Fibroblast activating protein alpha-cleavable propeptides and methods of use

Abstract

Fibroblast activating protein alpha (FAP alpha) cleavable propeptides are provided for use in the treatment of conditions associated with FAP alpha expression. The FAPalpha cleavable propeptides are also useful for imaging localization of FAPalpha activity in vivo. In some embodiments, the propeptides of the present disclosure comprise a membrane interaction domain, a masking domain, and a FAP [alpha] cleavage site. The masking domain is effective to inhibit the interaction of the membrane interaction domain with the phospholipid bilayer when linked to the membrane interaction domain. The FAP alpha cleavage site is disposed between the membrane interaction domain and the masking domain. The membrane interaction domains may be conjugated to one or more therapeutic agents, non-limiting examples of which include radioisotopes. Also provided are methods of treating a condition associated with FAP alpha expression in a subject in need thereof, such methods comprising administering to the subject an effective amount of a propeptide of the present disclosure.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 401,008, filed on August 25, 2022, which is incorporated herein by reference in its entirety.

[0003] Government support statement

[0004] This invention was made with government support under R01 CA258297 and R01 AI161027 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Incorporation by Reference into the Sequence Listing

[0006] A Sequence Listing is provided herein as a Sequence Listing XML, "UCSF-680WO_SEQ_LIST," created on August 24, 2023, and having a size of 48,677 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. Background Art

[0007] Recently, the FDA approved Lu 177dotatate iobenguane I 131 and 177 Lu-PSMA 617 This, combined with the emergence of promising experimental agents in clinical trials, highlights the growing enthusiasm for investigating targeted radiation therapy (TRT) as a cancer treatment modality. However, clinical experience with TRT suggests that they are rarely curative and that tumor responses are often transient and / or vary between patients. Therefore, new strategies are needed to maximize the therapeutic benefit of TRT for cancer patients.

[0008] Over the past 10 years, the field of nuclear medicine has prioritized the development of low MW radioligand therapies (RLTs) that rapidly exit the bloodstream to minimize host toxicity but remain potent antitumor agents by targeting highly overexpressed oncoproteins. This shift has been driven by 30 years of clinical experience with various high MW radioligands such as immunoglobulins, which demonstrated that their long serum half-lives (3-7 days) resulted in radiation exposure to sensitive normal tissue compartments (e.g., bone marrow), which narrowed or eliminated the therapeutic index. The evolution of radioligands targeting prostate-specific membrane antigen (PSMA) stands out as an instructive case study for the impact of radioligand pharmacokinetics (PK) on the therapeutic index. While the inclusion of 177 Various radiolabeled forms of IgG J591, including Lu-J591, have been hampered in clinical trials due to dose-limiting toxicity, but ( 177 Lu-PSMA 617), a low MW radioligand with weaker affinity for PSMA and lower tumor uptake than J591, is still FDA-approved for prostate cancer treatment in 2022, largely due to its better safety profile.

[0009] However, treating cancer with low MW RLTs is challenging for many reasons. First, in order to deliver adequate doses to tumors, the field is limited to a small number of highly overexpressed proteins in cancer that can extract sufficient RLTs from the circulation as the drug rapidly leaves the body. Indeed, prominent RLT drug targets such as PSMA, somatostatin receptor type 2, carbonic anhydrase 9, and bombesin receptor are all very highly overexpressed on cancer cells (at least 10 per cell). 5 Second, the ligand / receptor complex is biologically intrinsically unstable and undergoes dissociation or degradation following endocytosis. In fact, longitudinal PET studies in patients have shown that RLTs are typically cleared from tumors within 96 hours, and in some extreme cases (e.g., FAPIPET), the radioisotope can leave the tumor within a few hours.

[0010] Fibroblast activation protein-α (FAPα) is a type II integrative serine protease expressed by activated fibroblasts. Tumor-associated fibroblasts (CAFs) in tumor stroma show abundant and stable FAPα expression, which plays an important role in promoting tumor growth, invasion, metastasis and immunosuppression. The protein encoded by the human FAPα gene is a single-pass type II transmembrane protein of 760 amino acids, which consists of a short cytoplasmic N-terminal part (6 amino acids), a transmembrane region (amino acids 7-26) and a large extracellular domain. FAP has enzymatic activity as a homodimer. It exhibits post-proline dipeptidyl peptidase and endopeptidase activity, both of which depend on the catalytic triad containing Ser624 Asp702His734 in human and mouse FAPα. Due to the unique structure of proline, most proteases do not cut peptide bonds adjacent to it. In several cases, the presence of proline therefore serves as a mechanism to prevent protein degradation or cleavage.

[0011] Normal tissues have low and usually undetectable levels of FAPα expression. However, FAPα is overexpressed in many tumor tissues, including breast cancer, colorectal cancer, pancreatic cancer, lung cancer, brain cancer, intrahepatic bile duct cancer, and ovarian cancer. In addition, high levels of FAPα expression can be detected in some tumors derived from non-epithelial tissues, such as melanoma and myeloma. In these tumors, FAPα overexpression is often observed in the stroma, which leads to FAPα being considered a universal marker for CAFs, although FAPα expression can also be detected in gastric cancer, pancreatic cancer, and melanoma cells. Details on FAPα expression in human malignancies can be found in, for example, Busek et al. (2018) Frontiers In Bioscience, Landmark 23: 1933-1968. Summary of the invention

[0012] Provided is a fibroblast activation protein alpha (FAPα) cleavable propeptide for treating a condition associated with FAPα expression. FAPα cleavable propeptides are also used for imaging localization of FAPα expression in vivo. In some embodiments, the propeptide of the present disclosure comprises a membrane interaction domain, a masking domain, and a FAPα cleavage site. The masking domain effectively inhibits the interaction of the membrane interaction domain with the phospholipid bilayer when connected to the membrane interaction domain. The FAPα cleavage site is disposed between the membrane interaction domain and the masking domain. The membrane interaction domain can be conjugated to one or more therapeutic agents, non-limiting examples of therapeutic agents include radioisotopes. Also provided is a method for treating a condition associated with FAPα expression in a subject in need thereof, such method comprising administering to the subject an effective amount of the propeptide of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Top: Schematic diagram of a propeptide (sometimes referred to herein as a "restricted interaction peptide" or "RIP") according to an embodiment of the present disclosure. RIPs are low MW (approximately 4 kDa) peptides that contain three domains from N-terminus to C-terminus: (i) a membrane interaction domain (in this example, a membrane-bound antimicrobial peptide (AMP)) coupled to a payload (e.g., a chelator bound to a radioisotope); (ii) an endoprotease cleavage site, e.g., spanning P4-P4'; and (iii) a peptide "masking domain" that prevents the AMP from adopting a helical conformation required for membrane binding. Bottom: In this example, upon cleavage of the propeptide by a target endoprotease in vivo, the radiolabeled AMP is released, spontaneously adopts a helical conformation and attaches to a nearby phospholipid membrane.

[0014] Figure 2: A schematic diagram showing the proposed advantages of RIP-based targeted radiotherapy (TRT). The left side shows typical blood and tumor activity curves for large molecular weight targeted radiotherapy (e.g., immunoglobulins). High MW targeted radiotherapy has high tumor uptake, but has dose-limiting toxicity due to slow clearance from the blood. Low MW targeted radiotherapy (e.g., small molecule radioligand therapy) benefits from improved safety due to rapid serum clearance, but rapid clearance itself limits tumor exposure. RIP achieves both the desired safety due to rapid clearance and the renewable catalytic amplification due to isotope accumulation in tumors, as well as the high tumor AUC caused by the persistent binding of membrane-interacting peptides to cell membranes.

[0015] Figure 3 : Discovery of unique candidate P4-P4' fibroblast activation protein alpha (FAPα) substrate sequences via multi-substrate analysis using mass spectrometry (MSP-MS). Top: Icelogo representing the cumulative amino acid preferences of recombinant human FAPα. Bottom: Heatmap summarizing the most frequently incorporated amino acids at each site in the cleavage product. Light colors indicate enriched amino acids. X = no preferred amino acid.

[0016] Figure 4A-4C : Kinetic data for the three top P4-P4' 8-mer peptides emerging from MSP-MS assays. The kinetic properties were determined using FRET donor / acceptor substrate adducts and human recombinant FAPα.

[0017] Figure 5 : Schematic representation of a propeptide (SEQ ID NO: 53) according to an embodiment of the present disclosure. From N-terminus to C-terminus in this non-limiting example (sometimes referred to herein as "FRIP2"): (1) 64 Cu-tagged membrane interaction / binding domain; (2) FAPα cleavage site; and (3) masking domain.

[0018] Figure 6A-6B Representative coronal PET / CT images from male nu / nu mice bearing subcutaneous U87 tumors show that FRIP1, FRIP3, and two negative control RIPs, PAR1 (cleaved by thrombin) and GRIP B (cleaved by granzyme B), are expressed compared to FRIP1, FRIP3, and two negative control RIPs, PAR1 (cleaved by thrombin) and GRIP B (cleaved by granzyme B). 64 Cu-FRIP2 has higher tumor uptake. These images were acquired 24 hours after injection. The mean tumor SUV for each mouse cohort is shown on the right. *P<0.01.

[0019] Figure 7 Compared with tumor models lacking FAPα, 64Evaluation of Cu-FRIP2 uptake. The data demonstrate the specificity of FRIP2 for FAPα.

[0020] Figure 8 : Injection of male nu / nu mice bearing subcutaneous U87 MG xenografts 64 The biodistribution data collected 24 hours after Cu-FRIP2 showed high uptake in tumors compared to normal tissues. The normal tissues with the highest uptake were liver and kidney.

[0021] Figure 9A-9B : Demonstrating that compared to FAPI-46, the labeled propeptide of the present disclosure (in this example 64 Cu-FRIP2) for excellent tumor uptake.

[0022] Figure 10A-10B : Shows PC3-PIP tumors 64 Cu-FRIP2 and 64 Longitudinal PET / CT study of Cu-FAPI 46 uptake in a model with low FAPα expression. ROI analysis of tumor uptake ( Fig. 10A ) shows that 64 Compared with Cu-FAPI 46, 64 Cu-FRIP2 was significantly higher in tumors. Fig. 10B Representative transverse images showing tumor uptake of either radiotracer over time are shown in . The orange arrows indicate the location of the tumor.

[0023] Fig.11 : Schematic diagram showing the advantages of the RIP-based approach for targeted radiation therapy (TRT).

[0024] Figure 12A-12C : 67 Cu-FRIP2 anti-tumor evaluation data. Fig. 12A : From the carrier or 67 Relative changes in tumor volume in a cohort of male nu / nu mice bearing subcutaneous U87 xenografts treated with Cu-FRIP2. 67 Mice (n=8 / group) were treated with a single IV bolus of Cu-FRIP2 (1 mCi / mouse). Figure 12B-Figure 12C : IHC images of FAPα staining of U87 xenografts. Staining of HEK-FAP tumors (HEK293 stably overexpressing human FAPα) is shown to put the staining levels in U87 into context.

[0025] Figure 13A-13B :Other 67 Cu-FRIP2 anti-tumor evaluation data. Fig.13A : Survival curve for each cohort. The end point is (1) more than 2000mm 3 tumor volume or (2) greater than 20% body weight loss. Fig. 13B : Relative changes in body weight of mice in the cohort.

[0026] Fig.14 : Spider plots showing changes in individual tumor volumes for the mice represented.

[0027] Figure 15A-15B :From the medium, 67 Cu-FAPI 46 or 67 Spider plot of tumor volume for antitumor evaluation study in male nu / nu mice bearing subcutaneous U87 tumors treated with Cu-FRIP2 ( Fig.15A ). On day 0 of the study, mice received 1 mCi of radioactivity in a single IV administration. Fig. 15B : Relative tumor volumes from each cohort (n=8 mice / group) at day 13, the latest date in the study where all mice were still alive. 67 Cu-FRIP2 ratio 67 Cu-FAPI 46 more effectively inhibited tumor growth. *P<0.01.

[0028] Fig.16 : The survival curves for each cohort are shown in Figure 15. The endpoint is (1) more than 2000 mm 3 tumor volume or (2) greater than 20% body weight loss.

[0029] Figure 17A-17B : Utilized in PC3-PIP 67 Radioligand therapy of Cu-FRIP2. Fig.17A : Anti-tumor evaluation data on day 69, which showed 67 Effect of Cu-FRIP2 treatment on PC3-PIP subcutaneous xenografts (P<0.01). 67 Cu-FRIP2 (N=9) treated mice. Fig. 17B : Survival data plotted on Kaplan-Meier curves showed significantly prolonged survival of the treatment group compared to the group receiving vehicle (P<0.001).

[0030] Fig.18 : On day 0, vehicle or 1 mCi 67 Weight changes in mice in both cohorts treated with Cu-FRIP2. No unsafe weight changes were observed in the treatment groups.

[0031] Figure 19A-19B:Used in U87 MG 64 Radioligand therapy of Cu-FRIP2. Fig.19A : Anti-tumor evaluation data on day 27, which showed 64 Effect of Cu-FRIP2 treatment on U87 MG subcutaneous xenografts (P<0.01). 64 Cu-FRIP2 (N=8) treated mice. Fig.19B : Survival data plotted on Kaplan-Meier curves showed significantly prolonged survival of the treatment group compared to the group receiving vehicle (P<0.01). DETAILED DESCRIPTION

[0032] Before describing the propeptides and methods of the present disclosure in more detail, it should be understood that the propeptides and methods are not limited to the specific embodiments described, and therefore these embodiments can certainly vary. It should also be understood that the terminology used herein is only used to describe specific embodiments and is not intended to be limiting, as the scope of the propeptides, compositions and methods is limited only by the appended claims.

[0033] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit and any other stated or intervening value in the stated range is encompassed in the propeptides, compositions and methods unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed in the propeptides, compositions and methods, subject to any specifically excluded limits in the stated ranges. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed in the propeptides, compositions and methods.

[0034] Certain ranges are given herein where a numerical value is preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that provides a substantial equivalent of the specifically recited number in the context in which it appears.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the propeptides, compositions and methods belong. Although any propeptides and methods similar or equivalent to the propeptides and methods described herein can also be used in the practice or testing of the propeptides and methods, representative illustrative propeptides and methods are now described.

[0036] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present propeptides, compositions and methods are not entitled to antedate such publications, as the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0037] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "only," "only," and the like, in connection with the recitation of claim elements or as a "negative" limitation.

[0038] It should be understood that, for the sake of clarity, certain features of the propeptides, compositions and methods described in the context of separate embodiments may also be provided in a single embodiment in combination. Conversely, for the sake of brevity, various features of the propeptides, compositions and methods described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments are specifically included by the present disclosure and are disclosed herein as if each combination were individually and explicitly disclosed, in the sense that such combinations include operable methods and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically included by the present propeptides, compositions and methods and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

[0039] Those skilled in the art will appreciate upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the present method. Any recorded method can be performed in the order of events recorded or in any other order that is logically possible.

[0040] Propeptide

[0041] The present disclosure provides a FAPα-cleavable propeptide. According to some embodiments, the propeptide comprises a membrane interaction domain and a masking domain, which, when linked to the membrane interaction domain, effectively inhibits the interaction of the membrane interaction domain with a phospholipid bilayer. Such propeptides further comprise a FAPα cleavage site disposed between the membrane interaction domain and the masking domain.

[0042] exist Figure 1A schematic diagram of a propeptide (sometimes referred to herein as a "restricted interaction peptide" or "RIP") according to an embodiment of the present disclosure is provided in Figure 1. A RIP can be a low MW (approximately 4 kDa) peptide consisting of three domains from the N-terminus to the C-terminus: (i) a membrane interaction domain (in this example, a membrane-bound antimicrobial peptide (AMP)) coupled to a payload (e.g., a chelator bound to a radioisotope); (ii) an endoprotease cleavage site, e.g., spanning P4-P4'; and (iii) a peptide "masking domain" that prevents the AMP from adopting a helical conformation required for membrane binding. Bottom: In this example, after cleavage of the propeptide by a target endoprotease in vivo, the radiolabeled AMP is released, spontaneously adopts a helical conformation and immediately attaches to a nearby phospholipid membrane.

[0043] The propeptides of the present disclosure can be used in various situations. For example, when the membrane interaction domain is stably associated with (e.g., conjugated to) one or more therapeutic agents (e.g., radioisotopes), the propeptides are used to treat conditions associated with FAPα expression in a subject in need thereof. Non-limiting examples of such conditions include cancer. Figure 2 A schematic diagram illustrating the advantages of targeted radiotherapy (TRT) based on RIP is shown. The typical blood and tumor activity curves of large molecular weight targeted radiotherapy (e.g., immunoglobulin) are shown on the left. High MW targeted radiotherapy has high tumor uptake, but has a limited toxic dose due to slow clearance from the blood. Low MW targeted radiotherapy (e.g., small molecule radioligand therapy) benefits from improved safety due to rapid serum clearance, but rapid clearance itself limits tumor exposure. RIP achieves both the desired safety due to rapid clearance and the renewable catalytic amplification due to isotope accumulation in tumors, as well as the high tumor AUC caused by the persistent binding of membrane-interactive peptides to cell membranes.

[0044] Also by way of example, when the membrane interaction domain is detectably labeled (e.g., labeled with a radioisotope), the propeptide is used to assess FAPα activity in a subject, e.g., by in vivo imaging of cells labeled with the detectably labeled membrane interaction domain. As demonstrated in the experimental section herein, the propeptides of the present disclosure surprisingly exhibit superior biodistribution compared to active site-directed FAPα radioligands currently in clinical trials. Details regarding the propeptides of the present disclosure will now be described.

[0045] The terms "polypeptide", "peptide" or "protein" are used interchangeably herein to refer to a linear series of amino acid residues interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The amino acids may include the 20 "standard" genetically encodable amino acids, amino acid analogs, or combinations thereof. The series of amino acid residues may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and peptides with modified peptide backbones.

[0046] As used herein, the term "propeptide" refers to a peptide whose activity is limited because the separate parts of the peptide are linked together, thereby limiting or restricting the activity that the separate parts may have when they are not linked to each other. The activity of the separate parts of the propeptide is released after the cleavage or destruction of one or more bonds holding the separate parts together.

[0047] According to some embodiments, the propeptides of the present disclosure comprise 10 to 50 amino acids. For example, in some embodiments, the propeptides of the present disclosure comprise at least 10 amino acids, but 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less amino acids.

[0048] In certain embodiments, the propeptide of the present disclosure comprises, from the N-terminus to the C-terminus: a membrane interaction domain, a FAPα cleavage site, and a masking domain. As used herein in the context of the structure of a polypeptide, "N-terminus" and "C-terminus" refer to the terminal amino and carboxyl termini of a polypeptide, respectively, while "N-terminus" and "C-terminus" refer to the relative positions toward the N-terminus and the C-terminus in the amino acid sequence of a polypeptide, respectively, and may include residues at the N-terminus and the C-terminus, respectively. "Immediately adjacent to the N-terminus" or "immediately adjacent to the C-terminus" refers to the position of a first amino acid residue relative to a second amino acid residue, wherein the first amino acid residue and the second amino acid residue are covalently bound to provide a continuous amino acid sequence.

[0049] membrane interaction domain

[0050] In certain embodiments, the membrane interaction domain (sometimes referred to herein as a "membrane binding domain") comprises a plurality of non-polar hydrophobic amino acid residues and, when not constrained by a masking domain, comprises an alpha-helical structure capable of interacting with a phospholipid bilayer (such as a cell membrane). Such secondary structures may appear before, during, or after the membrane interaction domain is inserted into the phospholipid bilayer. The composition of the membrane interaction domain as described herein is not strictly limited to non-polar hydrophobic amino acid residues, as such peptides may also include different types of amino acid residues, for example, polar uncharged amino acid residues, polar basic amino acid residues, or polar acidic amino acid residues.

[0051] Thus, in some instances, the membrane interaction domain comprises an alpha-helical structure capable of being inserted into a phospholipid bilayer when separated from the masking domain. The alpha helix is ​​a common motif in the secondary structure of proteins and generally comprises a right-handed coil or helical conformation stabilized by hydrogen bonds, wherein the NH group of the first amino acid residue forms a hydrogen bond with the C=O group of an amino acid residue located four residues outside the polypeptide chain. A typical alpha helix contains approximately 3.6 amino acid residues per turn of the helix and is a tightly packed structure. The side chains of the amino acid residues constituting the alpha helix face the outside of the helix. Different amino acid sequences have different tendencies to form alpha helices, which is partly due to the different chemical properties of the amino acid side chains.

[0052] According to some embodiments, the membrane interaction domain comprises about 5 to about 30 amino acid residues. For example, in some embodiments, the membrane interaction domain comprises at least 5 amino acids, but 30 or less, 25 or less, 20 or less, or 15 or less amino acid residues.

[0053] In certain embodiments, the membrane interaction domain comprises an antimicrobial peptide (AMP) or its part.AMP or its part can be incorporated into the propeptide of the present disclosure in its naturally occurring form, or can be modified to change its chemical nature and adapt it to required purposes.For example, the membrane interaction potentiality of the antimicrobial peptide can be enhanced or weakened by, for example, adding, eliminating or replacing some amino acid residues in the protein sequence.Can carry out this type of addition, elimination or replacement, for example to introduce charged amino acid residues, eliminate charged amino acid residues, introduce hydrophobic amino acid residues, eliminate hydrophobic amino acid residues etc.

[0054] According to some embodiments, the antimicrobial peptide sequence can be changed by chemically modifying the peptide with a disulfide bond or other chemical modifications (e.g., amidation).Many antimicrobial peptides are naturally produced by such modifications to improve their effectiveness in interacting with phospholipid membranes and resistance to proteolysis.

[0055] In some embodiments, the membrane interaction domain comprises a protein from the Temporin family. The length of proteins in the Temporin family is generally in the range of about 10 to about 14 amino acids. The consensus sequence of proteins in the Temporin family showing the most abundant amino acids found at each position is: FLP (I / L) IAS LL (S / G) KLL (SEQ ID NO: 1). The consensus sequence of proteins in the Temporin family showing the general amino acid types found at each position is: X a X b X c X d X e Xf Y a X g X h Y b Y * X i X j , where X a , X b , X c , X d , X e , X f , X g , X h , X i and X j is a hydrophobic amino acid residue, Y a and Y b is a hydrophilic amino acid residue, and Y * is a charged amino acid residue. The following table shows the amino acid sequences of several Temporin and Temporin-like peptides useful in the propeptides and methods of the present disclosure.

[0056] As mentioned above, the antimicrobial peptide sequence can be changed by eliminating or replacing one or more amino acid residues. For example, in some embodiments, the membrane interaction domain comprises Temporin-L, and its amino acid sequence is FVQWFSKFLGRIL (SEQ ID NO:2). In other embodiments, the membrane interaction domain comprises a derivative of Temporin-L with the amino acid sequence FVQWFSKFLGKLL (SEQ ID NO:3), wherein the amino acid residues R and I at the position 11 and 12 of the Temporin-L sequence have been substituted by amino acid residues K and L respectively.

[0057] Table 1: Amino acid sequences of Temporin and Temporin-like peptides of 13 amino acids in length. Longer and shorter members of this family are also described but are not included in this table.

[0058]

[0059]

[0060] In some embodiments of the present disclosure, the membrane interaction domain comprises a Temporin or Temporin-like peptide listed in Table 1, or a conservative amino acid substitution thereof. In some embodiments of the present disclosure, the membrane interaction domain comprises the sequence of Temporin-L (FVQWFSKFLGRIL; SEQ ID NO: 2), or a conservative amino acid variant thereof.

[0061] In some embodiments of the present disclosure, the membrane interacting domain comprises a Protonectin having the amino acid sequence ILGTILGLLKGL (SEQ ID NO: 33), or a conservative amino acid variant thereof.

[0062] In some embodiments, the membrane interaction domain may comprise a Japonicin or a Japonicin-like peptide. In some embodiments of the present disclosure, the membrane interaction peptide comprises the sequence of Japonicin-1 (FFPIGVFCKIFKTC; SEQ ID NO: 34), or a conservative amino acid variant thereof. Japonicin can be obtained naturally from the skin of the Japanese brown frog Rana japonica, and ranges in length from about 14 to about 21 amino acid residues.

[0063] Masking domain

[0064] As summarized above, the propeptides of the present disclosure comprise a masking domain that, when linked to a membrane interacting domain, is effective to inhibit the interaction of the membrane interacting domain with the phospholipid bilayer.

[0065] In certain embodiments, the masking domain comprises 5 to 30 amino acids. For example, in some embodiments, the masking domain comprises at least 5 amino acids, but 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less amino acid residues.

[0066] According to some embodiments, the masking domain comprises a peptide derived from protease activated receptor-1 (PAR-1). "Derived from..." in the context of an amino acid sequence or a polynucleotide sequence is meant to indicate that the polypeptide or nucleic acid has a sequence based on the sequence of a reference polypeptide or nucleic acid, and is not meant to limit the source or preparation method of the protein or nucleic acid.

[0067] In certain embodiments, when the masking domain comprises a peptide derived from PAR-1, the masking domain comprises the amino acid sequence QDPNDQYEPF (SEQ ID NO: 35). In other embodiments, when the masking domain comprises a peptide derived from PAR-1, the masking domain comprises the amino acid sequence RNPNDKYEPF (SEQ ID NO: 36).

[0068] FAPα cleavage site

[0069] As summarized above, the propeptides of the present disclosure comprise a FAPa cleavage site disposed between a membrane interaction domain and a masking domain. According to some embodiments, the FAPa cleavage site comprises the amino acid sequence YHGPLAHX (SEQ ID NO: 37), HIGPTAAY (SEQ ID NO: 38), XXIPTNIR (SEQ ID NO: 39), HQGPFWML (SEQ ID NO: 40), XXGPKLTY (SEQ ID NO: 41), HYGPTVNK (SEQ ID NO: 42), XXWPMGMY (SEQ ID NO: 43), XXFPNMWS (SEQ ID NO: 44), XXGSQVFS (SEQ ID NO: 45), XXMPEEVA (SEQ ID NO: 46), or XXHPTKSF (SEQ ID NO: 47), wherein X is any amino acid.

[0070] In certain embodiments, the FAPα cleavage site comprises the FAPα specific motif GP in the P2-P1 position. For example, the FAPα cleavage site that can be incorporated into the propeptide of the present disclosure includes, but is not limited to, a FAPα cleavage site comprising the sequence HQGPFWML (SEQ ID NO: 40), XXGPKLTY (SEQ ID NO: 41), or HYGPTVNK (SEQ ID NO: 42). According to some embodiments, the FAPα cleavage site comprises the sequence HQGPFWML (SEQ ID NO: 40). As demonstrated in the experimental section herein and in FIG. 4, the kcat / Km of the FAPα cleavage site comprising the sequence HQGPFWML (SEQ ID NO: 40) was found to be about 80,000 M -1 sec -1 , which is the highest turnover number reported so far for a FAPα substrate.

[0071] Therapeutic agents

[0072] As described above, when the membrane interacting domain of the propeptides of the present disclosure is stably associated with (e.g., conjugated to) one or more therapeutic agents, the propeptides are useful for treating a condition associated with FAPa expression (e.g., a cancer associated with FAPa expression) in a subject in need thereof.

[0073] Therefore, in certain embodiments, there is provided a propeptide comprising a membrane interaction domain stably associated with one or more therapeutic agents (e.g., conjugated thereto). As used herein, a "therapeutic agent" is a physiological or pharmacologically active substance that can produce a desired biological effect in a target site of an animal such as a mammal or a human. The therapeutic agent can be any inorganic compound, organic compound, radioactive isotope, etc. The therapeutic agent can reduce, inhibit, weaken, reduce, prevent or stabilize the development or progress of a disease, illness or cell growth of an animal such as a mammal or a human. Examples include, but are not limited to, peptides, proteins, nucleic acids (including siRNA, miRNA and DNA), polymers, small molecules and radioactive isotopes. When the function of a cell / tissue is pathological, a therapeutic agent that reduces the function of a cell / tissue can be used. In certain embodiments, a membrane interaction domain is stably associated with an agent that reduces the function of a target cell / tissue by inhibiting cell proliferation and / or killing cells / tissues. Such agents can vary and include radioactive isotopes, cell inhibitors and cytotoxic agents, for example, agents that can kill target cell tissues and be internalized into target cells or not internalized into target cells.

[0074] In certain embodiments, "stably associated" means a physical association between two entities, wherein the average half-life of the association is one day or more in PBS at 4°C. In some embodiments, the physical association between the two entities has an average half-life of one day or more, one week or more, one month or more, including six months or more, such as 1 year or more in PBS at 4°C. According to some embodiments, the stable association results from a covalent bond between the two entities, a non-covalent bond (e.g., an ionic bond or a metallic bond) between the two entities, or other forms of chemical attraction, such as hydrogen bonding, van der Waals forces, etc. In certain embodiments, the membrane interaction domain is conjugated to one or more therapeutic agents.

[0075] According to some embodiments, the one or more therapeutic agents include a cytotoxic agent, a toxin, a radiosensitizer, a radioisotope, or any combination thereof.

[0076] In certain embodiments, the therapeutic agent is a cytotoxic agent selected from the group consisting of enediynes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, and vinca alkaloids. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, maytansine DM1, maytansine DM4, DM-1, auristatin or other dolastatin derivatives such as auristatin E or auristatin F, AEB (AEB-071), AEVB (AE 5-benzoylvaleric acid ester), AEFP (antibody-endostatin fusion protein), MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), pyrrolobenzodiazepine class (PBD), eleutherobin, netropsin or any combination thereof.

[0077] According to some embodiments, the pharmaceutical agent is a toxin, such as a protein toxin selected from hemiasterlin and hemiasterlin analogs such as HTI-286 (see, e.g., USPN 7,579,323; WO 2004 / 026293; and USPN 8,129,407, the entire disclosures of which are incorporated herein by reference), abrin, strychnine, cicutoxin, diphtheria toxin, batrachotoxin, botulinum toxin, Shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin B1, fumonisin B2, aflatoxin, maurotoxin, agitoxin, scorpion to ... xin, charybdotoxin, margatoxin, slotoxin, scorpion toxin, hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin, gelonin, lotaustralin, ocratoxin A, patulin, ricin, strychnine, trichothecenes, zearalenone, and tetrodotoxin. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, Dianthus caryophyllus proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, neomycin and trichothecenes.

[0078] In certain embodiments, the agent is a radiation sensitizer. As used herein, a "radiation sensitizer" is an agent that enhances the ability of radiation to kill cells (e.g., tumor cells). Non-limiting examples of radiation sensitizers that can be conjugated to a membrane interaction domain include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like.

[0079] In certain embodiments, the agent is a radioactive isotope, for example, which can be used for therapy and / or detection (e.g., imaging). Non-limiting examples of radioactive isotopes that can be conjugated to the membrane interacting domain include, but are not limited to225 And 111 Ag 114 Ag 71 As 72 As 77 As 211 And 198 I 199 I 212 Hello 213 Hello 75 Bro 76 Bro 11 C、 13 C、 55 Co. 62 Cu、 64 Cu、 67 Cu、 165 Dy 166 Dy 169 Err 18 F、 19 F、 52 Fe 59 Fe 66 Ga 67 Ga 68 Ga 72 Ga 154-158 God 157 God 159 God 166 Ho 120 I 121 I 123 I 124 I 125 I 131 I 110 in 111 in 113m in 194 Ir 81m Cr 177 sun 51 Mn 52 Mn 99 For 13 N 15 N 15 Or 17 Or 32 Q 33 Q 211 Pb、 212 Pb、 109 pd 149 PM 151 PM 142 Mr. 143 Mr. 191 PT. 193mPT, 195m Pt, 223 Ra, 142 Rb, 186 Re, 188 Re, 189 Re, 105 Rh, 47 Sc, 75 Se, 153 Sm, 117m Sn, 121 Sn, 83 Sr. 89 Sr. 161 Tb, 94 Tc, 99 Tc, 99m Tc, 227 Th, 201 Tl, 172 Tm, 127 Te, 90 Y. 169 Yb, 175 Yb, 133 X and 89 According to some embodiments, the membrane interaction domain is 64 Cu(Copper-64), 177 Lu(Lutetium-177) or 225 Ac(Actin-225) conjugation.

[0080] In certain embodiments, the radioisotope is conjugated to the membrane interaction domain via a chelator. Non-limiting examples of chelators that can be employed include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), MACROPA, and the like.

[0081] Methods for producing propeptides

[0082] The propeptide of the present disclosure can be produced by any suitable method, including recombinant methods and non-recombinant methods (such as chemical synthesis). In the case where the polypeptide is chemically synthesized, the synthesis can be carried out via liquid phase or solid phase. Solid phase synthesis (SPPS) allows the incorporation of non-natural amino acids, peptide / protein backbone modifications. Various forms of SPPS (such as Fmoc and Boc) can be used to synthesize the propeptide of the present disclosure. The details of chemical synthesis are known in the art (such as Ganesan A.2006 "Mini Rev. Med Chem. Review" 6:3-10 and Camarero JA et al. 2005 "Protein and Peptide Express (Protein Pept Lett.)" 12:723-8). In short, small insoluble porous beads are treated with functional units on which peptide chains are constructed. After repeated cycles of coupling / deprotection, the free N-terminal amine of the peptide or amino acid attached to the solid phase is coupled to a single N-protected amino acid unit. The unit is then deprotected, showing a new N-terminal amine, to which additional amino acids can be attached. The peptide remains immobilized on the solid phase and undergoes a filtration process before being cleaved.

[0083] In a non-limiting example, the propeptide of the present disclosure can be synthesized by Fmoc solid phase synthesis on a Biotage SyroII peptide synthesizer at ambient temperature. Using pre-loaded lysine (2-dinitrophenyl) Wang resin, the synthesis scale can be 12.5 μM, wherein the DNP quencher can be connected to the ε nitrogen of lysine. The coupling reaction can be carried out for 8 minutes with 4.9 equivalents of HCTU (O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) in 500 μL N, N-dimethylformamide (DMF), 5 equivalents of Fmoc-amino acid-OH and 20 equivalents of N-methylmorpholine (NMM) while shaking. Each amino acid position can be double coupled, and the subsequent Fmoc deprotection can be carried out for 10 minutes with 500 μL of 40% 4-methylpiperidine in DMF, followed by 6 washings with 500 μL DMF for 3 minutes. While shaking, the propeptide was cut from Wang's resin with 500 μL of a solution consisting of 95% trifluoroacetic acid, 2.5% water and 2.5% triisopropylsilane for 1 hour. The crude propeptide product can then be precipitated in 30 mL of cold 1:1 ether: hexane, and then dissolved in a 1:1:1 mixture of DMSO: water: acetonitrile. The dissolved crude product can be purified by high performance liquid chromatography (HPLC) using Agilent Pursuit 5C18 columns (5 mm bead size, 150 x 21.2 mm) on Agilent PrepStar 218 series preparative HPLC. Mobile phases A and B can be water+0.1% TFA and acetonitrile+0.1% TFA, respectively. Solvents can be removed under a reducing atmosphere, and the purified peptide product can be dissolved in DMSO stock with a desired final concentration (e.g., 10 mM). Purity can be confirmed by liquid chromatography-mass spectrometry, and the stock solution is stored at -20 ° C.

[0084] Where the propeptide is produced using recombinant techniques, it may be produced as an intracellular or secreted protein using any suitable construct and any suitable host cell, which may be a prokaryotic or eukaryotic cell, such as a bacterial (e.g. E. coli) or yeast host cell, respectively.

[0085] Other examples of eukaryotic cells that can be used as host cells include insect cells, mammalian cells and / or plant cells. In the case of using mammalian host cells, cells can include one or more of the following: human cells (e.g., HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., X3, NIH3T3, pancreatic ductal adenocarcinoma 2.1, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos 7, and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

[0086] According to standard procedures known in the art, a wide range of host-vector systems suitable for expressing the subject propeptide can be used. See, for example, Sambrook et al., 1989 "Current Protocols in Molecular Biology", Cold Spring Harbor Press, New York, and Ausubel et al., 1995 "Current Protocols in Molecular Biology", edited by Wiley and Sons. Methods for introducing genetic material into host cells include, for example, conversion, electroporation, conjugation, calcium phosphate methods, etc. Methods for transfer can be selected to provide stable expression of the nucleic acid encoding the polypeptide introduced. The nucleic acid encoding the polypeptide can be provided as a heritable episomal element (e.g., plasmid), or can be genomic integration. A variety of suitable vectors for producing a polypeptide of interest are commercially available.

[0087] The carrier can provide extrachromosomal maintenance in the host cell, or can provide integration into the host cell genome. Expression vectors provide transcription and translation regulatory sequences, and can provide inducible or constitutive expression, wherein the coding region is operably connected under the transcriptional control of the transcription initiation region and the transcription and translation termination region. Generally speaking, transcription and translation regulatory sequences can include but are not limited to promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences and enhancer or activator sequences. The promoter can be constitutive or inducible, and can be a strong constitutive promoter (such as T7, etc.). The expression construct usually has a convenient restriction site located near the promoter sequence, to provide the insertion of the nucleic acid sequence encoding the protein of interest. There can be operable selective markers in the expression host, to promote the selection of the cell containing the carrier. In addition, the expression construct can include other elements. For example, the expression vector can have one or two replication systems, thereby allowing it to be maintained in an organism, such as maintaining for expression in a mammal or insect cell, and maintaining for cloning and amplification in a prokaryotic host. In addition, the expression construct may contain a selectable marker gene to allow selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.

[0088] The separation and purification of proteins can be accomplished according to methods known in the art. For example, proteins can be separated from lysates of cells genetically modified to express proteins constitutively and / or after induction, or from synthetic reaction mixtures, by immunoaffinity purification, which generally involves contacting the sample with anti-protein antibodies, washing to remove non-specifically bound materials, and eluting specifically bound proteins. The separated proteins can be further purified by other methods commonly used in dialysis and protein purification methods. In one embodiment, metal chelate chromatography methods can be used to separate proteins. Proteins of the present disclosure may contain modifications to facilitate separation.

[0089] The subject propeptides can be prepared in substantially pure or isolated form (e.g., free of other polypeptides). The propeptides can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified propeptides can be provided so that the propeptides are present in a composition that is substantially free of other expressed proteins, e.g., less than 98%, less than 95%, less than 90%, less than 80%, less than 60%, or less than 50% of the composition consists of other expressed proteins.

[0090] DOTA-FRIP can be first synthesized using solid phase peptide synthesis conditions as outlined above. The resin-bound peptide with an N-terminal hexanoic acid can then be triple coupled with 2 equivalents of dota-NHS, 5 equivalents of HCTU, and 20 equivalents of N,N-diisopropylethylamine (DIPEA) for 12 hours to synthesize DOTA-FRIP.

[0091] Composition

[0092] Aspects of the present disclosure also include compositions. According to some embodiments, the subject compositions comprise any propeptide of the present disclosure, including but not limited to any propeptide described in the Propeptide section above and in the Experimental section below.

[0093] In certain aspects, the compositions of the present disclosure include propeptides present in a liquid medium. The liquid medium can be an aqueous liquid medium, such as water, a buffer solution, etc. One or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), 2-(N-morpholino) ethanesulfonic acid sodium salt (MES), 3-(N-morpholino) propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS) etc.), solubilizers, detergents (e.g., nonionic detergents such as Tween-20 etc.), nuclease inhibitors, protease inhibitors, glycerol, chelating agents etc. can be present in such compositions.

[0094] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure comprises a propeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0095] The propeptide can be incorporated into various formulations for treatment, diagnosis or therapeutic diagnosis. More particularly, the propeptide can be formulated into a pharmaceutical composition by combining with an appropriate pharmaceutically acceptable excipient or diluent, and can be formulated into a preparation in solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and sprays.

[0096] Formulations of propeptides for administration to an individual (eg, suitable for human administration) are generally sterile, and, depending on the route of administration selected, may further be free of detectable pyrogens or other contaminants that would contraindicate administration to a patient.

[0097] In pharmaceutical dosage forms, the propeptides may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone or in appropriate association, and in combination with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are by no means limiting.

[0098] For oral formulations, the propeptide can be used alone or in combination with suitable additives to make tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if necessary, with diluents, buffers, wetting agents, preservatives and flavoring agents.

[0099] The propeptide can be formulated for parenteral administration (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, intratumoral, etc.). In certain aspects, the propeptide is formulated for injection by dissolving, suspending or emulsifying the propeptide in an aqueous or non-aqueous solvent, such as a vegetable oil or other similar oil, an ester of a synthetic aliphatic acid glyceride, a higher aliphatic acid ester or propylene glycol; and if necessary, the propeptide is dissolved, suspended or emulsified together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0100] Pharmaceutical compositions comprising the propeptide can be prepared by mixing the propeptide having the desired purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride or a combination thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, thiazolinone ... acid, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other sugars; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine and neuraminic acid; and / or nonionic surfactants, such as Tween, Brij Pluronics, Triton-X or polyethylene glycol (PEG).

[0101] The pharmaceutical composition can be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is reconstituted with a sterile solution before administration. The standard method for reconstitution of a lyophilized composition is to add back a volume of purified water (usually equivalent to the volume removed during the lyophilization process); however, a solution containing an antibacterial agent can be used to produce a pharmaceutical composition for parenteral administration.

[0102] Aqueous formulations of the propeptides can be prepared in a pH buffered solution, for example, in the pH range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or about 5.5. Examples of buffers suitable for pH in this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on, for example, the desired tonicity of the buffer and formulation.

[0103] A tonicity agent may be included to adjust the tension of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerol, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, but hypertonic or hypotonic solutions may also be suitable. The term "isotonic" refers to a solution having the same tension as some other solution compared thereto, such as a physiological saline solution or serum. The tonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0104] Surfactants can also be added to the formulation to reduce aggregation and / or minimize the formation of particles in the formulation and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamers, Pluronic) and sodium lauryl sulfate (SDS). An example of a suitable polyoxyethylene sorbitan-fatty acid ester is polysorbate 20 (known under the trademark Tween 20 TM Polysorbate 80 (sold under the trademark Tween 80 TM Examples of suitable polyethylene-polypropylene copolymers are marketed under the name F68 or Poloxamer 188 TM Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij TM Exemplary concentrations of surfactants may range from about 0.001% to about 1% w / v.

[0105] Lyoprotectants may also be added to protect the propeptide from unstable conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants may be included in an amount of about 10 mM to 500 nM.

[0106] In some embodiments, the pharmaceutical composition comprises the propeptide of the present disclosure and one or more of the components identified above (e.g., surfactants, buffers, stabilizers, tonicity agents) and is substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, the preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0107] Reagent test kit

[0108] Aspects of the present disclosure also include kits. According to some embodiments, the subject kits comprise any propeptide of the present disclosure, including but not limited to any propeptide described in the propeptide section above and in the experimental section below.

[0109] In certain embodiments, the kits are used to practice the methods of the disclosure, e.g., methods for treating a condition associated with FAPa expression in a subject in need thereof, methods for assessing FAPa activity in a subject, and the like.

[0110] Thus, in certain embodiments, a kit of the present disclosure may comprise any of the propeptides or pharmaceutical compositions of the present disclosure, and instructions for administering the propeptide or pharmaceutical composition to a subject in need thereof.

[0111] The kit of the present disclosure may include a certain amount of propeptide or pharmaceutical composition, which is in a unit dose, such as an ampoule or a multiple dose form. Therefore, in certain embodiments, the kit may include a composition of one or more (e.g., two or more) unit doses (e.g., ampoules), and the composition includes the propeptide of the present disclosure. As used herein, the term "unit dose" refers to a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of composition calculated in an amount sufficient to produce the desired effect. The amount of a unit dose depends on a variety of factors, such as the specific propeptide used, the effect to be achieved, and the pharmacodynamics associated with the propeptide in the subject. In yet other embodiments, the kit may include a composition of the amount of a single multiple dose.

[0112] The instructions (e.g., instructions for use (IFU)) included in the test kit can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. Therefore, the instructions can be present in the test kit as a package insert, in the label of the container of the test kit or its components (i.e., related to packaging or subpackaging). In other embodiments, the instructions exist as an electronic storage data file present on a suitable computer-readable storage medium, such as a portable flash drive, DVD, CD-ROM, floppy disk, etc. In yet other embodiments, the actual instructions are not present in the test kit, but a method for obtaining instructions from a remote source, such as via the Internet, is provided. An example of this embodiment is a test kit comprising a website, in which the instructions can be viewed and / or can be downloaded from the website. As with the instructions, the method for obtaining the instructions is recorded on a suitable substrate.

[0113] How to use

[0114] Aspects of the present disclosure include methods of using the propeptides of the present disclosure. The methods can be used in a variety of situations, including in vitro and / or in vivo studies and / or clinical applications.

[0115] In certain aspects, methods of treating a condition associated with FAPa expression in a subject in need thereof are provided, the method comprising administering to the subject an effective amount of a propeptide of the present disclosure.

[0116] In some embodiments, the condition associated with FAPα expression is cancer. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals, which is usually characterized by unregulated cell growth / proliferation. The subject method can be used to treat a variety of cancers. Normal tissues have low and usually undetectable levels of FAPα expression. However, FAPα is overexpressed in many tumor tissues, including breast cancer, colorectal cancer, pancreatic cancer, lung cancer, brain cancer, intrahepatic bile duct cancer, and ovarian cancer. In addition, high levels of FAPα expression can be detected in some tumors derived from non-epithelial tissues, such as melanoma and myeloma. In these tumors, FAPα overexpression is often observed in the stroma, which leads to FAPα being considered a universal marker for CAF, although FAPα expression can also be detected in gastric cancer, pancreatic cancer, and melanoma cells. Details about FAPα expression in human malignancies can be found in, for example, Busek et al. (2018) Frontiers of Biological Sciences-Milestones 23: 1933-1968.

[0117] Thus, in some embodiments, when the condition associated with FAPa expression is cancer, the cancer may be characterized by expression of FAPa on cancer cells, cancer-associated fibroblasts, or both. In certain embodiments, the cancer comprises a solid tumor. Examples of solid tumors that can be treated by the methods of the present disclosure include carcinomas, lymphomas, blastomas, and sarcomas. Non-limiting examples of cancers that can be treated by the methods of the present disclosure include basal cell carcinoma, squamous cell carcinoma of the skin, squamous cell carcinoma of the oral cavity, melanoma, esophageal cancer, gastric cancer, colorectal cancer, pancreatic adenocarcinoma, hepatocellular carcinoma, non-small cell lung cancer, mesothelioma, breast cancer, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, glioma, parathyroid cancer, sarcoma, and myeloma.

[0118] In certain embodiments, when the condition associated with FAPa expression is cancer, the cancer may include a hematological malignancy. According to some embodiments, the hematological malignancy is a leukemia, a lymphoma, or multiple myeloma.

[0119] In addition to cancer, conditions associated with FAPa expression may also be treated by the methods of the present disclosure. For example, the condition associated with FAPa expression may be a fibrotic disease. In some embodiments, the fibrotic disease includes liver fibrosis. According to certain embodiments, the fibrotic disease is an interstitial lung disease.

[0120] The propeptides of the present disclosure can be administered in a therapeutically effective amount as a composition. "Therapeutically effective amount" means a dose sufficient to produce a desired result, for example, an amount sufficient to achieve a beneficial or desired therapeutic (including preventive) result, such as a reduction in symptoms of cancer, fibrotic disease, or other conditions associated with FAPα expression, compared to a control. With regard to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow the growth of a tumor, reduce the size of a tumor, etc. An effective amount can be administered in one or more administrations.

[0121] As described above, aspects of the present disclosure include methods for treating a condition associated with FAPα expression in a subject in need thereof. "Treatment" or "treating" means at least ameliorating one or more symptoms associated with a condition associated with FAPα expression (e.g., cancer, fibrotic disease, etc.), wherein improvement is used in a broad sense to refer to at least reducing the magnitude of a parameter (e.g., symptom) associated with the condition being treated. Thus, treatment also includes situations in which the condition or at least one or more symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least no longer suffers from the symptoms characterizing the condition.

[0122] The propeptides of the present disclosure can be administered to a subject alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for the treatment of conditions associated with FAPα expression (such as cancer, fibrotic diseases, etc.).

[0123] The present disclosure also provides a method for detectably labeling the phospholipid bilayer of a cell in the presence of FAPα activity. Such methods include contacting a propeptide of the present disclosure with a FAPα that contributes to FAPα activity. The membrane interaction domain is detectably labeled, and the FAPα cleavage site of the propeptide is cleaved by FAPα to release a cleavage product containing a detectably labeled membrane interaction domain, so that the detectably labeled membrane interaction domain interacts with the phospholipid bilayer of the cell, and detectably labels the phospholipid bilayer of the cell in the presence of FAPα activity.

[0124] Aspects of the present disclosure further include methods for assessing FAPα activity in a subject. Such methods include administering a propeptide of the present disclosure to a subject, wherein the membrane interaction domain is detectably labeled, wherein at the site of FAPα activity in the subject, the FAPα cleavage site of the propeptide is cleaved by FAPα to release a cleavage product comprising the detectably labeled membrane interaction domain, and wherein the cleavage product interacts with the phospholipid bilayer of a cell at the site of FAPα activity in the subject. Such methods further include assessing the presence or absence of cells labeled with the cleavage product, wherein the presence of cells labeled with the cleavage product indicates FAPα activity in the subject.

[0125] In certain embodiments, the membrane interaction domain is detectably labeled with a labeling agent that is used for in vivo imaging, such as near infrared (NIR) optical imaging, single photon emission computed tomography (SPECT) ± CT imaging, positron emission tomography (PET) ± CT imaging, nuclear magnetic resonance (NMR) spectroscopy, etc. Labeling agents used in such applications include, but are not limited to, fluorescent labels, radioisotopes, etc. In certain aspects, the labeling agent is a multimodal in vivo imaging agent that allows in vivo imaging using two or more imaging methods (e.g., see Thorp-Greenwood and Coogan (2011) Dalton Trans. 40: 6129-6143).

[0126] In certain embodiments, the labeling agent is an in vivo imaging agent for near infrared (NIR) imaging applications. Such agents include, but are not limited to, Kodak X-SIGHT dyes, Pz 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 dyes, IRDye 680 and 800CW Fluors. According to some embodiments, the labeling agent is an in vivo imaging agent for SPECT imaging applications, non-limiting examples of which include 99m Tc, 111 In, 123 I. 201 Tland 133 In certain embodiments, the labeled agent is an in vivo imaging agent for PET imaging applications, such as 11 C. 13 N. 15 O. 18 F. 64 Cu, 62 Cu, 124 I. 76 Br, 82 Rb, 68 Ga, 177Lu, 225 Ac et al.

[0127] It will be appreciated by those having the benefit of this disclosure that the propeptides of the present disclosure are useful in diagnostic applications. For example, when the membrane interacting domain is treated with a radioactive isotope (e.g. 64 Cu(Copper-64), 177 Lu(Lutetium-177) or 225 When labeled with Ac (actinium-225), the propeptide can be used in a method for treating a condition associated with FAPα expression (e.g., cancer, fibrotic disease, etc.) in a subject in need thereof, wherein the method further comprises utilizing a radioisotope for in vivo imaging, such as by PET / CT and / or PET / MR to assess FAPα activity in the subject.

[0128] The following embodiments are offered by way of illustration and not by way of limitation.

[0129] experiment

[0130] Example 1 - Identification of unique FAPα substrate sequences using multiplex substrate profiling by mass spectrometry (MSP-MS)

[0131] Although much is known about the substrate preference of P4-P1 for FAPα, the optimal P4-P4' substrate sequence for FAPα has never been defined. In this example, multiple substrate analysis by mass spectrometry (MSP-MS) was performed to identify candidate FAPα P4-P4' substrate sequences for possible inclusion in a FAPα-targeted restricted interaction peptide (or "FRIP").

[0132] MSP-MS resulted in several potential substrates, almost all of which carried a Gly-Pro at the P2-P1 position, which was previously shown to be required for FAPα proteolysis. Figure 3 The Icelogo representing the cumulative amino acid preference of recombinant human FAPα is shown at the top. Figure 3 The bottom shows a heat map summarizing the most frequently incorporated amino acids at each site in the cleavage products. Light colors indicate abundant amino acids. X = no preferred amino acid.

[0133] The list of top P4-P4' peptides cleaved by human FAPα as determined using MSP-MS substrate analysis techniques is shown in the table below, where X indicates undefined amino acid positions.

[0134]

[0135]

[0136] Three hits, HIGPTAAY (SEQ ID NO: 38), HQGPFWML (SEQ ID NO: 40), and HYGPTVNK (SEQ ID NO: 42), were selected for further analysis. 8-mer peptides carrying FRET donor-acceptor dyes at the N- and C-termini were synthesized and evaluated as substrates for recombinant human FAPa (RND system) under Michaelis-Menten conditions. The kcat / Km were determined to be approximately 120,000, approximately 80,000, and approximately 15,000 M, respectively. -1 sec -1 .

[0137] Provided below is a table summarizing the kinetic properties of the three top P4-P4' 8-mer peptides that emerged from the MSP-MS assays, where the kinetic properties were determined using FRET donor / acceptor substrate adducts and human recombinant FAPα.

[0138]

[0139] The data showing the kinetic properties of probe 1, probe 2, and probe 3 are shown in Figure 4A , Figure 4B and Figure 4C Available in.

[0140] Example 2 - Development of FAPα-targeted restricted interaction peptides (FRIPs) and selection of FAPα-positive tumors in vivo Sexual intake

[0141] In this example, the three top cleavage sequences from the previous example were incorporated into full-length RIPs with N-terminal DOTA for radiolabeling (referred to herein as "FRIP1," "FRIP2," and "FRIP3"). Figure 5 A schematic diagram of an exemplary FRIP with a probe 2 sequence is provided at the top of FIG. 64 Cu-labeled membrane interaction / binding domain; (2) FAPα cleavage site; and (3) masking domain. In this example, the membrane interaction domain is an antimicrobial peptide (AMP), in particular Temporin L peptide (FVQWFSKFLGK; SEQ ID NO: 51) as an AMP. The FAPα cleavage site is a hot site identified in Example 1. In this example, the masking domain is a peptide derived from protease activated receptor 1 (PAR1) having the sequence QDPNDQYEPF (SEQ ID NO: 35).

[0142] DOTA-FRIP was radiolabeled with Cu-64 using a protocol developed for DOTA-GRIP B (Zhao et al. (2021) ACS Cent Sci. 7(10):1638-49). The radiochemical yield was always >95% and the radiochemical purity was >99%. Human FAPα was tested on HPLC for each 64 Cleavage of Cu-FRIP. 64 Cu-FRIP2 was the most efficient cleavage, with complete conversion to product peaks within 3 h. 64 The in vitro serum stability of Cu-FRIP2 was determined and was found to be >95% stable over 12 hours at room temperature.

[0143] Next, mice bearing U87 xenografts were 64 Cu-FRIP2 uptake and 64 Cu-FRIP1, 64 Cu-FRIP3 and 64 Cu-GRIP B was used for comparison. 24 hours after injection, 64 The tumor uptake of Cu-FRIP2 was significantly higher than that of other probes ( Figure 6A-6B ), emphasizing that efficient proteolytic cleavage is required for tumor retention of RIP.

[0144] Next test 64 Whether Cu-FRIP2 is selectively taken up by FAPα-positive tumors in vivo. 64 Cu-FRIP2 (approximately 400 mCi / mouse, n=4 per group) was administered to different cohorts of mice bearing subcutaneous U87 MG (FAPα-positive human glioblastoma), HT1080 (FAPα-negative human fibrosarcoma), or MIA Paca-2 tumors (FAPa-negative human pancreatic cancer). At 24 hours post-injection, compared with models lacking FAPα, Cu-FRIP2 increased in U87 tumors. 64 The uptake of Cu-FRIP2 was significantly higher ( Figure 7 Biodistribution data collected 24 hours after injection ( Figure 8 ) showed that U87 tumors had the highest uptake of the radiotracer compared to normal tissues. The liver and kidneys also showed modest uptake of the radiotracer. As with GRIP B, uptake in normal mouse tissues was overall low. The molecule is cleared by the kidneys with some retention in the liver.

[0145] Male and female C57Bl6 mice 64 Formal dosimetry studies of Cu-FRIP2 have shown that the effective whole-body dose of the tracer is 64Cu-GRIP B and other commonly used tracers such as 18 The liver and kidneys experience the highest doses, as we have previously described. 64 Cu-GRIP B recorded.

[0146] Example 3 - FRIP exhibits superior biodistribution compared to FAPI 46

[0147] In this example, a longitudinal PET study was performed to compare 64 Tumor uptake of Cu-FRIP2 and FAPI-46, an active site-directed FAPα radioligand currently in clinical trials (Meyer et al. (2020) J Nucl Med. 61(8):1171-7). DOTA-FAPI-46 was purchased from MedChemExpress and used similarly to 64 Protocol for Cu-FRIP2 coupled with Cu-64. Radiochemical yield and purity >95%.

[0148] Will 64 Cu-FRIP2 and 64 Cu-FAPI 46 was administered to mice bearing subcutaneous U87 tumors at the same dose and specific activity. PET studies ( Fig.9A )show, 64 Tumor uptake of Cu-FRIP increases between 2 and 24 hours after injection and reaches approximately 9% ID / cc of SUV 平均值 ( Fig. 9B ). In contrast, 64 Cu-FAPI 46 levels were significantly lower during the same time interval (SUV 平均值 is about 1% ID / cc). It is worth noting that although 64 Tumor uptake of Cu-FRIP2 was significantly higher than 64 Cu-FAPI 46, but the clearance of each radiopharmaceutical was virtually identical. 64 Cu-FRIP2 and 64 Cu-FAPI 46 tumor uptake was evaluated in a separate cohort of mice bearing PC3 PIP xenografts, which have lower FAP compared to U87. Figure 10A-10B ) shows that the tumor 64 Cu-FRIP2 uptake was significantly higher than 64 Cu-FAPI 46. As observed in the U87 cohort, 64 Tumor area under the curve ratio of Cu-FRIP2 64Cu-FAPI 46 was approximately 5-fold higher in PC3 PIP tumors compared to U87 tumors, as expected. 64 The uptake of Cu-FRIP2 was significantly lower.

[0149]

[0150] The following table provides estimates based on mouse dosimetry studies. 64 Human absorbed dose of Cu-FRIP2, where the values ​​were calculated using OLINDA EXM software.

[0151]

[0152]

[0153] Example 4 67 Cu-FRIP2 effectively inhibits tumor growth

[0154] In light of the data presented in the preceding examples herein, it is expected that RIPs (including FRIPs) will be useful for targeted radiation therapy (TRT). In contrast to RLTs, RIPs accumulate in tumors through a renewable catalytic mechanism, but also persist in tumors because cancer cells do not have an effective mechanism to disrupt the peptide / membrane complex ( Fig.11 In this example, a non-limiting exemplary FRIP ( 67 Cu-FRIP2) demonstrated effective inhibition of tumor growth by FRIP.

[0155] By using 67 Cu-FRIP2 was used to treat mice bearing U87 MG tumors for anti-tumor evaluation. Anti-tumor evaluation studies were piloted with Cu-67 because it is a beta emitter, isosteric to Cu-64, and this isotope has clinical significance, such as the recent use of Cu-67 in the United States. 67 Cu-SARTATE was demonstrated in a trial targeting SSTR2-positive neuroendocrine tumors (NCT04023331). 67 Cu-FRIP2, used in 64 The same protocol for Cu-FRIP2 will be 67 CuCl2 was coupled to DOTA-FRIP2. The radiotracer was synthesized to >95% radiochemical yield and purity.

[0156] Male athymic nu / nu mice bearing subcutaneous U87 MG tumors received a single IV dose of 67 Cu-FRIP2 (1 mCi / mouse) or vehicle (n=8 mice / group). Data are shown in Figure 12A-12C , Figure 13A-13B and Fig.14 In. Single dose 67 Cu-FRIP2 significantly inhibited the growth of U87 MG tumors ( Fig. 12A ) and increase survival rate ( Fig.13A ). Survival rate using (1) more than 2000mm 3 The treatment was well tolerated as no significant decrease in body weight was detected ( Fig. 13B ).At Fig.14 Spider diagrams showing changes in individual tumor volumes for the represented mice are shown in .

[0157] Next test with 67 Compared with Cu-FAPI 46, 67 Cu-FRIP2 is more effective in inhibiting tumor growth. Figure 15A-15B and Fig.16 Use vehicle or a single IV dose of 67 Cu-FRIP2 (1 mCi / mouse) or 67 Mice bearing subcutaneous U87 MG xenografts were treated with Cu-FAPI46 (1 mCi / mouse). 67 Compared with Cu-FAPI 46 or vehicle, 67 Cu-FRIP2 more effectively inhibited tumor growth.

[0158] Next, we evaluated the 64 Cu-FRIP2 can inhibit tumor growth. 64 Male athymic nu / nu mice (n=8 mice / group) bearing subcutaneous U87 MG xenografts were treated with Cu-FRIP2 (1.5 mCi / mouse). 67 Cu-FRIP2 inhibited tumor growth ( Fig.19A ). The survival data plotted on the Kaplan-Meier curve showed that the survival of the treatment group was significantly prolonged compared with the vehicle group (P<0.01) ( Fig.19B ).

[0159] Therefore, the above only illustrates the principle of the present disclosure.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language narrated in this article are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventor to promote this area, and should be interpreted as not being limited to such specific examples and conditions of narration.In addition, all statements of the principles, aspects and embodiments of the present invention and its specific examples are narrated herein and are intended to cover both its structural equivalents and functional equivalents.In addition, it is intended that such equivalents include both currently known equivalents and future development equivalents, i.e. any element of the performance of the same function developed, regardless of the structure.Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. A propeptide comprising: membrane interaction domain; a masking domain that, when linked to the membrane interaction domain, is effective to inhibit interaction of the membrane interaction domain with a phospholipid bilayer; and A fibroblast activation protein alpha (FAPα) cleavage site is disposed between the membrane interaction domain and the masking domain.

2. The propeptide of claim 1, wherein the membrane interaction domain comprises about 5 to about 30 amino acid residues.

3. The propeptide of claim 1 or claim 2, wherein the membrane interacting domain, when separated from the masking domain, comprises an alpha-helical structure capable of inserting into a phospholipid bilayer.

4. The propeptide of claim 3, wherein the membrane interaction domain comprises a protein from the Temporin family.

5. The propeptide of claim 4, wherein the membrane interaction domain comprises the amino acid sequence X a X b X c X d X e X f Y a X g X h Y b Y * X i X j , where X a , X b , X c , X d , X e , X f , X g , X h , X i and X j is a hydrophobic amino acid residue, Y a and Y b is a hydrophilic amino acid residue, and Y * It is a charged amino acid residue.

6. The propeptide of claim 5, wherein the membrane interaction domain comprises the amino acid sequence FVQWFSKFLGRIL (SEQ ID NO: 2).

7. The propeptide of claim 5, wherein the membrane interaction domain comprises the amino acid sequence FVQWFSKFLGKLL (SEQ ID NO: 3).

8. The propeptide according to any one of claims 1 to 7, wherein the masking domain comprises a peptide derived from protease activated receptor-1 (PAR-1).

9. The propeptide of claim 8, wherein the masking domain comprises the amino acid sequence (X a )(X b )PND(X c )YEPF (SEQ ID NO: 52), wherein X a Is R or Q, X b is N or D, and X c It is K or Q.

10. The propeptide of claim 9, wherein the masking domain comprises the amino acid sequence QDPNDQYEPF (SEQ ID NO: 35).

11. The propeptide of claim 9, wherein the masking domain comprises the amino acid sequence RNPNDKYEPF (SEQ ID NO: 36).

12. The propeptide according to any one of claims 1 to 11, wherein the FAPa cleavage site comprises the FAPa specific motif GP in the P2-P1 position.

13. The propeptide of claim 12, wherein the FAPα cleavage site comprises the amino acid sequence YHGPLAHX (SEQ ID NO: 37), HIGPTAAY (SEQ ID NO: 38), XXIPTNIR (SEQ ID NO: 39), HQGPFWML (SEQ ID NO: 40), XXGPKLTY (SEQ ID NO: 41), HYGPTVNK (SEQ ID NO: 42), XXWPMGMY (SEQ ID NO: 43), XXFPNMWS (SEQ ID NO: 44), XXGSQVFS (SEQ ID NO: 45), XXMPEEVA (SEQ ID NO: 46), or XXHPTKSF (SEQ ID NO: 47), wherein X is any amino acid. 14 . The propeptide of claim 13 , wherein the FAPa cleavage site comprises the amino acid sequence HQGPFWML (SEQ ID NO: 40), XXGPKLTY (SEQ ID NO: 41), or HYGPTVNK (SEQ ID NO: 42).

15. The propeptide of claim 14, wherein the FAPa cleavage site comprises the amino acid sequence HQGPFWML (SEQ ID NO: 40). 16 . The propeptide according to claim 1 , comprising, from N-terminus to C-terminus: the membrane interaction domain, the FAPa cleavage site and the masking domain.

17. The propeptide of any one of claims 1 to 16, wherein the membrane interacting domain is conjugated to one or more therapeutic agents.

18. The propeptide of claim 17, wherein the one or more therapeutic agents comprise a cytotoxic agent, a toxin, a radiosensitizer, a radioisotope, or any combination thereof.

19. The propeptide of claim 18, wherein the one or more therapeutic agents comprises a radioactive isotope.

20. The propeptide of claim 19, wherein the radioisotope is selected from the group consisting of actinium-225, astatine-211, bismuth-212, bismuth-213, bromine-76, bromine-77, calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, copper-64, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-14, -111, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, lead-212, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, radium-224, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, thorium-226, thorium-227, xenon-133, or yttrium-9.

21. The propeptide of claim 20, wherein the radioisotope is copper-64, lutetium-177 or actinium-225.

22. The propeptide of any one of claims 19 to 21, wherein the radioisotope is conjugated to the membrane interacting domain via a chelating moiety.

23. The propeptide of claim 22, wherein the chelating moiety comprises 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

24. A composition comprising the propeptide according to any one of claims 1 to 23 in a liquid medium.

25. A pharmaceutical composition comprising: The propeptide according to any one of claims 1 to 23; and A pharmaceutically acceptable carrier.

26. A method of treating a condition associated with FAPa expression in a subject in need thereof, the method comprising administering to the subject an effective amount of the propeptide of any one of claims 17 to 23.

27. The method of claim 26, wherein the condition associated with FAPa expression is cancer.

28. The method of claim 27, wherein the cancer is characterized by expression of FAPa on cancer cells, cancer-associated fibroblasts, or both.

29. The method of claim 27 or claim 28, wherein the cancer comprises a solid tumor.

30. The method of claim 29, wherein the solid tumor is a carcinoma, lymphoma, blastoma, or sarcoma.

31. The method of any one of claims 27 to 30, wherein the cancer is basal cell carcinoma, squamous cell carcinoma of the skin, squamous cell carcinoma of the oral cavity, melanoma, esophageal cancer, gastric cancer, colorectal cancer, pancreatic adenocarcinoma, hepatocellular carcinoma, non-small cell lung cancer, mesothelioma, breast cancer, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, glioma, parathyroid cancer, sarcoma, or myeloma.

32. The method of claim 27, wherein the cancer comprises a hematological malignancy.

33. The method of claim 32, wherein the hematological malignancy is leukemia, lymphoma or multiple myeloma.

34. The method of claim 26, wherein the condition associated with FAPa expression is a fibrotic disease.

35. The method of claim 34, wherein the fibrotic disease comprises liver fibrosis.

36. The method of claim 34, wherein the fibrotic disease is interstitial lung disease.

37. The method of any one of claims 26 to 36, wherein the membrane interaction domain comprises a detectable label, and wherein the method further comprises performing in vivo imaging of cells in the subject labeled with the membrane interaction domain.

38. The method of claim 37, wherein the one or more therapeutic agents comprises the detectable marker.

39. The method of claim 38, wherein the one or more therapeutic agents comprises a radioisotope, wherein the radioisotope is the detectable marker.

40. The method of any one of claims 37 to 39, wherein the in vivo imaging comprises in vivo positron emission tomography (PET).

41. A method for detectably labeling a phospholipid bilayer of a cell in the presence of FAPα activity, the method comprising: contacting the propeptide according to any one of claims 1 to 23 with a FAPα that contributes to the activity of said FAPα, wherein: The membrane interacting domain is detectably labeled, and The FAPα cleavage site of the propeptide is cleaved by FAPα to release a cleavage product comprising a detectably labeled membrane interaction domain, such that the detectably labeled membrane interaction domain interacts with the phospholipid bilayer of the cell and detectably labels the phospholipid bilayer of the cell in the presence of FAPα activity.

42. A method for assessing FAPα activity in a subject, the method comprising: administering to the subject a propeptide according to any one of claims 1 to 23, wherein the membrane interacting domain is detectably labeled, wherein at the site of FAPα activity in the subject, the FAPα cleavage site of the propeptide is cleaved by FAPα to release a cleavage product comprising the detectably labeled membrane interacting domain, and wherein the cleavage product interacts with the phospholipid bilayer of the cell at the site of FAPα activity in the subject; as well as assessing the presence or absence of cells labeled with said cleavage product, The presence of cells labeled with said cleavage product indicates FAPa activity in the subject.

43. The method of claim 42, wherein the evaluating comprises in vivo imaging.

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