connectivity of peptide ML-PD-05. Cysteine modifications identified are highlighted by light gray circles with black outline for N-ethylmal eimide or dark gray circles with gray outline for iodoacetamide. FIG. 15B shows pairwise disulfide bonds of ML-PD-05 identified by TCEP reduction and pH specific Cys- alkylation. Disulfide bonds are represented as light sticks. Cysteines are numbered in order from the N-terminus. Cysteine modifications identified are highlighted by light gray circles with black outline for N-ethylmaleimide or dark gray circles with gray outline for iodoacetamide. FIG. 15C shows an MS2 diagram showing the b (bottom) and y (top) fragmentation ions identified by PMI Byos for peptide ML-PD-05 (SEQ ID NO:28). Byonic scores indicate peptide spectrum match correctness, with scores >400 considered very good with low rates of false PSM assignment.

[0023] FIG. 16A-16E: FIG. 16A shows the synthetic strategy to functionalize DCPs with RESCA. FIGS. 16B-16E show positron emission tomography (PET) imaging data of RESCA-18F-modified ML-PD-03 peptide (i.e., ML-PD-03 -RESCA- 18F or [18F]A1F- RESCA-ML-PD-03) in tumor-bearing mice and a schematic of the method. FIG. 16B shows a schematic of the method used. FIG. 16C shows an image taken 60 minutes after administration of the DCP in a representative mouse. The tumor is highlighted with a white dotted box. FIG. 16D and FIG. 16E show graphs and bar plots, respectively, depicting the concentration of radioactivity in various regions of interest.

[0024] FIGS. 17A-17H: FIG. 17A shows DCP ML-YSD-07 functionalized at the N- terminal amide with the chelating ligand RESCA, which captures ([18F]A1F)2+. The kinetic constants konand koff, and the calculated equilibrium dissociation constant KD for RESCA- ML-YSD-07 binding to muLRRC15 are shown as average ± s.d. (N = three independent experiments). FIG. 17B shows surface plasmon resonance (SPR) sensorgrams and dose response binding of RESCA-ML-YSD-07 to immobilized muLRRC15. Experimental curves are shown in black. Kinetic binding fits, based on 1 : 1 model, are shown in red. Associated binding kinetics konand koff, and subsequent equilibrium dissociation constant KD are shown as average ± s.d. (N = 3 independent experiments). FIGS. 17C-17D show positron emission tomography (PET) / computed tomography (CT) imaging with [18F]A1F-RESCA-ML-YSD-O7 of KPR3070 pancreatic ductal adenocarcinoma (PDAC) tumors in mice. FIG. 17C shows PET / CT images from two mice growing subcutaneous KPR3070 tumors taken 45-60 min after [18F]A1F -RESCA-ML-YSD-07 administration. The tumors are highlighted with a white dotted box. B = bladder; K = kidneys. FIG. 17D shows radioactivity concentrations in the tumor the regions of interest (tumor and muscle) were measured as a percentage of the injected dose per gram (% ID / g) during the 60 minute dynamic PET scan (N = five mice). FIG. 17E shows PET / CT images from all five KPR3070 tumor-bearing mice (see FIG. 17C) at 45-60 min after [18F]A1F -RESCA-ML-YSD-07 administration. The tumors are highlighted with a white dotted box. B = bladder; K = kidneys. FIGS. 17F-17H show biodistribution of [18F]A1F-RESCA-ML-YSD-O7 imaged in mice growing KPR3070 pancreatic ductal adenocarcinoma (PDAC) tumors. FIG. 17F shows binding potential (BPND) of [18F]A1F- RESCA-ML-YSD-07 to tumor tissue using the Logan graphical analysis, referenced to healthy muscle tissue. FIG. 17G shows radioactivity concentrations of [18F]A1F-RESCA-ML- YSD-07 at 45-60 minutes post administration in the regions of interest measured as a percentage of the injected dose per gram (% ID / g). FIG. 17H shows radioactivity concentrations of [18F]A1F-RESCA-ML-YSD-O7 in the regions of interest during a 60 minute dynamic positron emission tomography (PET) scan. (N = 5 mice).

[0025] FIGS. 18A-18B show the binding affinity of select DCPs for huLRRC15. Figs. 18A-18B shows representative SPR sensorgrams showing dose response binding of ML- YSD-07 (FIG. 18 A) and ML-PD-03 (FIG. 18B) to immobilized huLRRC15.DETAILED DESCRIPTIONI. DEFINITIONS

[0026] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0027] In this application, the use of “or” means “and / or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0028] As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an” element herein means one or more than one such element, unless the context clearly dictates otherwise.

[0029] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0030] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0031] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acids linked by peptide bonds. The term “peptide” as used herein refers to a relatively short polypeptide chain of generally two to fifty amino acids. Examples of peptides herein can comprise, for instance, amino acid chains of 2 to 50, 2 to 40, 2 to 35, 10 to 35, 15 to 35, 20 to 35, 20 to 30, 25 to 35, 30 to 35, 30 to 32, or 28 to 32 amino acids in length. In some embodiments, a peptide can be chemically modified, such as by the presence of C-terminal or N-terminal modification, such as a C-terminal amidation, or a side-chain modification.

[0032] A “disulfide-constrained peptide” or “DCP” refers to a peptide comprising orpredicted to comprise at least one disulfide bond between two cysteine residues within the peptide. In some cases, such a DCP may comprise or be predicted to comprise more than one disulfide bond, such as two or three or four disulfide bonds. A DCP that is “predicted to” comprise a disulfide bond is one that molecular modeling, chromatography, mass spectrometry, or similar modeling or experimental data indicates comprises at least one disulfide bond.

[0033] As used herein, “LRRC15” or “leucine-rich repeat-containing 15” protein refers to a particular cell-surface receptor protein. In certain embodiments, the LRRC15 is the murine LRRC15 protein also called “muLRRC15” herein. An exemplary muLRRC15 sequence is that of SEQ ID NO: 1 herein, which provides the immature form of the protein including the N-terminal signal sequence of residues 1-21. An exemplary mature form of muLRRC15, thus is provided by SEQ ID NO: 1 from position 22 to the C-terminal end.

[0034] The terms “domain” or “region” of a protein refer to a portion of a protein, that may in some instances have a particular function. Examples of domains include, for example, portions of a protein that resemble specific proteins or protein domains, such as a growth factor domain, or such as an antibody constant region domain, or the like.

[0035] An “extracellular domain” or “extracellular region” of a polypeptide is a domain of a transmembrane protein that is found in the extracellular space. An exemplary extracellular domain of muLRRC15 comprises the amino acid sequence of SEQ ID NO: 45. An N-terminal portion of the muLRRC15 extracellular domain is also shown in SEQ ID NO: 2, or FIG. 13C.

[0036] A “signal sequence” or “signal peptide” or “leader sequence” or “leader peptide” herein refers to a short N-terminal domain of a translated polypeptide that directs the polypeptide to the cellular membrane or to an intracellular membrane. The signal sequence is typically cleaved from the polypeptide to form the mature form of the polypeptide.

[0037] In this disclosure, “binds” or “binding” or “specific binding” and similar terms, when referring to a molecule such as a peptide that “binds” to an LRRC15 protein or protein domain or other molecule, for example, means that the binding affinity is sufficiently strong that the interaction between the members of the binding pair cannot be due to random molecular associations (i.e. “nonspecific binding”). Thus, the binding is selective or specific and is not due to simple nonspecific aggregation.

[0038] A “label” herein refers to a molecule that may be used for detecting the presence of a molecule, such as LRRC15 or a peptide herein, for example, in a detection assay. A label may be attached covalently to a molecule or noncovalently, either directly or indirectlyvia an intervening molecule such as a linker. Or it may be incorporated into a molecule such as being included in one or more amino acids or atoms therein or at the N- or C-terminus of a peptide or protein. Examples of labels include radioisotopes, fluorescent molecules, dyes, haptens, and the like, as well as molecules that specifically bind to or react with detectable molecules such as dyes, fluorescent molecules, and the like. Labels herein, for example, may in some cases comprise one or more “prosthetic group,” which refers to a non-amino acid component that facilitates attachment of a radionuclide to the protein or peptide.

[0039] A “detection assay” herein refers to an experiment intended to detect the presence of a particular molecule, such as LRRC15 or a peptide binding to LRRC15 or the like.

[0040] Other definitions are included in the sections below, as appropriate.II. PEPTIDES BINDING TO LRRC15 AND METHODS OF MAKING PEPTIDES

[0041] The disclosure relates, inter alia, to disulfide-constrained peptides (DCP) that bind to murine leucine-rich repeat containing 15 (muLRRC15). In some embodiments, the DCPs bind to muLRRC15 with a Kd of 500 nM or less, 250 nM or less, 150 nM or less, 100 nM or less, 50 nM or less, 25 nM or less, or with a Kd of 10 nM to 500 nM, or 100-500 nM, or 25- 500 nM, or 25-250 nM, or 10-100 nM, or 10-250 nM, or 10-50 nM, or 50-100 nM, or 50-250 nM, or 100-250 nM. In some embodiments, they bind to muLRRC15 with a Kd of 20 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less, such as a Kd of 0.1 nM to 20 nM, or 0.2-20 nM, or 0.1-5 nM, or 1-25 nM, or 1-20 nM, or 1-10 nM, or 0.1-10 nM, or 0.1-1 nM. In some embodiments, the Kd of a DCP to muLRRC15 is determined by surface plasmon resonance (SPR).

[0042] In some cases, the muLRRC15 is on the surface of a cell, such as a fibroblast cell. Thus, in some cases, the DCP binds to muLRRC15 on the surface of fibroblast cells. In some cases, the fibroblast cells are found in a cell or tissue sample from an animal, such as a mouse. In other cases, the fibroblast cells are in culture.

[0043] In some cases, the DCP binds to the extracellular domain of muLRRC15, such as comprising the amino acid sequence of SEQ ID NO: 45. In some cases, the DCP binds to a portion of muLRRC15 extracellular domain comprising the amino acid sequence of SEQ ID NO: 2, which comprises an N-terminal portion of the muLRRC15 extracellular domain, as shown in Figs. 13B-13C. For example, hydrogen deuterium exchange mass spectrometry (HDX-MS) and cross-linking experiments indicated that DCPs herein bind to a portion of the muLRRC15 extracellular domain comprising residues 82-91, 107-112, 134-138, and 154-160 of SEQ ID NO: 2 or SEQ ID NO: 45, based on protection from hydrogen-deuteriumexchange in an HDX-MS assay at those locations. (See Figs. 13A-13C). Furthermore, crosslinking experiments indicated presence of a cross-linking site at residue K86 of SEQ ID NO: 2 or SEQ ID NO: 45, in the residue 82-91 segment.

[0044] In some embodiments, the DCP has a length of 25-35 amino acid residues, such as 28-33 amino acid residues. In some embodiments, the DCP has a length of 30-32 amino acid residues, or of 30-31 amino acid residues. In some cases, the DCP has a length of 30 amino acid residues. In other cases, the DCP has a length of 31 amino acid residues. In some cases, the C-terminus is modified, such as to provide stability or as a result of in vitro peptide synthesis. In some cases, the C-terminus of the peptide is amidated. In other cases, the C- terminus is not amidated. In some cases, the N-terminus may be modified, such as with an N-terminal cap, for example, to provide added stability or as a result of peptide synthesis in vitro. In other cases, the N-terminus is not modified.

[0045] The disclosure herein further relates to DCPs binding to muLRRC15 comprising an amino acid sequence of any one of SEQ ID NO: 35-41, as shown in Table 1 below. In some such cases, the DCP is 30-32 amino acids in length. Those sequences, for example, show library sequences in which particular portions of a starting peptide sequence are randomized and can be any amino acid except for cysteine. In some embodiments, a DCP has an amino acid sequence of any one of SEQ ID NOs: 35-37. In other cases, the DCP has an amino acid sequence of SEQ ID NO: 38. In other cases, the DCP has an amino acid sequence of any one of SEQ ID NOs: 39-41.

[0046] The instant disclosure also relates to DCPs having specific peptide sequences as shown in Table 2A and SEQ ID NOs: 3-34 herein. For instance, in some embodiments, the DCP has an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 26, 18, 30, 3-17, 19-25, 27-29, or 31-34. In some such cases, the DCP is 30-32 amino acids in length. In some cases, the DCP is 30 amino acids in length. In some cases, the DCP is 31 amino acids in length.

[0047] In some cases, the DCP comprises or consists of an amino acid sequence as shown in Table 2A herein, and further binds to muLRRC15 with a Kd of, for example, 50 nM or less. Examples of such DCPs include those comprising or consisting of the amino acid sequences of SEQ ID NOs: 8, 9, 15-18, 21-22, and 25-34. In some cases, the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 9, 15-18, 21-22, or 25-34, and has a Kd of 30 nM or less. In some cases, the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 9, 16-18, 21-22, or 25-31, or 34, and has a Kd of 20 nM or less. In some cases, the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 9, 18,26-31, or 34, and has a Kd of 10 nM or less. In some cases, the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 18, 26, 28, 30, or 31, and has a Kd of 5 nM or less. In some cases, the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 18 or 26 and has a Kd of 1 nM or less.

[0048] In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 26. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 18. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 30. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 31. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 9. In some cases, the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0049] In some cases, the DCP comprises at least four cysteine residues. In some cases, the DCP comprises at least six cysteine residues. In some such cases, the DCP may comprise up to three cysteine-cysteine disulfide bonds. In some cases, cysteine residues are located at each of positions 2, 11, 17, 21, 23, and at one of positions 29, 30, or 31. In some cases, cysteine residues are located at each of positions 2, 11, 17, 21, 23, and 29. In some such cases, the DCP has 30-32 residues in length, such as 30 residues. In some such cases, the DCP has a specific peptide sequences as shown in Table 2A and SEQ ID NOs: 3-34 herein. For instance, in some embodiments, the DCP has an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 26, 18, 30, 3-17, 19-25,27-29, or 31-34. In some cases, the DCP has an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID Nos: 13-34, e.g., SEQ ID NO: 18 or SEQ ID NO: 28, as well as, for example, disulfide bonds between the first and fourth, second and third, and fifth and sixth cysteine residues of the sequence.

[0050] In some cases, the DCP further comprises or is attached to a label. In some cases, the label may be incorporated into the DCP, or alternatively may be a fluorophore or dye, which may be covalently or noncovalently attached to the peptide.

[0051] The disclosure herein also encompasses a complex comprising the DCP as described herein and muLRRC15 or the extracellular domain of muLRRC15 such as comprising the amino acid sequence of SEQ ID NO: 45. In some cases, the complex comprises a DCP herein in complex with a portion of muLRRC15 comprising the amino acid sequence of SEQ ID NO: 2, or comprising an amino acid sequence comprising residues 82- 91, 107-112, 134-138, and 154-160 of SEQ ID NO: 2.

[0052] The disclosure herein also comprises a polynucleotide encoding the DCP as described herein. Such a polynucleotide in some embodiments is a DNA. In other cases it is an RNA. In some cases, the polynucleotide may be incorporated into a vector, such as a plasmid or viral vector. The disclosure herein further encompasses a cell comprising a polynucleotide or vector encoding a DCP herein, such as a host cell allowing for expression of the DCP, such as a bacterial cell, a yeast cell, or a eukaryotic cell, such as a mammalian cell.

[0053] The present disclosure further encompasses a method of making a DCP as described herein. In some cases, a DCP may be chemically synthesized in vitro. In other cases, the DCP is produced in a host cell, such as a bacterial cell, a yeast cell, or a eukaryotic cell, such as a mammalian cell. For example, such a method may comprise incubating the host cell such that the cell expresses the DCP. Such a method may further comprise isolating the DCP from the host cell.III. USES OF PEPTIDES BINDING TO LRRC15

[0054] The present disclosure additionally encompasses, inter alia, methods of using the DCPs herein, for example, for detecting muLRRC15. In some embodiments, such methods comprise detecting muLRRC15 in vitro, such as in a cell or tissue sample. For example, such a method may comprise contacting the muLRRC15 with a DCP herein. For example, in some cases, the DCP may comprise or may be attached to a label, such as a fluorescent label or dye, which label may be used to detect the muLRRC15, such as in a cell or tissue sample, such as on the surface of cells in the sample. As muLRRC15 is expressed on the surface of fibroblast cells, in some embodiments, the cell or tissue sample comprises fibroblast cells or other cells that are expected to express muLRRC15 on their surface under certain conditions.

[0055] In other embodiments, detection methods may be conducted in vivo, such as in a mouse or other animal. For example, a DCP herein may be used in conjunction with a mouse model, in order to detect muLRRC15 in vivo. For instance, a DCP herein may be used to detect muLRRC15 in vivo, in a method comprising administering the DCP to an animal in anamount sufficient to detect muLRRC15 in the animal or in tissues obtained from the animal.In some cases, the animal is a mouse. In some cases, the DCP may be formulated for injection into the animal, such as systemically, such as by hydrodynamic tail vein injection or the like. In some cases, the DCP may be formulated for direct administration to the site of the expected muLRRC 15 -expressing cells such as by administration directly to tumor cells. In some cases, the mouse may be used in a mouse model of a disease, such as cancer, or another disease that may be associated with expression of muLRRC 15 on the surface of cells, such as fibroblast cells. Thus, for example, as muLRRC 15 may be expressed on the surface of fibroblast cells, for instance in a tumor microenvironment, such methods may be used for detection of muLRRC 15-expressing fibroblast cells. In some cases, detection methods include, for example positron emission tomography (PET) imaging. In some cases, for instance, for imaging experiments, a DCP herein may be attached to another molecule such as a label. In some embodiments, the label is or comprises a fluorophore, dye, prosthetic group, and / or radiolabel. In some embodiments, the DCP herein may be attached directly or indirectly to a radiolabel comprising fluorine 18 (18F) such as aluminum(III) [18F]fluoride ([18F] A1F2+) (also designated interchangeably herein as A118F). In some embodiments, the DCP is attached to the radiolabel via a RESCA or NAZL prosthetic group.

[0056] Embodiments herein include, for example, methods of detecting muLRRC 15 in vivo, in an animal. In some embodiments, such a method comprises (a) administering a DCP to an animal; and (b) detecting binding of the DCP to muLRRC 15 in the animal or in tissues obtained from the animal, wherein the detection of the binding indicates the presence of muLRRC15 in the animal or in tissues obtained from the animal. In some embodiments, the detecting comprises performing a positron emission tomography (PET) scan or positron emission tomography / computed tomography (PET / CT) scan of the animal. In some embodiments, the animal is a mouse. The DCP in some such methods, for example, may comprise the amino acid sequence of any one of SEQ ID NOs: 26, 18, 30, 3-17, 19-25, 27-29, or 31-34, or an amino acid sequence of any one of SEQ ID NOs: 35-41.IV. KITS

[0057] The present disclosure also includes a kit comprising a DCP herein, and optionally further components. For example, a kit may comprise one or more buffers for use in incubating a sample with the DCP. A kit may optionally further comprise a label and / or detection assay reagents. Kits may also comprise control molecules and reagents to be used with control molecules. For instance, a kit may comprise a further control peptide, such as anegative control that does not bind to muLRRC15. Or a kit may comprise a sample comprising muLRRC15 and / or a sample comprising a mutant muLRRC15 that does not recognize the DCP, which may respectively act as positive and negative controls. In some cases, for instance, for use in imaging experiments, a DCP in a kit herein may be attached to a label, or the kit may comprise reagents for labeling the DCP. In some embodiments, the label is or comprises a fluorophore, dye, prosthetic group, and / or radiolabel. In some embodiments, a DCP herein is attached, indirectly or directly, to a radiolabel comprising fluorine 18 (18F) such as aluminum(III) [18F]fluoride ([18F]A1F2+) (aka., A118F). In some embodiments, the DCP is attached to the radiolabel via a RESCA or NAZL prosthetic group. In some cases, more than one label may be attached to a DCP. In some embodiments, kits may also comprise directions for use.

[0058] Further nonlimiting embodiments of the disclosure are provided in the Examples that follow.EXAMPLES

[0059] The following are examples of methods and compositions of the disclosure. It is understood that these Examples are not meant to limit the disclosure, but only to exemplify it, and that various other embodiments may be practiced, given the general description provided above.Example 1: Identification of DCPs Binding to Murine LRRC15

[0060] DCPs have several advantages due to their small size and stability, which makes them attractive as potential protein binding molecules for diagnostic uses in vitro and in vivo. For instance, DCPs show good tissue penetration, high chemical and thermal stability, and tolerance to molecular engineering. And because they are relatively small peptides, they can be produced by chemical synthesis, including under good manufacturing process (GMP) standards, which allows for significant savings in both the cost and time in production. However, developing a DCP library and identifying DCPs that bind specifically to a given protein target from an initial DCP library can be difficult and time consuming. Here, a particular DCP library was coupled with a machine learning and yeast surface display approach to identify DCPs that bind specifically to murine LRRC15 (muLRRC15), and several DCPs were identified through this method that bind to muLRRC15 with particularly high affinity.Materials and MethodsRecombinant protein expression and purification

[0061] Mouse LRRC15 protein (Uniprot ID Q80X72; residues Y22-S475) with a C- terminal AviTag and His6 tag were expressed in Trichoplusia ni cells. Harvested supernatant containing crude protein was purified by nickel affinity chromatography by eluting with 300 mM imidazole in 50 mM Tris pH 7.5 0.3 M NaCl. Proteins were further purified by size exclusion chromatography (SEC) using a Sephacryl S-200 column (GE Healthcare) equilibrated with 20 mM HEPES pH 7.2 100 mM NaCl. Protein containing fractions were combined and concentrated, and stored at -80 °C.

[0062] Mouse LRRC15 was site-specifically biotinylated at the AviTag using the BirA500 biotin-protein ligase kit (Avidity LLC). Briefly, mouse LRRC15 was concentrated to 13 pM (2 mL) and 200 pL BirA buffer A and 240 pL BirA buffer B was added before BirA (1 mg / mL, 8 pL) was added. Reaction was left for two hours at room temperature, and biotinylated mouse LRRC15 was purified by SEC as described above.Phage library construction

[0063] All phage libraries were constructed as previously described (Tonikian et al Nat Prot 2007) using the phagemid pComb3 template. The Kunkel mutagenesis approach was used to build the Ecballium elaterium trypsin inhibitor II (EETI-II) libraries for panning. In Library 1, only loop 1 (residues 3-8) was randomized with 6, 8, or 10 amino acids (SEQ ID NOs: 35-37; Table 1). In Library 2, only loop 5 (residues 22-26) was randomized at each amino acid position without changing the native loop length (SEQ ID NO: 38; Table 1). Library 3 comprised both loop 1 and loop 5 amino acid randomizations (SEQ ID NOs: 39-41; Table 1). Residues were randomized with the degenerate codon encoding 19 natural amino acids except cysteine. Oligonucleotides for mutagenesis were synthesized using 28 custom mixes of trimer phosphoramidites encoding for 19 amino acids at equimolar concentration (Glen Research, Sterling, VA). The libraries were displayed on the M13 phage as a fusion to gene 8 for selection against biotinylated mouse LRRC15 immobilized on Dynabeads™ MyOne™ Streptavidin T1 (Invitrogen). After four iterative rounds of selection and enrichment, single phage clones were amplified and analyzed in phage ELISA (Tonikian et al Nat Prot 2007).Table 1: EETI-II phage library sequences. X represents a randomized residue and X followed by a number indicates the number of randomized residues (i.e., X6 for a string ofsix randomized residues). The libraries were displayed on the M13 phage as a fusion to gene 8.

[0064] Shotgun-scanning Ala library was constructed as previously described (Weiss et al, PNAS 2000). DNA degeneracies are represented by IUB code: K=G / T, M=A / C, N=A / C / G / T, R=A / G, S=G / C, W=A / T, Y=C / T. The library was displayed on the M13 phage as a fusion to gene 8 for selection against biotinylated mouse LRRC15 immobilized on Dynabeads™ MyOne™ Streptavidin T1 (Invitrogen).

[0065] Oligonucleotides for the 2nd generation phage libraries mutagenesis were synthesized as described above (Genelink and IDT). The libraries were displayed on the Ml 3 phage as a fusion to gene 3 for selection against biotinylated mouse LRRC15 immobilized on Dynabeads™ MyOne™ Streptavidin T1 (Invitrogen). After three iterative rounds of selection and enrichment, phage pools after every round were subject to amplicon sequencing targeting the variable peptide region on the Illumina MiSeq™ platform. The probability of residues occupying each position of the variable region was visualized with WebLogo 3.

[0066] Oligos for the 50,000 member machine learning-designed library were purchased from Twist Bioscience. Peptides were fused to gene 3 for selection against biotinylated mouse LRRC15 immobilized on Dynabeads™ MyOne™ Streptavidin T1 (Invitrogen).Machine learning (ML) analysis

[0067] The ML-library was generated using an oligomer pool ordered from TWIST Bioscience. This target-focused library was designed by feeding NGS enrichment scores from the 2ndgeneration phage panning and SPR affinity data of the primary phage panning into a machine-learning model conducted by DeepSeq.AI (Sacramento, CA).Yeast surface display (YSD)

[0068] Yeast surface display was done following a standard protocol (Chao et al, Nat Prot 2006) with four rounds of panning. Target protein concentrations used in four rounds of panning were 500 nM, 250 nM, 100 nM, and 100 nM, respectively. Next generation sequencing (NGS) was then performed.Phage display ofM-library

[0069] Peptides were fused to gene 3 for selection against biotinylated mouse LRRC15 immobilized on Dynabeads™ MyOne™ Streptavidin T1 (Invitrogen). Three rounds of panning were performed and phage outputs were analyzed by NGS.Peptide synthesis and functionalization

[0070] Peptides were synthesized using solid-phase peptide synthesis by CS Bio and Wuxi. Fluorescently labeled peptide Dl-7-6[D8E]-A488 was synthesized using CuAAC. AF- Dye-488 alkyne (Lumiprobe) (100 pL, 15 mM) was diluted to 1000 pL 50 mM sodium phosphate pH 7.0. In a separate tube, CuSO4 (100 pL, 20 mM) was mixed with THPTA (100 pL, 40 mM) and added to the AF-Dye-488 alkyne solution. Dl-7-6[D8E] (100 pL, 10 mM) was immediately added, before the addition of sodium ascorbate (100 pL, 100 mM). The reaction was mixed for 1 h at room temperature and reaction progress was monitored by LC- MS. The reaction was purified by HPLC - 5 pm Cl 8 100 A pore size, 21.2 x 100 mm column (Phenomenex) using a gradient of 20-70% acetonitrile over 20 min.Surface plasmon resonance

[0071] Surface plasmon resonance (SPR) experiments were performed using a Biacore® T200 instrument at 25 °C. Biotinylated mouse LRRC15 was captured to a level of 1200 RU on a streptavidin sensor chip (GE healthcare). All peptides were prepared in HBS-EP buffer (0.01 M Hepes pH 7.4 0.15 M NaCl 3 mM EDTA 0.005% v / v Surfactant P20). A reference channel was subtracted from the HTRA-captured channel. Five serial three-fold dilutions of peptide were flowed using a single cycle kinetics model. Peptide maximum concentrations were as follows: 1st generation DCPs, 10 pM; 2nd generation DCPs, 100 nM; ML-YSDs, 100 nM, ML-PDs, 30 nM. Data were analyzed using GE BIAevaluation® software using a 1 : 1 Langmuir binding model.Isothermal titration calorimetry

[0072] All measurements were performed on a MicroCai 200 iTC instrument 50 mM Tris pH 7.5 150 mM NaCl 3% (v / v) DMSO. Mouse LRRC15 was loaded in the sample cell at a concentration of 20 pM and EE-D1 was loaded into the syringe at 200 pM.ResultsIdentification of 1st generation DCPs

[0073] The Ecballium elaterium trypsin inhibitor II (EETI-II) scaffold has previously been engineered using combinatorial libraries to identify binders against biological relevant protein targets (Thakur et al. ACS Chem Biol 2023). This study used a strategy that combined high throughput screening with machine learning (ML) to generate potent DCP binders to LRRC15 (FIG. 1). EETI phage libraries (Table 1) were pooled together and screened against the extracellular region of mouse LRRC15 (FIG. 2). After four rounds of panning, enrichment was observed and five representative DCP sequences were chosen from round four, based on sequence diversity and ELISA signal strength, for solid-phase peptide synthesis and oxidative folding. All DCP sequences were derived from EETI library 3 (Table 1; SEQ ID NOs: 39-41).

[0074] Surface plasmon resonance (SPR) experiments were performed and determined that four of the DCPs synthesized had weak affinity for mouse LRRC15 (KD 0.4-1.5 pM) (FIGS. 3A-3D). DCP EE-B6 (SEQ ID NO: 6) showed no binding to mouse LRRC15 at 10 pM (data not shown). Interestingly, three of the four DCPs that bound to mouse LRRC15 had an identical amino acid motif in loop 1 (WWXWE; SEQ ID NO: 42) suggesting a conserved binding mechanism. As hypothesized, the four DCPs all competed with each other for mouse LRRC15 binding in a phage competition binding assay (FIG. 3E). DCP EE-D1 (SEQ ID NO: 3) was the most potent DCP from SPR analysis, and thermodynamic binding analysis, using isothermal calorimetry (ITC), confirmed that the EE-D1 binding to mouse LRRC15 was enthalpically driven (AH = -7.6 kcal / mol) (FIG. 4).

[0075] To identify the EE-D1 residues responsible for mouse LRRC15 binding, a combinatorial Ala-scanning strategy was implemented on phage where each residue, except the Cys, were preferentially allowed to vary as the wild type or Ala (Weiss et al, PNAS 2000). EE-Dl-Ala library was panned against mouse LRRC15 and phage outputs were subject to NGS sequencing to determine the wild type / Ala ratio at each position. As shown in FIG. 5 A, loop 1 wild type residues were determined to be important for mouse LRRC15 binding as the wild type / Ala ratio was positive. Alternatively, loop 5 residues were indicated to not be contributing to mouse LRRC15 binding as wild type / Ala ratio. Interestingly, residues in loops 2, 3, and 4 had a positive wild type / Ala ratio, suggesting that they may either be forming binding contacts with mouse LRRC15 or they are important for peptide folding and structure. Based on the SPR and ITC binding analysis, EE-D1 was selected as the scaffold to design 2nd generation phage libraries.Screening 2nd generation phage libraries based on EE-D1 scaffold

[0076] 2nd generation phage libraries were designed to identify improved binders by investigating additional peptide loops. The libraries focused on the simultaneous mutation of peptide loops using either a hard or soft randomization approach. Libraries were fused to gene 3 and panned against mouse LRRC15. Following three rounds of selections, phage outputs were analyzed using MiSeq next-generation sequencing (NGS).Machine-learning (ML) high throughput screening to identify potent mouse LRRC15 binders

[0077] The peptide sequence and enrichment data from the NGS was used to train a ML language model that can generate a predicted binding score for a given sequence using the assumption that enrichment is positively correlated to improved mouse LRRC15 binding. It is important to note that no information regarding mouse LRRC15 or peptide structure was provided to the model. The accuracy of the model was tested by comparing the predicted binding scores of the 1st generation DCP sequences with their respective experimental determined affinities (FIG. 6A). Despite the small sample size of the test data set, there was a positive trend in predicted binding vs affinity, providing confidence in the performance of the ML model.

[0078] The study computationally generated one million unique DCP sequences, that were not present in the training or test data set, that had a target predicted binding score > 2.1, and a non-specific binding score < -0.1 (FIG. 6B). The non-specific binding score was determined based on NGS enrichment data from negative selections against streptavidin only during the 2nd generation phage panning. The computationally generated sequences were biased towards peptide length of 30 residues (compared to 31 and 32 residues) based on the weight of the NGS data used to train the model. These one million sequences were filtered based on peptide length and predicted binding score to leave 50,000 peptide sequences (40,000 30-mers, 7,500 31-mers, and 2,500 32-mers) that were referred to the ML-designed library (FIG. 6C). Non-specific binding scores for the ML-library sequences were also determined (FIG. 6D). A 2D-UMAP analysis plot, where the distance between any two points approximates their embedding-based similarity in a high-dimensional space, showed that the ML-library contained DCP sequences with different properties than the enriched DCPs from the 2ndgeneration libraries (Figures 6E-6F). This highlighted the ability of the model to combine enrichment data from the different libraries to generate distinct sequences. The model observed a Y / F5WWE / DWE10motif in loop 1, which was very similar to the EE-D1 sequence (Y5WWDWE10), in predicted binders for muLRRC15. In addition, the ML-library contained no mutations for this segment, suggesting that this motif is important for bindingmuLRRC15. The ML-designed library incorporated target-focused mutations across 12-13 residues (depending on the overall sequence length, see FIG. 6G) and was subject to high throughput screening, using both yeast surface display and phage display.

[0079] Yeast surface display offers several advantages for protein directed evolution. For example, the target-binding signal can be normalized for expression due to the quantitative FACS two-color screening analysis and displayed proteins are folded in the endoplasmic reticulum of the yeast cells, taking advantage of ER chaperones. In comparison, phage display using gene 3 fusion offers the advantage of lower peptide valency. Therefore, there was an interest in how sequence enrichment would differ between both screening approaches. For yeast surface display, the ML-library was panned against mouse LRRC15 using a solution based selection process for 3 rounds, and phage outputs were analyzed using both NGS and single colony FACS analysis. 12 DCPs from the ML-yeast surface display (ML- YSDs) were selected for synthesis and binding affinity characterization. Additionally, five DCPs were selected that were enriched from the 2nd generation phage libraries prior to any ML analysis. These 2nd generation peptides, which had mouse LRRC15 KD values ranging from 1700 nM to 9 nM (Figures 7B-7F), would serve as a reference for evaluating any affinity improvements of the ML-designed sequences, compared to 1stgeneration EE-D1 (FIG. 7A). In general, the ML-YSD peptides synthesized had moderate nM affinity for mouse LRRC15 (Figures 8A-8E and 8G-8L), with the exception of ML-YSD-07 (SEQ ID NO: 18), which had sub-nanomolar affinity (KD 0.7 nM) (FIG. 8F). As shown in the SPR sensorgrams, the more potent affinity of ML-YSD-07 was a result of improved binding kinetics, in particular a slower dissociation rate.

[0080] The ML-library displayed on phage was panned against mouse LRRC15 using the same magnetic bead capture system performed for the 1st and 2nd generation phage panning experiments. Following NGS and single colony ELISA analysis, ten ML-phage display (ML- PD) peptides were selected for synthesis. SPR binding analysis of the ML-PDs showed that all peptides had affinity < 30 nM for mouse LRRC15 (Figures 9A-9J). Of particular interest was ML-PD-03 (SEQ ID NO: 26) which had a very potent affinity for mouse LRRC15 (KD 0.4 nM) (FIG. 9B). The discovery of subnanomolar binders to mouse LRRC15 from both the ML-YSD and ML-PD families of peptides highlight the potential of combining ML with high throughput screening techniques. The sequences and affinities of all peptides discovered are shown in Tables 2 A and 2B.Table 2A: Peptide sequence and SPR affinity data for all DCPs identified. Am = amidated C-terminus. Sequences from top to bottom are SEQID NOs: 3-34.Table 2B: Further affinity data of EETI-based DCPs identified during this study. Sequences from top to bottom are SEQ ID NOs: 3-7 and 13-34.DCPDCPs are grouped together based on their discovery. KD affinity measurements for binding to muLRRC15 were measured by SPR with either steady state or kinetic fitting models (see methods). KD are shown as average ± s.d. (N = 3-4 independent experiments).Example 2: Properties of ML-YSD-07 and ML-PD-03 Peptides

[0081] The drug-like properties of the DCPs were next investigated, including lipophilicity, stability and specificity. The DCPs ML-YSD-07 and ML-PD-03 had low lipophilicity and overall negative net charge). In addition, ML-YSD-07, ML-PD-03 and the 2nd generation DCP Dl-7-6[D8E] (SEQ ID NO: 9) had moderately sufficient stability in mouse plasma, with half lives of 332 and 362 min, respectively (Figures 10A-10C). Interestingly, ML-YSD-07 did not have reduced stability compared to ML-PD-03 despite having two additional Arg residues which are typically more susceptible to protease cleavage. Dl-7-6[D8E] was synthesized with an N-terminal azidolysine and labeled the peptide with A488 for cell-binding studies with NIH / 3T3 cells stably transfecting mouse LRRC15. Dl-7- 6[D8E]-A488 specifically bound to NIH / 3T3-muLRRC15 cell line, but showed no background binding to NIH / 3T3 (Figures 11 A-l IB), indicating that the DCP binds to functional mouse LRRC15 on cell surfaces. Methods were as follows:Peptide stability assays

[0082] Mouse plasma was thawed and centrifuged at 3750 rpm, 4oC for 15 min. Plasma supernatant was adjusted to pH 7.25 to 7.35. Peptides were added to 50 pL plasma to a final concentration of 1 pM and incubated for 0, 15 min, 1 h, and 4 h. At the respective time points, reactions were quenched with 22 pL acetonitrile with 5% (v / v) formic acid and left for 25 min. Samples were centrifuged at 3750 rpm, 4oC for 15 min before being analyzed by LC-MS / MS (ThermoFisher).Flow cytometry

[0083] Mouse LRRC15+ / - NIH / 3T3 cells were washed in FACS buffer (PBS supplemented with 0.5% BSA and 3 mM EDTA) and resuspended to 2-5 x 10A5 cells per sample. Cells were incubated with Dl-7-6[D8E]-A488 (10 pM) for 10 min at 4oC and washed twice in FACS buffer. Cells were analyzed using a BD FACSymphony™ and data was visualized using FlowJo™ software.Example 3: Binding Epitope of DCPs on Murine LRRC15Cross-linking mass spectrometry

[0084] To identify the mouse LRRC15 binding site of the DCPs, a cross-linking strategy was implemented. After incubating EE-D1 with mouse LRRC15, disuccinimidyl suberate (DSS, a bifunctional amine-reactive linker with a spacer arm length of 11.4 A, was added. The covalently linked complex could be seen by an increased MW protein band in the SDS- PAGE (FIG. 12, left panel). Protease digestion and LC-MS / MS analysis indicated a singlecross-linking site between residues K12 of EE-D1 and K86 of mouse LRRC15 (Figures 13A- 13C).

[0085] The method was as follows: Mouse LRRC15 (4 pM) was added to DD-E1 (50 pM) in PBS and left at 4°C for Ih. Disuccinimidyl suberate (DSS) (Thermo) was added (100 pM) and reaction was left for 30 min at room temperature. Reaction was stopped with addition of Tris pH 7.5 (50 pM). Reaction progress was monitored using SDS-PAGE analysis.

[0086] Bands of interest were excised, and each gel slice was subjected to in-gel digestion with trypsin (Promega), chymotrypsin (Roche) 1 :5, or Elastase (Promega) enzyme: substrate ratio in 25 mM ammonium bicarbonate pH 8.0 overnight at 37 °C. Peptides were extracted with a solution containing 50% acetonitrile and 1% formic acid. Extracted samples were dried under vacuum and reconstituted in 2% acetonitrile and 0.1% formic acid. The peptides were quenched and desalted using Cl 8 PhyTips (PhyNexus) and dried in a speed-vac.

[0087] The desalted peptides were suspended in 2% acetonitrile / 0.1% formic acid and 1 / 50 of each of the digests were subjected to LC-MS / MS analysis on a Thermo-Fisher Scientific Orbitrap® Eclipse Tribid mass spectrometer. The gradient was supplied using a Dionex® U3000 nLC system with cl 8 BEH column and consisted of 0 to 40% acetonitrile in 0.1% formic acid / 2% acetronitrile over one hour at 0.4 pL per minute. Data was acquired under data dependent mode with 2 Sec Duty Cycle acquisition with OT-HCD / CID fragmentation, the parent ion resolution was 240,000 FWHM and OT-CID / HCD fragmentation at resolution 30,000 FWHM. The raw data files were collected for data analysis. MeroX (Gbtze et al J Am Soc Mass Spectrom 2012) open-source software was used for data processing with search parameters: Enzyme = Trypsin, chymotrypsin or Elastase (max mis-cleavage 3), Variable modification = oxidized Met, Crosslinker = DSS (C16H20N2O8, 138.0681 monoisotopic mass addition), Max Distance = 11.4 A, Specificity = N-terminus, Lys (*secondary sites Lys,Ser,Thr,Tyr,Cys,His,Asp), Include Dead-end crosslinks and intrapeptidal cross-links, Precursor MSI = 20ppm, Fragment precision 0.02 Da.Hydrogen deuterium exchange-mass spectrometry (HDX-MS)

[0088] Next, hydrogen deuterium exchange-mass spectrometry (HDX-MS) was performed to identify specific mouse LRRC15 amino acids involved in binding. HDX-MS measures changes in mass associated with the isotopic exchange between amide hydrogens of the protein backbone and deuterium in the solvent (Engen et al. Anal Chem 2021). Amide protons involved in the buried interface upon ligand binding will have a slower exchange ratedue to increased shielding, hence, comparing the exchange rate of mouse LRRC15 residues in the absence and presence of DCP will allow binding residues to be identified. The HDX- MS protocol was as follows:

[0089] Unbound muLRRC15 samples prior to dilution into D2O were 30 pM. Bound samples contained 30 pM muLRRC15 and 33 pM Dl-7-6[D8E] (1 : 1.2 molar ratio). These samples (3 pL each) were diluted into 60 pL of deuterated labeling buffer with 10 mM HEPES 150 mM NaCl and a pDread of 7.1 to begin the labeling experiment. The exchange process was then slowed after variable labeling times by the 1 : 1 addition of quench buffer, composed of 4 M GdmCl and 1 M glycine at a pH of 2.5. The material was then injected into a temperature-controlled chamber at 1°C for online proteolytic digestion and chromatographic separation. Samples pass through a pepsin and fungal protease XIII 1 : 1 mixture column (NovaBioassays) before being loaded onto a BEH C8 Vanguard trap column (Waters) where they are washed for 3 minutes before being put online with a BEH C18 HPLC column (Waters Acquity UPLC) where they are separated by an acetonitrile gradient and then ionized by electro-spray into a ThermoScientific Q-Exactive™ HF-x instrument for measurement of carried deuterium. Experimental manipulations were performed using a custom-built Leap HDX Pal DHR platform by Leap Technologies, Morrisville, NC. Samples were analyzed using the method of fragment separation (Mayne et al. J Am Soc Mass Spectrom 2011), and processed using the ExMS2 program (Kan et al. Anal Chem 2019) and protection factors were determined using a previously reported empirical method (Walters et al. Anal Chem 2017).

[0090] Figures 13A-13C shows the groups of mouse LRRC15 residues that underwent slower exchange in the presence of Dl-7-6[D8E], Mapping these slower exchanging residues on mouse LRRC15 (structure determined by AlphaFol d2) shows that they are clustered together (Figures 14A-14D). The cross-linking mouse LRRC15 residue K86 is positioned within the HDX-MS determined binding epitope, therefore, both experiments were in agreement and strongly suggest the DCP binding site is on the concave face of mouse LRRC15.Example 4: Additional surface plasmon resonance analysis of binding to muLRRC15Surface plasmon resonance method

[0091] Surface plasmon resonance (SPR) experiments were performed using a Biacore® T200 instrument at 25 °C. Biotinylated mouse LRRC15 was captured to a level of 250 RU and 1000 RU for kinetic affinity and steady state affinity, respectively, on a streptavidin sensor chip (GE healthcare). All peptides were prepared in HBS-EP buffer (0.01 M HEPESpH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20). A reference channel was subtracted from the muLRRC 15 -captured channel. Six serial three-fold dilutions of peptide concentrations were injected for 120 s at 30 pL / min. Peptides where the affinities were derived from steady state affinity were tested using both a single cycle and multi cycle injection method (it is important to highlight that affinities derived from the different methods were in agreement with each other). Peptides where the affinities were derived from kinetic analysis were solely tested using the multi cycle injection method. Information regarding peptide maximum concentrations and the model used to determine affinities are reported in Table 3. Data were analyzed using GE BIAevaluation® software using a 1 : 1 Langmuir binding model. Data are shown in Table 4.Table 3: Compound maximum concentrations used for SPR binding analysis. Model (steadystate or kinetics) used to determine binding constants for each compound are shown.

[0092] Binding affinity results are shown in Table 4, below. In the table, Am = amidated C-terminus, binding data were determined by SPR analysis, and KD ± S.D. values were determined from at least three independent experiments.Table 4A: SPR binding affinity resultsTable 4B: Select SPR binding affinity resultsValues are shown as average ± s.d. (N = three independent experiments). N.D. = not determined.Example 5: Imaging of Mouse Fibroblast Cells by Fluorescence Microscopy

[0093] Fluorescence microscopy was conducted as follows. Mouse LRRC15+ / - NIH / 3T3 cells were plated in a black p-Plate 96 well plate (ibidi) at 20,000 cells / well and left to grow for 48 h at 37 °C, 5% CO2. Cells were treated with 500 nM of ML-PD-03-N488 in DMEM 10% FBS glutamax for 1 h and 4 h. Cells were washed with Hanks' Balanced Salt Solution (+ CaC12 + MgC12) (HBSS). Cells were then treated simultaneously with CellBrite® Steady 650 Membrane Staining reagent (1 : 10000 dilution in HBSS, Biotium) and NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342) (5 drops added to 10 mL HBSS) for 10 min to stain outer cell membranes and nuclei, respectively. Fluorescent images werecaptured using a Leica TCS SP8 confocal microscope with a HC PL APO CS2 63x / 1.40 Oil objective. Hoechst 33342 was imaged with excitation at 405 nm and emission collected from 413-486 nm. ML-PD-03-N488 was imaged with excitation at 488 nm and emission collected from 501-602 nm. CellBrite® Steady 650 was imaged with excitation at 633 nm and emission collected from 646-703 nm. Cells were incubated at 37 °C, 5% CO2 during imaging.

[0094] Representative histograms of flow cytometry plots are shown in FIG. 11C and fluorescence micrographs showing the location of ML-PD-03-N488 compared with staining of cell nuclei and outer membranes, for example, are shown in FIG. 1 ID. As shown in FIG.1 ID, the DCP co-localized to the outer membrane in the cells expressing muLRRC15 (bottom row), and did not bind to NIH 3T3 cells not expressing muLRRC15 (top row). Example 6: X-Ray Structural Analysis of the ML-YSD-07 Peptide muLRRC15 Interaction

[0095] The crystallization chaperone Fab-El, discovered using phage display (data not shown), was expressed in E. coli and purified by protein G Sepharose affinity chromatography using standard procedures. Fab-El and muLRRC15 (1.5: 1 molar ratio) were mixed for 1 h at 4 °C and the complex was purified using Superdex® 200 size-exclusion chromatography equilibrated with 20 mM HEPES pH 7.2, 100 mM NaCl. The purified muLRRC15:Fab-El complex was concentrated to 16 mg / mL and added to powdered ML- YSD-07 (3-fold molar excess) and mixed for 2 h at 4 °C. Sitting drop crystallization screens were conducted with 1 : 1 sample:precipitant ratio. Single crystals were obtained in 0.1 M Tris pH 8.5, 10% w / v PEG 8000 from hanging drop optimization screens. Harvested crystals were cryo- preserved in precipitant supplemented with 25% (v / v) glycerol. Diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-1 at 0.97737 A beam wavelength at a temperature of 100K. Data were processed to 2.569 A resolution cutoff using XDS. Phases and an initial structure solution were obtained by molecular replacement using an in-house X-ray crystal structure of muLRRC15 alone, and a model of Fab-El generated using MOE Biologies Software, with the constant and variable regions of Fab-El split and searched for separately. ML-YSD-07 was built de novo in COOT into clear difference density in the phased electron density map. The overall model improved with iterative rounds of building and refinement in COOT and PHENX respectively to produce the final model (Table 5). an R-work / R-free of 0.1968 / 0.2524. Of the modeled residues, 93.30% exhibit in Ramachandran favored angles, 6.48% allowed, and 0.22% outliers.Table 5. Data collection and refinement statistics for ML-YSD-07:muLRRC15:Fab-El complex crystal structureStatistics for the highest-resolution shell are shown in parentheses.

[0096] A diagram of the crystal structure of the ML-YSD-07:muLRRC15-Fab-El complex is provided in FIGS. 14A. The figure provides a cartoon surface representation of muLRRC15 (right) with ML-YSD-07 (top, ribbon cartoon adjacent to surface representation of muLRRC15) bound to the concave face and Fab-El (left, ribbon cartoon) bound to the LRR C-terminal domain. FIG. 14B shows a more detailed version of the cartoon surface representation depicted in FIG. 14A. The ML-YSD-07 binding interface with muLRRC15 is shown in FIG. 14C with muLRRC15 contact residues within 4 A of ML-YSD-07 are labeled in black italics. The ML-YSD-07 contact residues are represented as sticks. The N-terminus of ML-YSD-07 is solvent exposed, whereas the C-terminus of ML-YSD-07 is buried in the binding interface.

[0097] An overlay of binding locations on muLRRC15 provided in the crystal structure compared to those determined by hydrogen-deuterium exchange (HDX) analysis of binding of the peptide Dl-7-6[D8E] to muLRRC15 (see Example 3) is shown in FIG. 14D. The figure shows the change in the calculated log 10 HDX protection factors for mouse LRRC15 residues in the presence of DCP Dl-7-6[D8E] (loglOAPF), where positive loglOAPF values correspond to residues that more slowly exchange backbone amide hydrogen atoms with solvent deuterons in the presence of Dl-7-6[D8E], therefore, highlight the shielded binding site. The shaded regions denoted “no sequence coverage” denote uncertainty. The black trace labeled “slower H-D exchange residues” represents an empirical quantification from data. MuLRRC15 residues are numbered based on Uniprot. Residues that are within 5 A of ML- YSD-07 interface from the crystal structure are identified with a cross on the x-axis. FIG. 14E shows the disulfide bonding pattern of the ML-YSD-07 peptide in the crystal structure.

[0098] Ten ML-YSD-07 residues from loops 1, 2, and 5 are engaged in binding contacts with muLRRC15 (FIG. 14F). The Y5WWEWE10 motif in loop 1 and provide several key binding contributions. This motif is conserved in all of the ML-YSD and ML-PD peptides compared to EE-D1, as shown in Tables 2A and 2B above. Residue E10 is inserted into a deep pocket and forms key electrostatic interactions with K177, N179, and R203 of muLRRC15 (FIG. 14G). Residues W7 and W9 of ML-YSD-07 occupy hydrophobic patches on muLRRC15’s surface and the imidazole groups of W7 and W9 form electrostatic interactions with El 53 and QI 55, respectively (FIG. 14H). Although E8 was highly enriched during library screening, it is solvent exposed and may therefore be important for folding. Residue R4 packs closely with R249 of muLRRC15 and forms Arg- Arg 7t-stacking interaction (FIG. 141). Interestingly, Y5 and W6 do not form interactions with muLRRC15 residues and instead provide intramolecular packing interactions and support the hydrophobic core of the DCP (FIG. 14J). Y5 and W6 may replace the stabilizing effects of the cysteine knot, which contributes to the structural stability of DCPs.

[0099] Loop 5 residue W25 was highly enriched for all high affinity DCPs, and it occupied a hydrophobic pocket covering LRRs 2-4 (FIG. 14H). The majority of the binding interface was covered by W7, W9, and W25, covering 397 2 of the interface. The importance of W9 and W25 for binding was confirmed by the complete lack of muLRRC15 binding by ML-YSD-07 in which these two residues were mutated to Ala (FIG. 14N). T24, V27, and T28 form hydrophobic packing away from the binding interface and appear to stabilize the turn in loop 5 that positions W25 to optimally interact with muLRRC15 (FIG. 14K). This is supported by mutational analysis as ML-PD-05, which has three mutationscompared to ML-YSD-07 (E3D; solvent exposed, E24T and N28T; loop 5 packing), has 10- fold reduced binding for muLRRC15, highlighting the effect of non-interacting DCP residues on affinity. Loop 2 residues of ML-YSD-07 also form key interactions with muLRRC15, as R12 forms electrostatic interactions with E206 and E230, and Y13 forms a H-bond with Y157 in addition to 7t-stacking with Y205 (FIG. 14L). In addition, D14, T15, and D16 all form side-chain binding contacts with K273 (FIG. 14M), despite both D14 and D16 not being subject to randomization during library designs.

[0100] Overall, the binding affinity data of the ML-DCPs were in agreement with the experimental structure.Example 7: Disulfide Mapping of DCPs

[0101] Partial reduction for disulfide mapping of DCP peptides was performed as follows. One mg of ML-PD-05 was dissolved in 500 pL of a 10 mM Glycine buffer (pH 3.0) containing 20% Acetonitrile. Two equivalents of tris(2-carboxyethyl)phosphine (TCEP) was added and the solution was incubated at 37 °C for 30 min. LC-MS was performed and detected the formation of major [M+2] and [M+4] peaks. N-Ethylmal eimide (NEM) was then added to a final concentration of 50 mM to cap the free thiols at room temperature for 1 hour. The solution was desalted with a 1 mL SPE (Cl 8) column to remove the excess NEM, and the elution solution containing [M+2NEM] and [M+4NEM] was lyophilized to dryness.

[0102] The mixture of [M+2NEM] and [M+4NEM] samples was dissolved in about 100 pL 1 : 1 H2O: ACN and fully reduced with 20 mM DTT at 37 °C for 1 hour. Then 40 mM lodoacetomide was added to alkylate all the free thiols under pH 8.5. The sample was then analyzed by tandem mass spectroscopy (MS / MS) (FIG. 15C). Comparison of the MS data from the samples was then used to determine the locations of each disulfide bond in the peptides. A schematic of the experimental results is shown in FIGS. 15A and 15B, where FIG. 15B is a more detailed version of the same experimental results. Specifically, the data indicated that ML-PD-05 has disulfide bonds between Cysl-Cys4, Cys2-Cys3, Cys5-Cys6 (Cys are numbered from the N-terminus). The disulfide connectivity determined for ML-PD- 05 using the partial reduction method was in agreement with the disulfide connectivity determined for ML-YSD-07 from the crystallography data, shown in FIG. 14E.Example 8: Imaging of Murine Tissues In VivoRESCA-junctionalization of DCPs

[0103] Strategy 1 : (+)-RESCA-TFP was directly conjugated to the amino group of the N- terminal Glyl residue of DCPs ML-YSD-07 and ML-PD-03 (FIG. 16A). DCPs (ML-YSD-07 and ML-PD-03) were dissolved to 3 mM in DMF. 10 eq. DIPEA was added before adding 3eq. RESCA-TFP. Reaction was mixed at room temperature for 1 h. Reaction progress was monitored by LC-MS. The reaction was purified by HPLC - 5 pm Cl 8 100 A pore size, 21.2 x 100 mm column (Phenomenex) using a gradient of 20-70% acetonitrile over 20 min.

[0104] Strategy 2: RESCA-TFP (30 mM, 3 eq.) and BCN-PEG3-NH2 (15 mM, 1.5 eq.) were dissolved in DMF and 10 eq. DIPEA was added. The reaction was mixed for 1 h at room temperature and reaction progress was monitored by LC-MS. 200 pL of the crude RESCA-PEG3-BCN was added to 100 pL Nazl-ML-YSD-07 (10 mM) and the reaction was mixed for 2 h at room temperature. The reaction was purified by HPLC - 5 pm C18 100 A pore size, 21.2 x 100 mm column (Phenomenex) using a gradient of 20-70% acetonitrile over 20 min.

[0105] The following peptides were prepared, and the affinity of each peptide for muLRRC15 was determined by SPR according to the method of Example 4, with a 30 nM maximum concentration kinetics affinity model: ML-PD-03-RESCA (i.e., RESCA-ML-PD- 03), ML-YSD-07-RESCA (i.e., RESCA-ML-YSD-07), RESCA-PEG3-BCN-Nazl_YSD-07, and Nazl-ML-YSD-07. Efforts to radiolabel RESCA-ML-PD-03 with ([18F]A1F)2+were challenging and the tracer underwent fast defluorination (data not shown). In contrast, we were able to successfully radiosynthesize the [18F]A1F-RESCA-ML-YSD-O7 conjugate at a molar activity of 151.7 GBq / pmol and with a radiochemical purity of 96%. The binding affinities determined by SPR are shown in Tables 6 A and 6B.Table 6A: Binding affinity data by SPR. KD ± S.D. values were determined from at least three independent experiments. Nazi = N-terminal azido-lysineTable 6B: Select binding analysis of RESCA-functionalized DCPs to muLRRC15The kinetic constants konand ko / f, and the calculated equilibrium dissociation constant KD are shown as average ± s.d. (N = three independent experiments).Radiolabeling of DCP ML-PD-03-RESCA for PET Imaging

[0106] Peptide ML-PD-03-RESCA was radiolabeled with [18F] fluoride for positron emission tomography (PET) imaging. [18F]fluoride (29 ± 3 GBq) was inversely eluted from a QMA light cartridge (Waters, Milford, MA, USA, part no. WAT023525) with 0.2 mL of 0.9% sterile sodium chloride into a vial containing 10 pL 0.5 M sodium acetate, pH 4.8 and 0.7 mole equivalents (relative to RESCA) of 2 mM aluminum chloride (A1C13, Sigma- Aldrich, part no. 563919). After a 3-5 minute incubation, a mixture of ML-PD-03 -RESCA (i.e., 2 pL, 10 mM in DMSO), 10 pL 0.3 M sodium ascorbate, pH 4.8, and 30 pL of 75 mM histidine, 240 mM sucrose, pH 5.0 was added to the aluminum(III) [18F]fluoride ([18F]A1F2+) solution and incubated at ambient temperature for 15 min. The reaction mixture was diluted into 3 mL of sterile water, loaded onto a Strata-X SPE cartridge (30-60 mg, Phenomenex, CA), washed with 3 mL of sterile water, eluted with 1 mL of ethanol, and formulated with 10 mM histidine, 10 mM methionine, 240 mM sucrose, pH 7.0 such that the ethanol concentration in the final product vial was less than 5%. The final product was analyzed using HPLC Waters BEH C18, lOOA, 50 x 2.1 mm; 280 nm (Agilent 1290 HPLC); mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile with 0.1% formic acid; gradient: 1% B, 0-1 min, 1-100% B, 1-4 min.PET imaging ofML-PD-03-RESCA in mice

[0107] Female C57B1-6 mice weighing 17-25 g were purchased from Charles River Laboratories (Hollister, CA). Mice were acclimated in animal housing facilities for at least 1 week before the study and were 2 months old at the study start date.

[0108] The KPR mouse pancreatic adenocarcinoma cell line was generated according to the protocol described in Krishnamurty, A.T., Shyer, J. A., Thai, M. et al. LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity. Nature 611, 148— 154 (2022). Each mouse was inoculated subcutaneously with 100,000 KPR cells in the right flank. Once tumors reached approximately 400 mm3in size, the animals were grouped out for subsequent PET imaging sessions using the P-Cube™ PET scanner (Molecubes, Belgium) followed by a CT scan on an X-Cube™ CT scanner (Molecubes, Belgium).

[0109] Mice (n = 3 per group) were lightly anesthetized for restraint with 3.5% sevoflurane and transferred to the scanner bed. Mice were catheterized at the lateral tail vein and approximately 7.4-11.1 MBq (200-300 pCi, 34-111 pmol) of18F-labeled ML-PD-03- RESCA tracer ([18F]AlF-RESCA-ML-PD-03) was administered intravenously as a 1-minute short infusion via the lateral tail vein catheter. A 60-minute dynamic PET scan was acquired to capture the injection and subsequent biodistribution of the tracer. The respiration rate wasmonitored, and the body temperature was monitored and maintained under feedback control with an electric heating pad. Following the PET scan, a low-dose CT scan was acquired for attenuation correction and anatomical reference.

[0110] PET images were reconstructed from the list-mode data by using Molecubes- provided histogram and reconstruction software. Regions of interest (ROIs) were drawn on multiple axial slices of the images by using VivoQuant™ software (Invicro, MA). Radioactivity concentrations in the ROIs were measured as a percentage of the injected dose per gram (% ID / g), assuming 1 cc equivalency in 1 gram of soft tissue and decay-corrected to the starting time of the scan. The PET images shown are maximum intensity projections of the final frame of the dynamic scans averaged from 45 minutes - 60 minutes post-tracer administration and uniformly scaled from 0-30 %ID / g. A schematic of the method and the resulting data are shown in FIGS. 16B-16E. As shown in FIGS. 16D-16E, radioactivity concentrations in the regions of interest (ROIs) were measured as a percentage of the injected dose per gram (% ID / g) during a 60-minute dynamic PET scan. Higher tumor % ID / g compared to blood and muscle shows specific accumulation of ML-PD-03-RESCA-18F ([18F]AlF-RESCA-ML-PD-03) to the LRRC15-rich tumor environment. FIG. 16C shows a representative image from a mouse in the experiment taken one hour after administration of the DCP.Example 9: Imaging of Murine Tissues In Vivo with ML-YSD-07-RESCARadiolabeling of ML-YSD-07-RESCA for PET Imaging

[0111] ML-YSD-07-RESCA was prepared and characterized according to the methods described in Example 8. The chelating ligand RESCA can capture A1-18F, and the DCP ML- YSD-07 was functionalized with RESCA at the N-terminal amide, as shown in FIG. 17 A. [18F]fluoride (29 ± 3 GBq) was inversely eluted from a QMA light cartridge (Waters, Milford, MA, USA, part no. WAT023525) with 0.2 mL of 0.9% sterile NaCl into a vial containing 10 pL of 0.5 M sodium acetate, pH 4.8 and 0.7 eq. (relative to RESCA-ML-YSD- 07) of 2 mM AlCh (Sigma-Aldrich, part no. 563919). After a 3-5 minute incubation, a mixture of RESCA-ML-YSD-07 (2 pL of 10 mM in DMSO), 10 pL of 0.3 M sodium ascorbate, pH 4.8, and 30 pL of 75 mM histidine, 240 mM sucrose, pH 5.0 was added to the aluminum(III) [18F]fluoride ([18F]A1F2+) solution and incubated at room temperature for 15 min. The reaction mixture was diluted into 3 mL of sterile water, loaded onto a Strata-X SPE cartridge (30-60 mg, Phenom enex, CA), washed with 3 mL of sterile water, eluted with 1 mL of ethanol, and formulated with 10 mM histidine, 10 mM methionine, 240 mM sucrose, pH 7.0 such that the ethanol concentration in the final product vial was less than 5%. The finalproduct was analyzed using HPLC Waters BEH Cl 8, 100 A, 50 x 2.1 mm; 280 nm (Agilent 1290 HPLC); mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile with 0.1% formic acid; gradient: 1% B, 0-1 min, 1-100% B, 1-4 min. [18F]A1F-RESCA-ML-YSD-O7 was successfully radiosynthesized after 20 min at a molar activity of 151.7 GBq / pmol and with a radiochemical purity of 95.7%.

[0112] After successfully synthesis of radiolabeled ML-YSD-07-RESCA, the binding affinity of [18F]A1F-RESCA-ML-YSD-O7 for muLRRC15 was tested (FIG. 17B). [18F]A1F- RESCA-ML-YSD-07 retained subnanomolar affinity for muLRRC15 (0.62 nM).PET imaging of ML-YSD-07-RESCA in mice

[0113] PET / CT imaging with [18F]A1F-RESCA-ML-YSD-O7 was performed using a recently established subcutaneous pancreatic ductal adenocarcinoma (PDAC) cancer model to investigate its utility as an effective PET imaging agent, following methods similar to those described in FIG. 16B. Female C57B1-6 mice were implanted with KPR3070 PDAC tumor cells, which develop a LRRC15-expressing fibroblast tumor environment (Krishnamurty et al. (2022) Nature, 611 :148-154).

[0114] The animal studies were conducted in accordance with the guidelines of Genentech’s AAALAC-accredited Institutional Animal Care and Use Committee. Female C57B1-6 mice weighing 23-26 g were purchased from Charles River Laboratories (Hollister, CA). Mice were acclimated in Genentech’s animal housing facilities for at least 1 week, and were 3 months old at the study start date.

[0115] Each mouse was inoculated subcutaneously with 100,000 KPR3070 PDAC tumor cells in the right flank. Once tumors reached approximately 400 mm3in size, the animals were grouped out for subsequent PET imaging sessions using the P-Cube PET scanner (Molecubes, Belgium) followed by a CT scan on an X-Cube CT scanner (Molecubes, Belgium). Mice (n = 5 per group) were lightly anesthetized for restraint with 3.5% sevoflurane and transferred to the scanner bed. Mice were catheterized at the lateral tail vein and approximately 2.4-2.9 MBq (65-79 pCi, 16-19 pmol) of [18F]A1F-RESCA-ML-YSD-O7 tracer was administered intravenously as a 1 -minute short infusion via the lateral tail vein catheter. A 60-minute dynamic PET scan was acquired to capture the injection and subsequent biodistribution of the tracer. The respiration rate was monitored, and the body temperature was monitored and maintained under feedback control with an electric heating pad. Following the PET scan, a low-dose CT scan was acquired for attenuation correction and anatomical reference.

[0116] PET images were reconstructed from the list-mode data by using Molecubes- provided histogram and reconstruction software. Regions of interest (ROIs) were drawn on multiple axial slices of the images by using VivoQuant™ software (Invicro, MA). Radioactivity concentrations in the ROIs were measured as a percentage of the injected dose per gram (% ID / g), assuming 1 cc equivalency in 1 gram of soft tissue and decay-corrected to the starting time of the scan. The PET images shown in Figures 17C and 17E are maximum intensity projections of the final 15 min frame of the dynamic scans, uniformly scaled from 0- 20 % ID / g. Logan graphical analysis was performed as previously described (Logan et al. (1990) J. Cereb. Blood Flow Metab. 10:740-747), using healthy muscle tissue as a reference.Mouse tissue digestion

[0117] Tumor and lung tissue samples were prepared similarly as previously described in Krishnamurty, A.T., et al., Nature 611 : 148-154 (2022). Tissues were collected, weighed and minced into small pieces. All tissues were subsequently enzymatically digested using a cocktail of dispase (Life Technologies), collagenase P and DNasel (Roche) for 45 min at 37 °C to obtain a single-cell suspension. Red blood cells were removed from the lung samples using ammonium chloride-based red blood cell lysis buffer. Cells were counted using a Vi- CELL XR (Beckman Coulter). Cells were blocked with Fc block (2.4G2; 1 :200, 553142, BD Bioscience) for 10 min at 4 °C. Cells were labeled with the following monoclonal antibodies at 4 °C on ice for 20-30 min: CD45 (30-F11, 612802, 1 :200, BioLegend); EPCAM (G8.8, 740559, 1 : 100, BD Bioscience); CD31 (390, 612802, 1 :200, BD Bioscience); PDPN (8.1.1, 127418, 1 :200, BioLegend); CD24 (MI / 69, 48-0242-82, 1 : 100, Invitrogen); LRRC15 (M25, in-house, 1 :200) and PE anti-mouse IgG2a (RMG2a-62, 407108. 1 :500, BioLegend). Live cells were identified by incubation with calcein blue (Invitrogen, C1429, 1 : 1,000) after surface staining. Data were acquired using a BD FACSymphony flow cytometer and analyzed using FlowJo (v.10.10.0). Data were processed using Prism GraphPad. Statistical analyses were carried out by Student’s / -test.

[0118] In representative images from dynamic PET scans, the KPR tumors could be clearly visualized in the right flank of the animals (FIG. 17C). Biodistribution studies averaged from 45 to 60 minutes showed the tumor uptake of [18F]A1F-RESCA-ML-YSD-O7 was 3.50 ± 1.0 % ID / g (FIG. 17D) compared to 0.887 ± 0.207 % ID / g for healthy muscle (FIG. 17E). Logan graphical analysis (Logan et al. (1990) J. Cereb. Blood Flow Metab. 10:740-747), using healthy muscle as the reference tissue, estimated a binding potential (BPNB) for [18F]A1F-RESCA-ML-YSD-O7 to the tumor tissue of 2.7 ± 0.7 (FIG. 17F). Thetumor uptake was higher than blood, bone, and lung, which were 1.712 ± 0.441, 2.497 ± 0.546, and 2.261 ± 0.905 % ID / g, respectively (FIGS. 17G-17H). The tracer was predominately excreted through the renal pathway, as high levels of [18F]A1F-RESCA-ML- YSD-07 were present in the bladder and kidneys (FIGS. 17G-17H). Biodistribution over the course of the dynamic PET scans showed that the tumor uptake of the tracer was stable 60 minutes post injection (FIG. 17D), showing that the strong affinity of the DCP to muLRRC15 translated to excellent tumor retention in vivo.

[0119] These results demonstrated significant accumulation and excellent in vivo retention of18F-radiolabelled ML-YSD-07-RESCA in tumors using a mouse pancreatic cancer model that develops a LRRC15-expressing fibroblast tumor environment.Example 10: DCP binding affinity for huLRRC15

[0120] Following successful imaging of muLRRC15-expressing CAFs using the PDAC tumor model, the binding affinity of the DCPs EE-D1, ML-YSD-07, ML-PD-03, and ML-PD- 08, to human LRRC15 (huLRRC15) was examined to assess their cross-species activity.

[0121] Surface plasmon resonance (SPR) binding analysis showed that the three strongest muLRRC 15 -binding DCPs, ML-YSD-07, ML-PD-06, and ML-PD-08, had relatively low affinities to huLRRC15 (KD of 123.3 nM, 84.6 nM, and 232.3 nM, respectively), as shown in Table 7. Affinity measurements for binding to huLRRC15 are shown as average ± s.d. (N = 3 independent experiments) for select DCPs.Table 7. DCP binding affinity for huLRRC15

[0122] The ~150-250-fold reduced binding affinities were due to both slower association and faster dissociation binding kinetics (FIGS. 18A-18B).ReferencesC. K. Wang and D. J. Craik, Nature Chemical Biology 2018 Vol. 14 Issue 5 Pages 417-427. DOI: 10.1038 / s41589-018-0039-yG. A. Weiss, C. K. Watanabe, A. Zhong, A. Goddard and S. S. Sidhu, Proceedings of the National Academy of Sciences 2000 Vol. 97 Issue 16 Pages 8950-8954. DOI: doi : 10.1073 / pnas.160252097A. K. Thakur, S. E. Miller, N. P. D. Liau, S. Hwang, S. Hansen, F. de Sousa e Melo, et al., ACS Chemical Biology 2023 Vol. 18 Issue 4 Pages 772-784. DOI: 10.1021 / acschembio.2c00753D. Kwon, Z. Zhang, J. Zeisler, H. T. Kuo, K. S. Lin and F. Benard, Pharmaceutics 2022 Vol. 14 Issue 7. DOI: 10.3390 / pharmaceuticsl4071502R. Tonikian, Y. Zhang, C. Boone and S. S. Sidhu, Nature Protocols 2007 Vol. 2 Issue 6 Pages 1368-1386. DOI: 10.1038 / nprot.2007.151M. Gotze, J. Pettelkau, S. Schaks, K. Bosse, C. H. Ihling, F. Krauth, et al., J Am Soc Mass Spectrom 2012 Vol. 23 Issue 1 Pages 76-87. DOI: 10.1007 / sl3361-011-0261-2L. Mayne, Z. Y. Kan, P. S. Chetty, A. Ricciuti, B. T. Walters and S. W. Englander, J Am Soc Mass Spectrom 2011 Vol. 22 Issue 11 Pages 1898-905. DOI: 10.1007 / sl3361-011- 0235-4Z. Y. Kan, X. Ye, J. J. Skinner, L. Mayne and S. W. Englander, Anal Chem 2019 Vol. 91 Issue 11 Pages 7474-7481. DOI: 10.1021 / acs.analchem.9b01682B. T. Walters, Analytical Chemistry 2017 Vol. 89 Issue 2 Pages 1049-1053. DOI: 10.1021 / acs.analchem.6b03908G. Chao, W. L. Lau, B. J. Hackel, S. L. Sazinsky, S. M. Lippow and K. D. Wittrup, Nature Protocols 2006 Vol. 1 Issue 2 Pages 755-768. DOI: 10.1038 / nprot.2006.94J. R. Engen, T. Botzanowski, D. Peterle, F. Georgescauld and T. E. Wales, Analytical Chemistry 2021 Vol. 93 Issue 1 Pages 567-582. DOI: 10.1021 / acs.analchem.0c04281

[0123] The following table describes certain sequences referenced herein. In SEQ ID NOs: 35-41, residues denoted “X” denote any natural amino acid residue except cysteine. The peptides herein, in particular those of SEQ ID NOs: 3-7, as indicated in Table 2A, may optionally comprise an amidated C-terminus.SEQUENCE TABLE

[0124] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

WHAT IS CLAIMED IS:

1. A disulfide-constrained peptide (DCP) that binds to murine leucine-rich repeat containing 15 (muLRRC15), comprising the amino acid sequence of any one of SEQ ID NOs: 26, 18, 30, 3-17, 19-25, 27-29, or 31-34, or comprising an amino acid sequence of any one of SEQ ID NOs: 35-41.

2. The DCP of claim 1, wherein the DCP binds to muLRRC15 with a Kd of 500 nM or less, 250 nM or less, 150 nM or less, 100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less.

3. The DCP of claim 1 or 2, wherein the DCP binds to a portion of muLRRC15 comprising SEQ ID NO: 2.

4. The DCP of any one of claims 1-3, wherein the DCP binds to muLRRC15 on the surface of fibroblast cells.

5. The DCP of any one of claims 1-4, wherein the DCP is 30-32 amino acids in length.

6. The DCP of any one of claims 1-5, wherein the DCP comprises an amidated C-terminus.

7. The DCP of any one of claims 1-6, wherein the DCP comprises cysteine residues at positions 2, 11, 17, 21, 23, and 29, optionally wherein the DC comprises 30 amino acids in length.

8. The DCP of any one of claims 1-7, wherein the DCP further comprises or is attached to a label.

9. The DCP of claim 8, wherein the label is or comprises a fluorophore, dye, prosthetic group, and / or radiolabel.

10. The DCP of claim 9, wherein the radiolabel is a radionuclide, optionally wherein the radionuclide is18F.

11. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 26, 18, 30, 3-17, 19-25, 27-29, or 31-34.

12. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 26-31, 9, 18, or 34.

13. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 26.

14. The DCP of any one of claims 1-10, wherein the amino acid sequence of theDCP comprises or consists of the amino acid sequence of SEQ ID NO: 18.

15. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 30.

16. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 31.

17. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 28.

18. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 27.

19. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 34.

20. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 9.

21. The DCP of any one of claims 1-10, wherein the amino acid sequence of the DCP comprises or consists of the amino acid sequence of SEQ ID NO: 29.

22. The DCP of any one of claims 1-21, wherein the DCP comprises or consists of the amino acid sequence of any one of SEQ ID NO: 13-34, and wherein the DCP comprises six cysteine residues and wherein disulfide bonds occur between the first and fourth, second and third, and fifth and sixth cysteine residues of the sequence.

23. A disulfide-constrained peptide (DCP) that binds to murine leucine-rich repeat-containing 15 (muLRRC15) with a Kd of 500 nM or less, 250 nM or less, 150 nM or less, 100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less.

24. The DCP of claim 23, wherein the DCP binds to a portion of muLRRC15 comprising SEQ ID NO: 2.

25. The DCP of claim 23 or 24, wherein the DCP binds to muLRRC15 on the surface of fibroblast cells.

26. The DCP of any one of claims 23-25, wherein the DCP is 30-32 amino acids in length.

27. The DCP of any one of claims 23-26, wherein the DCP comprises an amidated C-terminus.

28. The DCP of any one of claims 23-27, wherein the DCP comprises six cysteine residues, wherein disulfide bonds occur between the first and fourth, second and third, and fifth and sixth cysteine residues of the sequence.

29. The DCP of any one of claims 23-28, wherein the DCP comprises cysteine residues at positions 2, 11, 17, 21, 23, and 29, optionally wherein the DC comprises 30 amino acids in length.

30. The DCP of any one of claims 23-29, wherein the DCP further comprises or is attached to a label.

31. The DCP of claim 30, wherein the label is or comprises a fluorophore, dye, prosthetic group, and / or radiolabel.

32. The DCP of claim 32, wherein the radiolabel is a radionuclide, optionally wherein the radionuclide is18F.

33. A complex comprising the DCP of any one of claims 1-32 and muLRRC15 or the extracellular domain of muLRRC15.

34. A method of detecting muLRRC15 in vitro, comprising contacting the muLRRC15 with the DCP of any one of claims 1-32.

35. The method of claim 34, wherein the muLRRC15 is expressed on the surface of a cell.

36. A method of detecting muLRRC15 in vivo, comprising administering the DCP of any one of claims 1-32 to an animal in an amount sufficient to detect muLRRC15 in the animal or in tissues obtained from the animal.

37. A method of detecting muLRRC15 in vivo, comprising: a. Administering the DCP of any one of claims 8-10 or 31-34 to an animal; and b. Detecting binding of the DCP to muLRRC15 in the animal or in tissues obtained from the animal, wherein the detection of the binding indicates the presence of muLRRC15 in the animal or in tissues obtained from the animal.

38. The method of claim 37 or 38, wherein the detecting comprises performing a positron emission tomography (PET) scan or positron emission tomography / computed tomography (PET / CT) scan of the animal.

39. The method of any one of claims 35-38, wherein the animal is a mouse.

40. A polynucleotide encoding the DCP of any one of claims 1-32.

41. A vector comprising the polynucleotide of claim 40.

42. A host cell comprising the polynucleotide or vector of claim 40 or 41.

43. A method of making a DCP of any one of claims 1-32, comprising incubating the host cell of claim 42 such that the cell expresses the DCP, and optionally isolating the DCP from the host cell.

44. A method of making a DCP of any one of claims 1-32, comprising chemicallysynthesizing the DCP.

45. A kit comprising the DCP of any one of claims 1-32, and optionally further comprising a label, detection assay reagents, and / or instructions for use.

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