Endometriosis tracer

Abstract

The present invention relates to a conjugate comprising a FAP targeting ligand and an effector for use in the treatment and / or diagnosis of endometriosis, as well as associated methods and kits.

Description

Technical Field

[0001] This invention relates to the fields of medicine, medical imaging, and molecular biology. In particular, this invention relates to novel endometriosis tracers for use in therapeutic and / or diagnostic methods. Background Technology

[0002] Endometriosis is a chronic condition affecting 10% of women of reproductive age. The ectopic endometrial tissue located outside the uterine cavity causes severe and life-altering pain during menstruation, sexual intercourse, bowel movements, and / or urination. Women with endometriosis often experience chronic pelvic pain and infertility. This chronic pelvic pain is usually accompanied by bloating, nausea, fatigue, and sometimes depression and anxiety.

[0003] Endometriosis can begin at a woman's first menstrual period and continue until menopause, during which time endometrial tissue resembling the uterine lining grows outside the uterine cavity. This leads to inflammation and scarring (adhesions, fibrosis) in the pelvic region and even other parts of the body. Several types of lesions have been described: superficial endometriosis, primarily located on the pelvic peritoneum; cystic ovarian endometriosis (endometrioma) located within the ovaries; deep endometriosis located in the rectovaginal septum, bladder, and intestines; and, in rare cases, endometriosis can also occur outside the pelvis.

[0004] The cause of endometriosis is unknown, there is currently no known way to prevent it, and there is no definitive cure. Symptoms can be treated with medication and / or, in some cases, surgery. Surgical removal of ectopic lesions and / or focusing on reducing estrogen-suppressing hormones (such as progestins, androgens, gonadotropin-releasing hormone (GnRH) agonists, and aromatase inhibitors) is currently the gold standard of treatment. However, both surgical and non-surgical approaches are associated with various side effects and high recurrence rates. For example, the fact that no reliable imaging procedure is currently available that allows visualization of endometriotic lesions with sufficient sensitivity and specificity complicates the removal of endometriotic lesions or endometriosis. Therefore, surgery is performed on the patient, and the lesions are identified by visual examination. Many lesions cannot be removed because they are not detected or are overlooked during surgery, leading to ineffective surgery, reoperation, or disease persistence. Therefore, a non-invasive imaging procedure capable of detecting endometriotic lesions is needed, which would be a significant improvement over the current situation where complete surgical removal is not possible in most cases. Summary of the Invention

[0005] In a first aspect, a conjugate comprising an FAP-targeting ligand and an effector is provided for use in the treatment and / or diagnosis of endometriosis.

[0006] In a second aspect, a method for identifying endometriosis lesions is provided, the method comprising the following steps: i) Introducing the conjugates as disclosed herein into subjects, ii) To make endometriosis lesions visible.

[0007] In a third aspect, a kit is provided comprising a conjugate as disclosed herein, and at least one of the following: a drug administration medium; and an instruction manual. Detailed Implementation

[0008] definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described and used herein can be employed in the practice of this invention. In fact, this invention is by no means limited to these methods.

[0009] In this document and its claims, the verb "comprising" and its inflections are used in their non-limiting sense to mean including the items that follow the word, but not excluding items not specifically mentioned. Furthermore, unless the context explicitly requires the presence of one / a and only one / a type of element, mentioning an element by the indefinite article "a / a (a or an)" does not preclude the possibility of more than one / a type of element. Therefore, the indefinite article "a / a (a or an)" generally means "at least one / a type".

[0010] As used herein, the term “and / or” indicates that one or more of the stated circumstances may occur individually or in combination with at least one of the stated circumstances, up to and including all of the stated circumstances.

[0011] As used in this article, “at least” a specific value means that specific value or more. For example, “at least 2” should be understood as the same as “2 or more” (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.).

[0012] The word “about” or “approximately” when used in conjunction with a numerical value (e.g., about 10) preferably means that the value can be a given value (10) plus or minus 0.1% of that value.

[0013] As used herein, an "effective amount" is the amount of a drug agent required to improve the symptoms of a disease relative to an untreated patient. The effective amount of one or more active agents used in the practice of this invention for therapeutic treatment (e.g., for therapeutic or medical imaging purposes) varies depending on the manner of administration, the subject's age, weight, and general health condition. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount, which can be determined, for example, as genome copies / kilogram (GC / kg) or GC / dose. Therefore, in the context of this disclosure, when referring to the administration of a drug that is "effective" against a disease or condition, this indicates that administration in a clinically appropriate manner would produce a beneficial effect on at least a statistically significant proportion of patients, such as improvement of symptoms, cure, reduction of at least one sign or symptom of the disease, increased lifespan, improved quality of life, or other effects generally considered positive by physicians familiar with treating that particular type of disease or condition.

[0014] As described in this document, the use of a substance as a pharmaceutical agent can also be interpreted as its use in the manufacture of a pharmaceutical agent. Similarly, whenever a substance is used for treatment or as a pharmaceutical agent, it can also be used to manufacture a pharmaceutical agent for treatment. Products used as pharmaceutical agents as described herein can be used in treatment methods, wherein such treatment methods include the application of the product for use.

[0015] The terms “homology,” “sequence identity,” etc., are used interchangeably herein. Sequence identity is defined herein as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. In the art, “identity” and “similarity” also mean the degree of sequence correlation between amino acid or nucleic acid sequences, which, as appropriate, can be determined by matching such sequences. “Identity” and “similarity” can be readily calculated using known methods.

[0016] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or nucleotide sequences using global or local alignment algorithms (depending on the length of the two sequences). Sequences of similar length are preferably aligned using global alignment algorithms (e.g., Needleman-Wunsch), which optimally align sequences across their entire length; while sequences of significantly different lengths are preferably aligned using local alignment algorithms (e.g., Smith-Waterman). Sequences (when optimally aligned using default parameters via, for example, the GAP or BESTFIT program) share at least a minimum percentage of sequence identity (as defined below) can be described as "substantially identical" or "comprehensively similar." GAP uses Needleman and Wunsch global alignment algorithms to align two sequences across their entire length (full length), maximizing the number of matches and minimizing the number of gaps. When two sequences have similar lengths, global alignment is suitable for determining sequence identity. Typically, the default GAP parameters are used, where the vacancy creation penalty = 50 (nucleotides) / 8 (proteins) and the vacancy extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff and Henikoff, 1992, PNAS [Proceedings of the National Academy of Sciences] 89, 915-919). Sequence alignment and sequence identity percentage scores can be determined as follows: using a computer program, such as GCG Wisconsin software package version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, 92121-3752, USA; or using open-source software, such as the programs "needle" (using the global Needman-Onsch algorithm) or "water" (using the local Smith-Waltman algorithm) in EmbossWIN version 2.10.0, using the same parameters as GAP above, or using the default settings (this applies to both "needle" and "water," and to both protein and DNA alignments; the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; for proteins, the default score matrix is ​​Blossum62, and for DNA, the default score matrix is ​​DNAFull). When sequences have significantly different total lengths, local alignment, such as local alignment using the Smith-Waltman algorithm, is preferred.

[0017] Alternatively, the similarity or identity percentage can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Therefore, the nucleic acid and protein sequences of this invention can be further used as "query sequences" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of this invention. BLAST protein searches can be performed using the BLASTx program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of this invention. To obtain vacancy alignments for comparative purposes, Gapped BLAST, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. When using BLAST and Gapped BLAST procedures, the default parameters of the corresponding procedures (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information (NCBI). http: / / www.ncbi.nlm.nih.gov / .

[0018] As used herein, the terms "selectively hybridizing," "hybridizes selectively," and similar terms are intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 66%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, or more preferably at least 99% homologous to each other typically maintain hybridization. That is, such hybridized sequences can enjoy at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, or more preferably at least 99% sequence identity.

[0019] Preferred, non-limiting examples of such hybridization conditions are hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing once or more in 1X SSC, 0.1% SDS at about 50°C, preferably at about 55°C, preferably at about 60°C, and even more preferably at about 65°C.

[0020] Highly stringent conditions include, for example, hybridization at approximately 68°C in 5x SSC / 5x Denhardt's solution / 1.0% SDS, followed by washing at room temperature in 0.2x SSC / 0.1% SDS. Alternatively, washing can be performed at 42°C.

[0021] Skilled technicians will know which conditions are suitable for stringent and highly stringent hybridization conditions. Further guidance on such conditions is readily available in the field, for example in the following literature: Sambrook et al., 1989, *Molecular Cloning, A Laboratory Manual*, Cold Spring Harbor Press, New York; and Ausubel et al. (eds.), Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual" (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York; and 1995, *Current Protocols in Molecular Biology*, (John Wiley & Sons, New York).

[0022] Of course, polynucleotides that hybridize only with poly(A) sequences (such as the 3' end poly(A) region of mRNA) or complementary segments of T (or U) residues will not be included in the polynucleotides of the present invention for specific hybridization with a portion of the nucleic acid of the present invention, because such polynucleotides will hybridize with any nucleic acid molecule containing a poly(A) segment or its complementary sequence (e.g., virtually any double-stranded cDNA clone).

[0023] The terms “protein” or “polypeptide” are used interchangeably and refer to molecules composed of chains of amino acids without specifying a particular mode of action, size, three-dimensional structure, or origin.

[0024] The term "gene" refers to a segment of DNA contained in a cell that is operatively linked to a suitable regulatory region (e.g., a promoter) and transcribed into an RNA molecule (e.g., mRNA). A gene typically contains several operatively linked segments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) containing a polyadenylation site. "Gene expression" refers to the process in which a DNA region operatively linked to a suitable regulatory region (particularly a promoter) is transcribed into RNA that is biologically active, i.e., capable of being translated into a biologically active protein or peptide.

[0025] The term "homologous," when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, should be understood to mean that in nature, the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species (preferably the same strain or lineage). If homologous to a host cell, the nucleic acid sequence encoding the polypeptide will typically (but not necessarily) be operatively linked to another (heterologous) promoter sequence and (if applicable) another (heterologous) secretory signal sequence and / or terminator sequence, distinct from its natural environment. It should be understood that regulatory sequences, signal sequences, terminator sequences, etc., can also be homologous to a host cell. In this context, the use of only "homologous" sequence elements allows for the construction of "self-cloning" genetically modified organisms (GMOs) (self-cloning is defined herein as in Annex II of European Directive 98 / 81 / EC). When used to indicate the relevance of two nucleic acid sequences, the term "homologous" means that a single-stranded nucleic acid sequence can hybridize with a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on a variety of factors, including the amount of identity between the sequences and hybridization conditions (such as temperature and salt concentration, discussed later).

[0026] The terms “heterologous” and “exogenous” when used with respect to nucleic acids (DNA or RNA) or proteins refer to nucleic acids or proteins that are not naturally present as part of the organism, cell, genome, or DNA or RNA sequence in which they are found, or that are found in one or more locations in a cell or genome or DNA or RNA sequence other than those found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell in which they are introduced, but are obtained from another cell or synthesized or recombined. Typically, although not always, such nucleic acids encode proteins that are not typically produced by the cell that transcribes or expresses DNA, i.e., exogenous proteins. Similarly, exogenous RNA encodes proteins that are typically not expressed in the cell in which the exogenous RNA is present. Heterologous / exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that a person skilled in the art would consider to be foreign to the cell that expresses it is covered herein by the terms heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two of the sequences are foreign relative to each other.

[0027] As used herein, the term "non-naturally occurring" when used to refer to an organism means that the organism possesses at least one genetic alteration that is generally not found in naturally occurring strains of the reference species (including wild-type strains of the reference species). Genetic alterations include, for example, modifications to expressible nucleic acids encoding proteins or enzymes, additions of other nucleic acids, deletions of nucleic acids, substitutions of nucleic acids, or other functional disruptions of the organism's genetic material. Such modifications include, for example, coding regions and functional segments of heterologous or homologous polypeptides of the reference species. Additional modifications include, for example, non-coding regulatory regions, where modifications alter the expression of genes or operons. Genetic modifications to nucleic acid molecules encoding enzymes or functional segments thereof can confer on non-naturally occurring organisms biochemical reactivity or metabolic pathway capabilities altered from their naturally occurring state.

[0028] Any references in this document to nucleotide or amino acid sequences accessible in public sequence databases refer to the sequence entries available as of the date of this document's submission.

[0029] Detailed description of the invention The inventors of this invention have unexpectedly identified fibroblast activating protein (FAP) as a target for imaging endometriosis. Fibroblast activating protein (FAP) (also known as fibroblast activating protein α, fibroblast activating protein, or prolyl endopeptidase FAP) is a type II transmembrane serine protease expressed on the surface of fibroblasts during epithelial carcinoma and soft tissue sarcoma (Dendl, K. et al., 2023; Dorst, DN et al., 2023). In recent years, ligands targeting FAP have been developed and can be used in cancer imaging and therapy, such as radiolabeled FAP ligands for PET imaging (positron emission tomography) (Imlimthan, S. et al., 2021; Kratochwil, C. et al., 2019; Sollini, M. et al., 2021; Backhaus, P. et al., 2021) and photodynamic therapy (PDT) (Dorst, DN et al., 2020; Dorst, DN et al., 2022). Therefore, the object of this invention is to provide FAP-targeting ligands for the diagnosis and / or treatment of endometriosis. For example, for detecting lesions in guided procedures, fluorescence-guided procedures, PDT, and therapies.

[0030] Given the benign nature of endometriosis, it is not anticipated that known malignant tumor markers identified from the tumor microenvironment will help address the significant clinical challenge of treating and / or diagnosing endometriosis (e.g., by identifying endometriotic lesions to guide surgical resection). In a first aspect, a conjugate comprising an FAP-targeting ligand and an effector is provided for use in the treatment and / or diagnosis of endometriosis.

[0031] As used herein, the term “compound” has the meaning from the art as a substance formed by a combination of two or more other substances.

[0032] As used herein, the term "ligand" has the meaning from the art as any molecule or atom that binds to a receptor protein molecule (also known as a receptor). Therefore, a "FAP-targeting ligand" is any molecule or atom that binds to FAP. FAP ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins (such as sugars). Suitable ligands include, for example, antibodies, such as full-length (intact) antibodies and their antigen-binding fragments. In embodiments where the ligand unit is a non-antibody target, the ligand unit may be a peptide or polypeptide, or a non-protein molecule (such as a FAP inhibitor (FAPI)).

[0033] FAP stands for fibroblast activation protein or fibroblast activation protein (the two terms are used interchangeably), but it is also called prolyl endopeptidase FAP (uniProt Q12884; Ensembl ENSG00000078098). FAP is a 97-kDA type II transmembrane serine protease member of the prolyl peptidase family, and is expressed almost exclusively under pathological conditions, including arthritis and cancer (Dendl et al., 2023, PET Clin [Pet Clin] 18 (2023) 345–351).

[0034] As used herein, the term “effectant” has the meaning from the art as a molecule that is relevant to a purpose or activity, such as a small molecule that selectively binds to a protein to modulate its biological activity or a molecule used for imaging purposes.

[0035] As used in this article, the term "endometriosis" refers to a condition in which tissue resembling the uterine lining grows outside the uterine cavity. Pain is the most common symptom of endometriosis. Women with endometriosis may experience pelvic or lower abdominal pain, painful menstruation (dysmenorrhea), painful intercourse (dyspareunia), and painful bowel movements (dyspareunia). Several locations of lesions (also known as endometriosis lesions or foci) have been described for endometriosis: superficial endometriosis, primarily located on the pelvic peritoneum; cystic ovarian endometriosis (endometrioma) located within the ovaries; deep endometriosis located in the rectovaginal septum, bladder, and intestines; and in rare cases, endometriosis can also be found outside the pelvis. Endometriotic lesions or foci are pro-inflammatory, unlike the tumor microenvironment, which is known to be immunosuppressive and evade detection by the immune system. Therefore, it cannot be expected that known cancer markers will provide the histological characteristics of endometriosis lesions as described in the examples in this article.

[0036] FAP ligands In some embodiments, the FAP-targeting ligand is selected from the group consisting of: antibodies; FAP inhibitors (FAPI); and peptides.

[0037] Antibody Human FAP was initially identified using the monoclonal antibody (mAb) F19 in cultured fibroblasts (Welt, S. et al., 1994, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. [American Society of Clinical Oncology Official Journal - Journal of Clinical Oncology], 12, 1193-1203). Sirolizumab / BIBH1 (a humanized form of the F19 antibody) and other humanized or fully human antibodies against the FAP antigen that exhibit specificity for the F19 epitope have been reported (Schmidt, A. et al., 2001, Eur. J. Biochem. [European Journal of Biochemistry] 268, 1730-1738; Mersmann, M. et al., 2001, Int. J. Cancer [International Journal of Cancer], 92: 240-248). OS4 is another humanized antibody derived from the F19 antibody (CDR transplanted) (Wuest, T. et al., 2001, J. Biotechnol. [Journal of Biotechnology], 92:159-168). Furthermore, mouse anti-FAP antibodies have evolved, including chimeric and humanized forms (Ostermann, E. et al., 2008, Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. [Official Journal of the American Association for Cancer Research - Clinical Cancer Research], 14: 4584–4592) (Imlimthan, S. et al., 2021, Pharmaceuticals [Drugs], 12: 1023). Therefore, the production of FAP antibodies and suitable derivatives thereof is known to those skilled in the art and is incorporated herein by reference.

[0038] As used herein, "antibody" or "antibody unit" includes, within its scope, any portion of an antibody structure, antigen-binding fragment, nanobody, or single-domain antibody. This unit can bind, reactively associate, or complex with a receptor, antigen, or other receptor unit present in a target cell population. An antibody can be any protein or protein molecule that can bind, complex with, or otherwise react with a portion of a cell population to be treated or bioengineered. Therefore, the antibody described herein is capable of specifically binding to the FAP antigen.

[0039] The antibody according to the present invention can be a monomeric antibody or a multimeric antibody. In particular, the antibody according to the present invention is a monomeric antibody. The antibody according to the present invention can be monoclonal or polyclonal. Preferably, the antibody according to the present invention is monoclonal. In another preferred embodiment, the targeting agent is an anti-FAP antibody or a peptide that binds to FAP.

[0040] As used herein, the term "fragment" refers to a region containing the antigen-binding region or variable region of an antibody. The fragment may be selected from, but is not limited to, the group consisting of: Fv, Fab, Fab′, F(ab)2, F(ab′)2, F(ab)3, Fv, single-chain Fv (scFv), di-scFv or sc(Fv)2, dsFv, Fd, dAb, CDR, VH, VL, VHH, V-NAR, nanobodies, microbodies, biantibodies, and multispecific antibodies formed from antibody fragments.

[0041] Antibody mimics, such as affinity molecules and designed ankyrin repeats (DARPin), were also considered. Such antibodies and antibody mimics are well known in the art and can be prepared using well-known methods and information. For convenience, antigen-related information well known in the art is listed below, including name, alias, and GenBank accession number. Nucleic acid and protein sequences corresponding to FAP antigens are available in public databases such as Ensembl. The antibody targets the corresponding FAP antigen, which includes all amino acid sequence variants and homologs that have at least 70%, 80%, 85%, 90%, or 95% homology with sequences identified in the references, or have identical biological properties and characteristics to FAP antigen sequences known in the art (see Fischer, E. et al., 2012, Clin. Cancer Res. Off. J.Am. Assoc. Cancer Res. [Official Journal of the American Association for Cancer Research - Clinical Cancer Research], 18, 6208–6218; Schlothauer, T. et al. 2016, Protein Eng., Des. Sel. [Protein Engineering, Design, and Selection], 29, 457–466). In some embodiments, the FAP antibody is selected from: ESC11 and ESC14; 28H1.

[0042] Fap inhibitors (FAPI) The term "inhibition" or "inhibition of..." refers to a reduction in a detectable amount. Therefore, FAPI or FAPi can be considered to have a useful application in endometriosis, where its application can be used to treat endometriosis. In some embodiments, FAP inhibition employs a small molecule inhibitor.

[0043] As used herein, “small molecule” or “micromolecule” includes any low molecular weight molecule (less than 1000 Daltons), including but not limited to lipids, monosaccharides, second messengers, other natural products and metabolites, and pharmaceuticals. The compatibility and design of FAP inhibitors (FAPIs) are well known in the art and can be prepared using well-known methods and information (as discussed in Pure, E. et al., 2018, Oncogene, 37: 4343–4357).

[0044] In some embodiments, FAPI is selected from, but not limited to, the group consisting of: tarabosilan, PT-100, liraliptin (Tradjenta), FAP inhibitors with an N-(4-quinolineyl)-Gly-(2-cyanopyrrolidine) scaffold (as disclosed in Jansen et al., ACS Med Chem Lett. [ACS Medicinal Chemistry Letters] May 9, 2013; 4(5): 491-496), MIP-1232 (as described in Mueller et al., J Biol Chem [Journal of Biochemistry] 1999; 274:24947-52), and FAP inhibitors (as described in FAPI-02 and FAPI-04, for example, as described in Lindner et al., EJNMMI Radiopharmacy and Chemistry [Radiopharmacy and Chemistry] (2019) 4:16 and Lindner et al., Journal of Nuclear Medicine [Journal of Nuclear Medicine] published April 6, 2018). In addition to Imlimthan, S. et al. 2021, Pharmaceuticals, 14 (10), In addition to those listed in 1023, there are, for example, FAPI-74; Glc-FAPI-04; FAPI-02; FAPI-04; FAPI-20; FAPI-21; FAPI-22; FAPI-31; FAPI-35; FAPI-36; FAPI-37; DOTA.(SA.FAPi)2; DOTAGA.(SA.FAPi)2; RPS-309; QCP02; FAPI-34; FL-L3; FAPI-04; FAPI-04; FAPI-02; FAPI-04; FAPI-46; RPS-309; OncoFAP; FAP-2286; FAPI-46; FAPI-04; and FAPI-46. For example, FAP inhibitors are described in WO 20081522, WO 20132661, WO 20245173, WO 21005125, WO 21005131, US2020246383, WO 19154859, WO 19083990, WO 19118932, WO 18111989, WO 17189569, WO13107820, WO 08116054 or WO 07085895 (the disclosures of which are incorporated herein by reference).

[0045] In a more preferred embodiment, the FAP inhibitor is selected from, but not limited to, the group consisting of: FAPI-74, Glc-FAPI-04, FAPI-02, FAPI-04, FAPI-20, FAPI-21, FAPI-22, FAPI-31, FAPI-35, FAPI-36, FAPI-37, FAPI-46, FAPI-74, DATA5m.SA.FAPi, DOTA.(SA.FAPi)2, DOTAGA.(SA.FAPi)2, RPS-309, QCP02, FAPI-34, FL-L3, RPS-309, OncoFAP, and FAP-2286. In even more preferred embodiments, the FAP inhibitor comprises a high-affinity FAP ligand for targeted applications with pan-tumor potential, such as OncoFAP. OncoFAP is known from Millul J et al., An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications. Proc National Acad Sci. 2021;118. In the most preferred embodiment, the FAP inhibitor comprises OncoFAP.

[0046] Further examples are provided in Giesel, FL et al. 2019. J Nucl Med. [Journal of Nuclear Medicine];60:386–92; Loktev A. et al. 2018. J Nucl Med. [Journal of Nuclear Medicine] 59:1423–9; Ballal S, 2021. EurJ Nucl Med Mol., I [European Journal of Nuclear Medicine and Molecular Imaging] (48):1915–31; and Baum RP et al., 2021. J Nucl Med. [Journal of Nuclear Medicine],120.259192.

[0047] connector In some embodiments, the conjugates described herein further comprise a linker between the FAP-targeting ligand and the effector.

[0048] As used herein, the terms "connector," "connector fragment," or "connector unit" refer to a chemical part or bond that is linked at one end to an FAP ligand as disclosed herein and at the other end to an effector, and can be linked to another effector or ligand after attachment of another connector. Connectors can be synthesized by methods known in the art, such as those described in US2005-0238649A1 for at least antibody-drug conjugates. Connectors can be "releasable," also known as "cleavable," thereby facilitating the release of the effector. For example, acid-instable connectors (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) connectors, photostable connectors, dimethyl connectors, or disulfide-containing connectors (US5,208,020, Identification of connectors for use with at least cytotoxic agents) can be used. Examples of "non-releasable" connectors (also known as "non-cleavable" connectors) according to the invention include, but are not limited to, thioethers. Thus, in some embodiments, the connector is either releasable or non-releasable.

[0049] In some embodiments, the adapter is selected from the group consisting of: acid-instable adapters, protease-sensitive adapters, light-instable adapters, dimethyl adapters, or disulfide-containing adapters. Suitable adapters compatible with OncoFAP can be found in Backhaus, P. et al. Translational imaging of the fibroblast activation protein (FAP) using the new ligand [ 68 Ga]Ga-OncoFAP-DOTAGA [Using new ligands] 68 [Ga]Ga-OncoFAP-DOTAGA conversion imaging of fibroblast activation protein (FAP) revealed this information.

[0050] Effector In some embodiments, the effector is an imaging agent and / or a therapeutic agent. In some embodiments, the effector is an imaging agent. In some embodiments, the effector is a therapeutic agent. In some embodiments, the effector is both an imaging agent and a therapeutic agent.

[0051] Therapeutic agents As used herein, the term "therapeutic agent" refers to a pharmaceutical agent used in the treatment or therapy of endometriosis. In some embodiments, the therapeutic agent comprises a photosensitizer or a cytotoxic agent.

[0052] As used herein, the term "photosensitizer" refers to a molecule capable of generating reactive oxygen species (ROS) upon irradiation at a specific wavelength, including suitable porphyrins, chlorines, and dyes. In some embodiments, photosensitizers are suitable for use in photodynamic therapy (PDT). PDT involves a combination of light and a photosensitizer (PS) that is activated by absorbing light at a specific wavelength, causing the generation of potentially toxic ROS that induces a cascade of intracellular molecular events, thereby leading to targeted tissue damage (Avci, P. et al., 2014, J. Biomed. Nanotechnol. [Journal of Biomedical Nanotechnology], 10, 1937-1952). In some embodiments, the photosensitizer has an excitation peak between 600 and 900 nm (e.g., between 680 and 800 nm). In some embodiments, the photosensitizer is selected from, but not limited to, the group consisting of: Allumera, Photofrin, Visudyne, Levulan, Foscan, Metvix, Hexvix, Cysview, Laserphyrin, and Spectrum. In some embodiments, the photosensitizer is IRDye 700DX.

[0053] Therefore, this invention also covers the possibility of anti-Fap targeted photodynamic therapy (TPT), as disclosed in Dorst, D. et al., 2022, Rheumatology, 61:2999-3009, and Dorst, D. et al., 2020, Rheumatology, 59:3952-3960, but which are applied herein to the treatment and / or diagnosis of endometriosis. Therefore, in some embodiments, the therapeutic agent may also be an imaging agent.

[0054] As used herein, the term "cytotoxic agent" refers to a molecule that, upon entering, contacting, and ultimately being internalized into a cell, adversely alters cellular function (e.g., cell growth and / or proliferation and / or differentiation and / or metabolism, such as protein and / or DNA synthesis) or causes cell death. As used herein, the term "cytotoxic drug" encompasses toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, radioactive isotopes, toxic drugs, chemotherapeutic agents, antibiotics, and lysozymes, preferably toxic drugs.

[0055] The cytotoxic drugs according to the present invention can be selected from the group consisting of: ceratosalis (such as ceratosalis 10, ceratosalis 15, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), methylauristatin F (MMAF), methylauristatin-D (MMAD), methylauristatin E (MMAE), and 5-benzoylvalerate-AE ester (AEVB)), maytansine (such as anserine, metansine (also known as emtansine)). DM1 and ravtansine (also known as soravtansine or DM4), anthracyclines (such as daunorubicin, epirubicin, pirarubicin, idarubicin, zorubicin, erythromycin, arubicin, doxorubicin, mitoxantrone, daunorubicin liposome, nemorubicin and PNU-159682), chachiin (such as chachiin β1Br, chachiin γ1Br, chachiin α21, chachiin α31, chachiin β11, chachiin γ1L, chachiin δ) 11 and ozomicin), esperamicins (such as esperamicin A1), neocarcinomacin, bleomycin, duocarymycins (such as CC-1065 and duocarymycin A), pyrrolobenzodiazepines (such as atracin, abenomycin, cipromycin, DC-81, methylaminopicrin, neopicarymycin A and B, porothramycin, prothracarcin, sibanamicin (DC-102), siberiamycin and tomatine), pyrrolobenzodiazepine dimer (or PBD), indoline-benzodiazepine, indoline-benzodiazepine dimer, α-amaminine, albumin-bound paclitaxel, actinomycin, interleukin, hexamethylmelamine, retinoic acid, acridine, anastrozole, Arsenic, asparaginase, azacitidine, azathioprine, bexarotine, bendamustine, bicalutamide, bortezomib, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, chloramphenicol, cyclosporine, cidofovir, coal tar-containing products, colchicine, dacarbazine, actinomycin D, danazol, dasatinib, diethylstilbestrol, dinoprostone, dithranol, dutazol andramine, dexrazoxen, docetaxel, deoxyfluorouridine, erlotinib, estradiol, etoposide, exemestane, finasteride, flutamide, fluorouridine, flucytosine, fludarabine, fluorouracil, ganciclovir, gefitinib, gemcitabine, goserelin, hydroxyurea, hydroxycarbamide, ifosfamide, irinotecan, imatinib, lenalidomideLeflunomide, Letrozole, Leuprorelin Acetate, Lomustine, Dichloromethyldiethylamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotan, Menotropins, Mifepristone, Napharelin, Nerapine, Nitrosourea, Oxaliplatin, Ozogamicin, Paclitaxel, Podophyllyn, Pemetrexed, Pentamidine, Pentostatin, Procarbazine, Raloxifene, Ribavirin, Raltitrexed, Rituximab, Romidixin, Sorafenib, Streptourea Zomectin, sunitinib, sirolimus, temozolomide, tesirobolimus, teniposide, thalidomide, thioguanine, thiotepa, topotecan, tacrolimus, taxotere, tafluposide, toremifene, retinoic acid, trifluuridine, triptorelin, valganciclovir, penoxuridine, vinblastine, vidaradine, vincristine, vinorelbine, vemurafenib, vemodil, vorinostat, zidovudine, vedotine, and their derivatives and combinations thereof. Cytotoxic drugs can also be radionuclides, such as lutetium-177, iodine-131, samarium-153, and yttrium-90, or astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223, thorium-227, rhenium-188, and indium-111.

[0056] developer As used herein, the term "imaging agent" is a pharmaceutical agent used in medical imaging techniques. Techniques or methods include, but are not limited to, X-rays, computed tomography (CT), magnetic resonance imaging (MRI), scintillation imaging, fluorescence, luminescence, ultrasound, etc. Therefore, in some embodiments, the imaging agent comprises fluorescent or radioactive molecules. In some embodiments, the imaging agent may also be a diagnostic agent. In some embodiments, the imaging agent comprises a fluorescent diagnostic agent. In some embodiments, the imaging agent comprises a radioactive diagnostic agent.

[0057] The use of imaging agents has specific applications when using imaging agents to assist or guide surgery. In some embodiments, the imaging agent is a fluorescent molecule used in fluorescence-guided surgery. As used herein, the terms "fluorescence-guided surgery" or "fluorescence image-guided surgery" refer to a medical imaging technique that detects fluorescently labeled structures during surgery for the purpose of guiding the surgical procedure. In some embodiments, the fluorescent molecule is selected from the group consisting of: fluorescein or its derivatives (such as FITC or Tokyo green), ASP (preferably 20 4-(4-(didecylamino)styryl)-N-methylpyridine iodide), rhodamine, cyanine 5 (also known as Cy5), cyanine 5.5 (also known as Cy5.5), cyanine 7 (also known as Cy7), sulfonated cyanine 7 (also known as sulfonated Cy7), cyanine 7.5 (also known as Cy7.5), IRDye 700DX, IRDye 800CW, IRDye 800ZW, Alexa660, Alexa680, Alexa700, Alexa750, Alexa790, Dylight 755, Dylight 800, Fluoprobes 752, Fluoprobes 782, calcein, any other Alexa marker, any other cyanine marker, and any sulfonated or otherwise modified variants of these fluorophores.

[0058] Combinations are also contemplated in this invention, and those will be known to those skilled in the art; for example, in some embodiments, the imaging agent is a fluorescent molecule, and the effector is a photosensitizer used in photodynamic therapy (PDT). Such combinations are covered in this invention and can be applied to methods for the removal of endometriosis. Such methods can be used alone or in combination with other diagnostic and / or therapeutic approaches. For example, in some embodiments, methods as disclosed herein are used in combination with hormone suppression focused on reducing estrogen, such as progestins, androgens, gonadotropin-releasing hormone (GnRH) agonists, and aromatase inhibitors.

[0059] As used herein, the term “radioactive molecule” also refers to “radioisotope” and “radionucleus” used in nuclear medicine (including imaging, diagnostics, and therapy, or any combination thereof).

[0060] In some embodiments, the radioactive molecules are selected from, but not limited to, the group consisting of: technetium-99m, iodine-131, chromium-51, lutetium-177, iodine-125, indium-111, iodine-123, thallium-201, copper-64, fluorine-18, gallium-67, carbon-11, xenon-133, iridium-192, tritium, krypton-81, molybdenum, palladium-103, gallium-68, molybdenum-99, holmium-166, radium-223, zirconium-89, or derivatives thereof. Radionuclides used for imaging and / or therapy are selected based on their properties regarding emitted radiation / particles (regarding imaging devices and probes), their half-life (in balance with the pharmacokinetics of ligands), decay products (regarding radiation exposure / therapeutic efficacy), and radiochemical properties (for effective labeling).

[0061] In some embodiments, the radioactive molecule is a gamma emitter, more preferably Tc-99m or In-111. Suitable applications of radioactive labeling, compatible connectors, and their use in this invention will be known to those skilled in the art.

[0062] In some embodiments, the radioactive molecule comprises a PET radioisotope (positron emitter) or a SPECT radioisotope (gamma emitter). In some embodiments, the radioactive molecule is... 68 Ga、 18 F, 11 C 89 Zr、 64 CU 99m Tc or 111 Therefore, in some embodiments, the PET developer contains a positron-emitting radionuclide, preferably 68 Ga、 18 F, 11 C 89 Zr、 64 CU, preferably 18 F or 68 In a more preferred embodiment, the PET developer contains Ga. 68 Ga.

[0063] Combinations are also contemplated in this invention, and those will be known to those skilled in the art, for example, in some embodiments, the FAP ligand is FAPI and the effector is a radionuclide (e.g., lutetium-177), such as the FAP inhibitor FAPI-2286 developed by Clovis Oncology (Zboralski, D. et al., 2022, Eur J NuclMed Mol Imaging [European Journal of Nuclear Medicine and Molecular Imaging]. Sep;49(11):3651-3667). Thus, in some embodiments, the FAP ligand is FAPI and the effector is a radionuclide. Other examples of radiolabeled FAPI include gallium-68, fluorine-18, carbon-11, zirconium-89, and copper-64-FAPI. The FAPIs disclosed herein may include one or more of the following: FAPI-74, Glc-FAPI-04, FAPI-02, FAPI-04, FAPI-20, FAPI-21, FAPI-22, FAPI-31, FAPI-35, FAPI-36, FAPI-37, FAPI-46, FAPI-74, DATA5m.SA.FAPi, DOTA.(SA.FAPi)2, DOTAGA.(SA.FAPi)2, RPS-309, QCP02, FAPI-34, FL-L3, RPS-309, OncoFAP, FAP-2286. The synthesis of such radiolabeled FAPIs will be known to those skilled in the art, as described in Imlimthan et al., 2021, Pharmaceuticals, 14:1023. Therefore, in a preferred embodiment, the FAP ligand comprises OncoFAP, and the effector comprises a PET imaging agent. In a more preferred embodiment, the FAP ligand comprises OncoFAP, and the effector comprises 68 In an alternative embodiment, the FAP ligand comprises OncoFAP, and the effector comprises... 18 F.

[0064] FAP ligands (which may be FAPI) can also be “linked” or “coupled” to radionuclide chelators such as the radiotherapeutic tracer DOTA (FAP-2286). Alternative chelators and methods are also known to those skilled in the art, for example, chelator-modified FAPI (Moon, ES, 2020, EJNMMI radiopharm. chem. [Radiopharmacology and Chemistry] 5:19), several other bifunctional chelators, and NOTA, DATA. 5mDOTA, NODAGA, and DOTAGA are known in the art. Therefore, in some embodiments, the effector comprises a radioactive molecule optionally attached to a chelating agent.

[0065] Chelating agents are known in the art (see, for example, Price and Orvig, DOi: 10.1039 / C3CS60304K) and can be used to associate ligands with radiolabels. Suitable chelating agents for radiolabeling are 1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-triacetic acid (NOTA), triazacyclononane-phosphonate (TRAP), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), N,N'-bis[2-hydroxy-5-10(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), and diethylenetriaminepentaacetic anhydride (DTPA) or their hydrolyzed forms. Additional chelating agents for radiolabeling may be based on 2,2',2",2"'-(ethane-1,2-diyldiazono)tetraacetic acid (EDTA), or 1,4,8,11-tetraazacyclotetradecane (cyclopamide), or 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane (DiamSar). In preferred embodiments, the chelating agent is selected from HYNIC (6-hydrazinoic acid-15-acylhydrazide), MAG3 (mercaptoacetyltriglycine), MAS3 (acetylmercaptoacetyltriserine), DTPA (diethylenetriaminepentaacetic acid), chelating agents for tricarbonyl radiolabeling (such as Tc-99m-tricarbonyl) (e.g., L1, L8, L9, and L10 from Banerjee et al., DOi: 10.1021 / jm400823w), DFO (deferroamine), DFO (See Patra et al., ChemCommun. [Chemical Communications] 2014;50:11523-5), TAFC (triacetylfusarine C), FSC (fusarine C), HBED-CC, THP (tris(hydroxypyridinone)), NODAGA (1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid), NOTA, DOTAGA (2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4, 7-Trimethyl)triacetic acid), CHX-A"-DTPA (such as [(R)-2-amino-3-(4-isothiocyanophenyl)propyl]-trans(S,S)-cyclohexane-1,2-diamine-pentaacetic acid), TCMC (such as 2-[(4-isothiocyanophenyl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide tetrahydrochloride), DO3AM (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid), NET A (({4-[2-(bis-carboxy-methylamino)-5-(4-nitrophenyl)pentyl]-7-carboxymethyl[1,4,7]triazanonane-1-yl}acetic acid), CB-DO2A (4, 10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DiamSar, NODA-MPAA (with a 1,4,7-triazacyclononane-1,4-diacetate (NODA) motif on a methylphenylacetic acid (MPAA) backbone, see DOi: 10.1021 / bc200175c) and RESCA (WO 2016 / 065435). Highly preferred chelating agents are DOTA and Nota, with DOTA being the most preferred. DOTA can also be called tetraxetan and contains compounds of the formula (CH2CH2NCH2CO2H)4. Preferred Tc-99m chelating agents are HYNIC, MAG3, MAS3, DTPA, and tricarbonyl Tc-99m chelating agents, more preferably HYNIC, MAG3, MAS3, and DTPA. Preferred Zr-89 chelating agents are DFO. Preferred Ga-68 chelating agents are HBED-CC, TAFC, FSC, THP, TRAP, DOTA, 35NODAGA, and NOTA. Preferred Ln-111 chelating agents are DTPA, DOTA, NOTA, DOTAGA, and CHXA"-DTPA. Preferred Pb-chelating agents are TCMC, DO3AM, and NETA. Preferred Cu chelating agents are DOTA, CB-DO2A, TETA, and Diamsar. Preferred AIF-18 chelating agents are NODA-MPAA, NOTA, and RESCA.

[0066] Chelating agents compatible with OncoFAP can be adapted from Backhaus, P. et al. Translational imaging of the fibroblast activation protein (FAP) using the new ligand [ 68 Ga]Ga-OncoFAP-DOTAGA [Using new ligands] 68 Ga-OncoFAP-DOTAGA for Transformational Imaging of Fibroblast Activating Protein (FAP). Eur J Nucl Med Mol Imaging. May 2022;49(6):1822-1832. Therefore, in a preferred embodiment, the FAP inhibitor comprises OncoFAP, and the chelating agent comprises DOTAGA or a suitable compatible alternative. In a more preferred embodiment, the FAP inhibitor comprises OncoFAP, the chelating agent comprises DOTAGA, and the effector comprises a PET imaging agent. In even more preferred embodiments, the FAP inhibitor comprises OncoFAP, the chelating agent comprises DOTAGA, and the effector comprises... 68 Ga, such as Ga-68-DOTAGA-OncoFAP.

[0067] Composition In some embodiments, the composition is used to treat (human) subjects. For this purpose, a pharmaceutical composition is provided comprising a physiological buffer (e.g., PBS or HEPES) and an additional stabilizer (e.g., sucrose). Such a composition is compatible with and suitable for, and intended for, use in subsequent intravenous, intramuscular, subcutaneous, intraperitoneal, intrauterine, intravaginal, intraurethral, ​​or other related administrations, or in organ-targeted vascular delivery or isolated perfusion.

[0068] Uses and methods The significant difference in FAP expression between endometriotic tissue and surrounding unaffected tissue offers the possibility of using FAP expression to detect endometriotic lesions. In one aspect, conjugates as described herein are provided for use in the treatment of endometriotic lesions.

[0069] In some embodiments, FAP inhibitors (FAPIs) or labeled FAPIs, as described herein, are provided for use in the treatment of endometriosis and / or in differentiating endometriotic tissue from surrounding non-endometriotic tissue. This can be a significant improvement over current diagnostic tools (ultrasound, CT, and MRI) that typically cannot detect these lesions, particularly for peritoneal endometriosis. Therefore, in some embodiments, labeled FAPIs, as described herein, are provided for use in differentiating peritoneal endometriotic tissue from surrounding non-endometriotic tissue. Typically, laparoscopic surgery is required to confirm the presence of endometriotic lesions in a patient. Detection of endometriosis using labeled FAPIs is non-invasive and can significantly reduce the time required for a pre-diagnosis diagnosis of endometriosis. This can lead to a significant reduction in both the physical and psychological burden on the patient.

[0070] Therefore, ligands that can be used for diagnosis and / or preoperative SPECT / PET / CT scans, intraoperative localization of endometriotic lesions with relevant probes (such as gamma probes) or portable SPECT cameras (radiation-guided surgery), and radioligand therapy (such as Lu-177 FAP), or that can be used for fluorescence imaging or PDT, are within the scope of this disclosure.

[0071] To create a probe that is visible to the naked eye and has high penetration depth in human tissue, fluorescence-guided surgery (FGS) and radiation-guided surgery (RGS) can be combined in a single compound according to the invention, wherein the probe contains a fluorescent marker and a chelating portion capable of carrying the radiolabeled marker. Preoperatively, the location of endometriotic lesions can be determined using PET or SPECT imaging with the aid of a radionuclide. During the surgical procedure, the physician can be guided to the endometriotic lesion based on the signal generated by the radionuclide until the fluorescent marker becomes visible. Once the surgical field is exposed, the fluorescent marker can accurately assess the resection margins, which can assist the surgeon in removing all diseased tissue. An associated method can be derived from the following trial: Onofrio Catalano and Diandrea Galloway. PET / MRI for Evaluation of Endometriosis. ClinicalTrials.gov ID NCT06377553.

[0072] Another application based on the difference in FAP expression between endometriotic and healthy tissues is the eradication of endometriotic lesions via photodynamic therapy (which can use photosensitizers as discussed above). For this approach, it is important that the target cells express FAP and are therefore susceptible to the effects of radiolabeled FAPI, while the surrounding tissue should lack FAP expression and remain largely unaffected. Therefore, in some embodiments, labeled FAP inhibitors (FAPI) as described herein are provided for use in the treatment of endometriosis via photodynamic therapy. In some embodiments, labeled FAP inhibitors (FAPI) in combination with photosensitizers as described herein are provided for use in the treatment of endometriosis via photodynamic therapy.

[0073] Because radioactive and fluorescent labels are linked to the target site, the compounds according to the invention are suitable for diagnosis. This diagnosis can be performed before treatment, but depending on the unique properties of the compounds, it can also be performed during or immediately after treatment. Therefore, the examples provide compounds for use according to the invention, wherein the agents are used for imaging, diagnosis, and / or treatment of endometriosis. Such a method is preferably used to achieve diagnosis via two parameters, such as via radioactive labeling and via fluorescent labeling.

[0074] The compounds and compositions according to the invention described herein are well suited for use in methods for imaging, diagnosing, or treating endometriosis in subjects in need, methods comprising the step of administering the compounds or compositions according to the invention to the subject.

[0075] "Subject" includes animals such as humans, monkeys, cattle, horses, cats, dogs, mice, or rats. Animals can be mammals, such as non-primates and primates (e.g., monkeys and humans). In one embodiment, the subject is a human. In other embodiments, the subject is not a human, such as a mouse or rat.

[0076] In some embodiments, conjugates as disclosed herein are provided for use in treating and / or diagnosing one or more subgroups of endometriotic lesions selected from: superficial (peritoneal) endometriosis; cystic ovarian endometriosis (endometrioma); and deep endometriosis.

[0077] In some embodiments, conjugates as disclosed herein are provided for use in treating and / or diagnosing pain associated with endometriosis. Examples of pain associated with endometriosis or endometriosis-related pain include, but are not limited to, pelvic pain, pain felt during menstruation, pain accompanied by abdominal swelling, bowel discomfort, malaise, uterine pain, and lower abdominal pain.

[0078] In one aspect, a method for identifying endometriotic lesions is provided, the method comprising the following steps: i) Introduce the conjugates described in this article into subjects. ii) To make endometriosis lesions visible.

[0079] Visualization methods will be known to those skilled in the art, and include, but are not limited to, implementations of fluorescence camera detection, gamma probe detection, and positron probe detection.

[0080] In some embodiments, the methods described herein further include the following steps: iii) Remove endometriosis lesions.

[0081] As used herein, the term “resection” in reference to endometriosis lesions includes, but is not limited to, any excision, destruction or removal by invasive and / or non-invasive means.

[0082] In certain embodiments, the present invention relates to the use of FAP inhibitors in treating subjects with endometriosis or for use in treating subjects with endometriosis.

[0083] In some embodiments, the methods disclosed herein are used to identify and / or visualize and / or remove one or more subgroups of endometriosis lesions selected from: superficial endometriosis; cystic ovarian endometriosis (endometrioma); and deep endometriosis.

[0084] In some embodiments, the methods disclosed herein are used to identify and / or visualize and / or remove endometriotic lesions associated with pain associated with endometriosis.

[0085] In another embodiment, the methods disclosed herein are used to identify, visualize, and subsequently remove endometriotic lesions associated with pain associated with endometriosis.

[0086] Reagent test kit In one aspect, a kit is provided comprising the conjugate as described herein, and at least one of the following: a drug administration medium; and an instruction manual.

[0087] The invention has been described above with reference to several exemplary embodiments shown in the accompanying drawings. Modifications and alternative implementations of some parts or elements are possible and are included within the scope of protection as defined in the appended claims. Attached Figure Description

[0088] Figure 1 . Peritoneal endometriosis slides. A, B, and C are whole slide images; D, E, and F are magnified views of the black box in A. A and D) H&E staining; B and E) FAP immunohistochemistry; C) FAP immunohistochemistry negative control; F) CD68 immunohistochemistry.

[0089] Figure 2 . Peritoneal endometriosis. A, B, and C are whole-section images; D, E, and F are magnified views of the black box in A. A and D) H&E staining; B and E) FAP immunohistochemistry; C) FAP immunohistochemistry negative control; F) CD68 immunohistochemistry.

[0090] Figure 3 Peritoneal endometriosis. A, B, and C are whole-section images; D, E, and F are magnified views of the black box in A. A and D) H&E staining; B and E) FAP immunohistochemistry; C) FAP immunohistochemistry negative control; F) CD68 immunohistochemistry.

[0091] Figure 4 . Ovarian endometriosis. A, B, and C are whole-section images; D, E, and F are magnified views of the black box in A. A and D) H&E staining; B and E) FAP immunohistochemistry; C) FAP immunohistochemistry negative control; F) CD68 immunohistochemistry.

[0092] Figure 5 . Ovarian endometriosis. A, B, and C are whole-section images; D, E, and F are magnified views of the black box in A. A and D) H&E staining; B and E) FAP immunohistochemistry; C) FAP immunohistochemistry negative control; F) CD68 immunohistochemistry.

[0093] Figure 6 Deep invasive endometriosis. A, B, and C are whole-section images; D, E, and F are magnified views of the black box in A. A and D) H&E staining; B and E) FAP immunohistochemistry; C) FAP immunohistochemistry negative control; F) CD68 immunohistochemistry.

[0094] Example method Sample collection and ethical approval Endometriosis tissue samples were collected from patients who underwent surgery for endometriosis at Radboud University Medical Center (Radboudumc). Informed consent was obtained before the tissue was used. Tissue fragments from patients aged 18 years and older with different subtypes of endometriosis were included. Samples were collected from the following three subtypes of endometriosis: peritoneal endometriosis (PER); ovarian endometriosis or endometrioma (OMA); and deep invasive endometriosis (DIE).

[0095] Sample preparation Tissue suspected of being endometriosis was removed during surgery by a gynecologist experienced in endometriosis procedures. After removal, the fragments were fixed with paraformaldehyde (PFA, 4% buffered formaldehyde) and subsequently embedded in paraffin. The presence of endometriosis was confirmed by a pathologist as part of routine diagnostic procedures in the Netherlands. For FAP expression analysis, eligible sections were first selected based on standard hematoxylin-eosin staining. If endometriosis was detected, sections were selected for further analysis based on the presence of endometrial-like stroma and glands, as well as fibrosis. Adjacent sections were used for immunohistochemistry. Immunohistochemistry was performed on 17 eligible samples.

[0096] Immunohistochemistry For FAP immunohistochemical staining, 4 μm thick sections were fixed onto IHC slides. The slides were dewaxed in xylene and dehydrated in ethanol. Subsequently, antigen retrieval was performed for 10 min in 96°C Tris / borate / EDTA buffer (TBE) containing 0.5% (v / v) Tween-20, followed by passive cooling to room temperature (RT) and washing twice in demineralized water and three times in phosphate-buffered saline (PBS). Endogenous peroxidase activity was then blocked by incubation with hydrogen peroxide (3% H2O2 in 10 mM PBS) for 10 min. After washing (2x demineralized water, 3x PBS), the slides were first incubated with avidin blocking solution (Vector, SP-2001, USA), washed twice in PBS, then incubated with biotin blocking solution (both incubations lasting 15 min), and then washed again. Subsequently, the slides were pre-incubated with normal goat serum (NGS, 20% NGS in PBS) for 30 min. Then, the slides were incubated with a primary antibody against fibroblast activation protein α (FAP, monoclonal rabbit anti-human antibody, diluted 1:500 in PBS + 1% BSA, Abcam, ab207178) at room temperature for 1 hour. After washing in PBS, the slides were incubated with a biotinylated secondary antibody (biotinylated goat anti-rabbit IgG, diluted 1:200 in PBS + 1% BSA, Vector Biotechnology, BA-1000).

[0097] For the negative control, slides were incubated without secondary antibody. After washing in PBS and incubating at room temperature for 30 min with a biotin / streptovir complex (Elite ABC kit, 1:100 diluted in PBS + 1% BSA, incubated for 30 min before use, Vectastain, PK-6100), the slides were stained with 3,3′-diaminobenzidine (Bright DAB, Immunologic, B-500) for 8 min, counterstained with hematoxylin for 2–5 seconds, then washed in tap water for 10 min and twice with demineralized water. Following the staining protocol, the sections were dehydrated in an alcohol and xylene bath and covered with Permount (P46, Fisher, SP15-500).

[0098] Immunohistochemistry for CD68 detection was performed according to standard clinical protocols of the Dutch Pathology Department. Staining was performed using a DAKOOMNIS staining machine with a primary antibody against CD68 (CD68-PG-M1, DAKO GA613).

[0099] Example 1 Endometriosis tissue samples were collected from patients who underwent surgery for endometriosis and prepared for histopathology and immunohistochemistry. The presence of endometriosis was confirmed by a pathologist as part of routine diagnostic procedures in the Netherlands.

[0100] Samples were derived from peritoneal endometriosis (PER) Figures 1 to 3 Ovarian endometriosis or endometrioma (OMA) Figure 4 and Figure 5 ) and deep invasive endometriosis (DIE) Figure 6 and Figure 7 ).

[0101] In areas where endometriosis was identified based on HE staining ( Figures 1 to 7 (A, D) Immunohistochemistry was used to assess the extent and pattern of FAP expression. Figures 1 to 7 (B, C, E).

[0102] FAP expression was identified in stromal cells directly beneath the epithelial cells. Figure 3 E solid black arrow) and myofibroblasts that are usually arranged in a cord-like pattern ( Figure 3 The strongest values ​​were found in the dashed black arrows (E). However, epithelial cells were negative for FAP staining ( Figure 3 E, hollow black arrow). It was also identified that the intensity of FAP staining decreased with increasing distance from the ectopic glandular tissue. Figure 5 E (solid black arrow).

[0103] In the peripheral portion of endometriosis lesions, FAP staining is weaker (e.g., Figure 5 B and Figure 5 (As seen in E).

[0104] In biopsies of healthy tissue surrounding endometriotic lesions, FAP expression was not detected in the healthy tissue. This finding... Figure 3 B ( ; Comparison identifier), 5B ( ; Comparison identifier) ​​and 7B ( It was most clearly observed in the comparison identifiers, but was also evident in all other samples of the three subtypes of endometriosis.

[0105] in conclusion Strong expression of FAP was identified in all of the following subtypes of endometriosis: PER, OMA, and DIE. The expression patterns were comparable across subtypes, showing strong expression in the lesion center and weaker expression towards the periphery. FAP expression was not detected in healthy tissue surrounding the lesion.

[0106] Example 2 A suitable FAP-binding ligand (such as OncoFAP) is labeled with a radionuclide (such as Ga-68) via a chelating agent (such as DOTAGA) attached to the FAP ligand. The resulting compound is, for example, Ga-68-DOTAGA-OncoFAP, synthesized as shown in Millul J et al., *An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications*. *Proc National Acad Sci.* 2021;118, and is available from Philogen. An appropriate amount (active, e.g., 100 MBq) of the compound is then injected intravenously into a patient diagnosed with endometriosis. After 1 hour of incubation with the radiotracer, the patient is scanned in a PET / CT or PET / MR scanner, and the localization of the endometriotic lesions is visualized on the resulting images. The localization of the lesion confirms the diagnosis (e.g., peritoneal endometriosis) and allows for the development of an individualized treatment plan (e.g., surgical resection). A suitable approach for translational imaging of the fibroblast activation protein (FAP) using Ga-68-DOTAGA-OncoFAP can be adapted from Backhaus, P. et al.'s "Translational imaging of the fibroblast activation protein (FAP) using the new ligand..." 68 Ga]Ga-OncoFAP-DOTAGA [Using new ligands] 68Ga]Ga-OncoFAP-DOTAGA for conversion imaging of fibroblast activation protein (FAP). Eur J Nucl Med Mol Imaging. May 2022;49(6):1822-1832, and / or methods studied and discussed in the following trials: Onofrio Catalano and Diandrea Galloway. PET / MRI for Evaluation of Endometriosis. ClinicalTrials.gov ID NCT06377553.

[0107] in conclusion FAP-PET / CT imaging was used to detect endometriotic lesions, including peritoneal endometriosis lesions. In a cohort of patients with endometriosis, [the following was observed / detected]. 68 PET imaging performed with Ga-oncoFAP detected endometriotic lesions, namely peritoneal lesions and other abdominal lesions. Therefore, we observed that... 68 Ga-oncoFAP PET imaging can identify endometriosis lesions in subjects suffering from endometriosis pain.

[0108] Example 3 Suitable FAP ligands are labeled with fluorescent dyes (for visualizing lesions during surgery) or radioactive isotopes (for detecting lesions by probes). Such compounds are, for example, FAP-34 labeled with IRDye800CW. 99m Tc-FAP-34. The compound was administered intravenously, and the patient underwent surgery within 24 hours.

[0109] Depending on the compound, the surgeon can visualize the fluorescent signal (e.g., using a fluorescence camera) or detect the radioactive signal using a suitable probe (with a defined field of view shield). The surgeon is guided to the endometriosis lesion and removes it.

[0110] in conclusion The use of fluorescent or radioactive FAP ligands helps identify endometriotic lesions that are previously difficult to diagnose using ultrasound and MRI. Furthermore, the use of radioactive FAP ligands can differentiate between active endometriotic lesions.

Claims

1. A conjugate comprising an FAP-targeting ligand and an effector for use in the treatment and / or diagnosis of endometriosis.

2. The conjugate according to claim 1, wherein the FAP-targeting ligand is selected from the group consisting of: antibodies; FAP inhibitors (FAPI); and peptides.

3. The conjugate for use according to claim 1 or claim 2, further comprising a linker between the FAP targeting ligand and the effector.

4. The conjugate for use according to claim 3, wherein the connector is a releasable connector or a non-releasable connector.

5. The conjugate for use according to claim 3 or claim 4, wherein the connector is selected from the group consisting of: acid-instable connectors, protease-sensitive connectors, photoinstable connectors, dimethyl connectors, or disulfide-containing connectors.

6. The conjugate for use according to any one of claims 1 to 5, wherein the effector is an imaging agent and / or a therapeutic agent.

7. The conjugate according to claim 6, wherein the therapeutic agent comprises a photosensitizer or a cytotoxic agent.

8. The conjugate for use according to claim 6 or claim 7, wherein the developer comprises a fluorescent molecule or a radioactive molecule.

9. The conjugate according to claim 8, wherein the radioactive molecule comprises a PET radioisotope (positron emitter) or a SPECT radioisotope (gamma emitter), preferably. 68 Ga、 18 F, 11 C 89 Zr、 64 CU 99m Tc or 111 In.

10. The conjugate according to claim 9, wherein the PET developer comprises a positron-emitting radionuclide, preferably. 68 Ga、 18 F, 11 C 89 Zr, or 64 CU.

11. The conjugate for use according to claim 9 or claim 10, wherein the effector is attached to the chelating agent.

12. The conjugate according to any one of claims 1 to 10, for use in the treatment of endometriosis lesions.

13. A method for identifying endometriosis lesions, the method comprising the following steps: i) Introducing the conjugate according to any one of claims 6 to 11 into the subject. ii) To make these endometriosis lesions visible.

14. The method of claim 13, further comprising the following steps: iii) Remove these endometriosis lesions.

15. A kit comprising a conjugate according to any one of claims 1 to 11, and at least one of the following: a drug administration medium; and an instruction manual.

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