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, and associated methods and kits.

Description

[0001] Endometriosis Tracer

[0002] Field of the invention

[0003] The present invention relates to the fields of medicine, medical imaging and molecular biology. In particular, the invention relates to a novel endometriosis tracer for use in methods for treatment and / or diagnosis.

[0004] Background of the invention

[0005] Endometriosis is a chronic disease affecting 10% of women in their fertile age in which ectopic endometrium-like tissue located outside the uterine cavity causes severe, life-impacting pain during periods, sexual intercourse, bowel movements and / or urination. Frequently, those with endometriosis develop chronic pelvic pain and infertility. Such chronic pelvic pain is often accompanied by abdominal bloating, nausea, fatigue, and sometimes depression and anxiety.

[0006] Endometriosis can start during a person’s first menstrual period and last until menopause when tissue similar to the lining of the uterus grows outside the uterine cavity. This leads to inflammation and scar tissue (adhesions, fibrosis) forming in the pelvic region and even elsewhere in the body. Several lesion types have been described: superficial endometriosis found mainly on the pelvic peritoneum; cystic ovarian endometriosis (endometrioma) found in the ovaries; deep endometriosis found in the recto-vaginal septum, bladder, and bowel; and, in rare cases, endometriosis has also been found outside the pelvis.

[0007] The cause of endometriosis is unknown, there is no known way to prevent endometriosis, and there is no definitive cure. Symptoms can be treated with medicines and / or, in some cases, surgery. Surgical removal of ectopic lesions and / or hormonal suppression focused on reducing estrogen, such as progestins, androgens, gonadotropin-releasing hormone (GnRH) agonists, and aromatase inhibitors, are the current gold standards of therapy. However, both surgical and non- surgical approaches are associated with various side effects and a high incidence of relapse. For example, the removal of the lesions of endometriosis, or endometriotic lesions, is complicated by the fact that to date, no reliable imaging procedure is available that allows the visualization of endometriosis lesions with sufficient sensitivity and specificity. Thus, patients are operated on and lesions are identified by visual inspection. Many lesions cannot be removed because they can either not be found or are overlooked during the operation, leading to futile surgery, repeated surgery or persistence of the disease. There is, therefore, a need for a non-invasive imaging procedure that enables the detection of endometriosis lesions, which would be a tremendous improvement over the current situation in which complete surgical removal cannot be achieved in most cases.

[0008] Summary of the Invention

[0009] In a first aspect, there is provided a conjugate comprising a FAP targeting ligand and an effector, for use in the treatment and / or diagnosis of endometriosis In a second aspect, there is provided a method of identifying endometriosis lesions comprising the steps of: i) introducing the conjugate as disclosed herein into a subject. ii) visualizing the endometriosis lesions.

[0010] In a third aspect, there is provided a kit comprising the conjugate according as disclosed herein, and at least one of: a drug administration vehicle and; an instruction manual.

[0011] Description of the invention

[0012] Definitions

[0013] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

[0014] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0015] As used herein, the term "and / or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0016] As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... ,etc.

[0017] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.

[0018] As used herein, "an effective amount" is the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of, for example for the purpose of treatment or medical imaging, varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount, which may be determined for instance as genome copies per kilogram (GC / kg) or as GC per dose. Thus, in connection with the administration of a drug which, in the context of the current disclosure, is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

[0019] The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatment, wherein such methods of treatment comprise the administration of the product for use.

[0020] The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" and “similarity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods.

[0021] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (e.g. Needleman Wunsch) which align the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using local alignment algorithms (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) I 8 (proteins) and gap 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 & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall length, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, at al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul at al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0022] As used herein, the term "selectively hybridizing", “hybridizes selectively” and similar terms are intended to describe conditions for hybridization and washing under which nucleotide sequences 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 remain hybridized to each other. That is to say, such hybridizing sequences may share 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.

[0023] A preferred, non-limiting example of such hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 1 X 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.

[0024] Highly stringent conditions include, for example, hybridization at about 68°C in 5x SSC / 5x Denhardt's solution I 1.0% SDS and washing in 0.2x SSC / 0.1 % SDS at room temperature. Alternatively, washing may be performed at 42°C.

[0025] The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook at al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel at al. (eds.), Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).

[0026] Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

[0027] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin.

[0028] The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3'-nontranslated sequence (3'-end) comprising a polyadenylation site. "Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

[0029] The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and / or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's) (self-cloning is defined herein as in European Directive 98 / 81 / EC Annex II). When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.

[0030] The terms "heterologous" and "exogenous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which they are introduced but have been obtained from another cell or are synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins, i.e. exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes for proteins not normally 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 one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the term 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 where at least two of the combined sequences are foreign with respect to each other.

[0031] As used herein, the term "non-naturally occurring" when used in reference to an organism means that the organism has at least one genetic alternation that is not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical reaction capability or a metabolic pathway capability to the non-naturally occurring organism that is altered from its naturally occurring state.

[0032] Any reference to nucleotide or amino acid sequences accessible in public sequence databases herein refers to the version of the sequence entry as available on the filing date of this document.

[0033] Detailed description of the invention

[0034] The present inventors have surprisingly identified Fibroblast Activating Protein (FAP) as a target for imaging of endometriosis. Fibrocyte Activating Protein (FAP), also known as Fibroblast Activating Protein alpha, Fibrocyte Activating Protein or Prolyl Endopeptidase FAP, is a type-ll transmembrane serine protease expressed on the surface of fibrocytes during epithelial cancers and soft tissue sarcomas (Dendl, K., at al., 2023; Dorst, D. N., at al., 2023). In recent years, ligands have been developed that target FAP and can be used for cancer imaging and therapy, like radiolabeled FAP ligands for PET imaging (Positron Emission Tomography) (Imlimthan, S., at al., 2021 ; Kratochwil, C., at al., 2019; Sollini, M., er al., 2021 ; Backhaus, P., at al., 2021) and photodynamic therapy (PDT) (Dorst, D. N., at al., 2020; Dorst, D. N., at al., 2022). It is, therefore, an object of the current invention to provide FAP targeting ligands for the diagnosis and / or treatment of endometriosis. For example, for the detection of lesions in guiding surgery, fluorescent guided surgery, PDT and therapy.

[0035] Given the benign nature of endometriotic lesions, it could not have been anticipated that a known malignant tumour marker, identified from the tumour microenvironment, would help to solve a major clinical challenge, the treatment and / or diagnosis of endometriosis, such as though the identification of endometriosis lesions for guiding surgical removal. In a first aspect, there is provided a conjugate comprising a FAP targeting ligand and an effector, for use in the treatment and / or diagnosis of endometriosis. The term “conjugate” as used herein takes its meaning from the art, i.e. a substance formed by the combination of two or more others.

[0036] The term “ligand” as used herein takes its meaning from the art, i.e. any molecule or atom that binds to a receiving protein molecule, otherwise 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 antigen-binding fragments thereof. In embodiments where the ligand unit is a non-antibody targeting agent, it may be a peptide or polypeptide, or a non-proteinaceous molecule, such as a FAP inhibitor (FAPI).

[0037] FAP refers to Fibrocyte Activating Protein or Fibroblast Activation Protein, interchangeably, but is also known as prolyl endopeptidase FAP (uniProt Q12884; Ensembl ENSG00000078098). FAP is a 97-kDA type-ll transmembrane serine protease member of the proplyl peptidase family, expressed almost exclusively in pathological conditions including arthritis and cancer (Dendl at al., 2023, PET Clin 18 (2023) 345-351).

[0038] The term “effector” as used herein takes its meaning from the art, i.e. a molecule that relates to a purpose or activity, such as small molecules that selectively bind to a protein to regulate its biological activity or molecules used for imaging purposes.

[0039] The term “endometriosis” as used herein means a disease in which tissue similar to the endometrium grows outside of the uterine cavity. Pain is the most common symptom of endometriosis. Women with endometriosis can experience pelvic or lower abdominal pain, pain with menses (dysmenorrhea), pain with intercourse (dyspareunia) and pain during bowel movements (dyschezia). Several locations for lesions, also known as endometriosis or endometriotic lesions, or foci, have been described for endometriosis: superficial endometriosis found mainly on the pelvic peritoneum; cystic ovarian endometriosis (endometrioma) found in the ovaries; deep endometriosis found in the recto-vaginal septum, bladder, and bowel; and in rare cases, endometriosis has also been found outside the pelvis. The endometriotic lesions, or foci, are pro-inflammatory, which is distinct from the tumour microenvironment, which is known to be immunosuppressive and evade detection from the immune system. Thus, it could not have been anticipated that a known cancer marker would give the histological profile within endometriosis lesions as described in the examples herein.

[0040] FAP ligands

[0041] In some embodiments, the FAP targeting ligand is selected from the group consisting of: an antibody; a FAP inhibitor (FAPI); and a peptide. Antibodies

[0042] Human FAP was originally identified in cultured fibroblasts using the monoclonal antibody (mAb) F19 (Welt, S„ at al., 1994, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol., 12, 1193-1203). Sibrotuzumab / BIBH1 , a humanized version of the F19 antibody as well as other humanized or fully human antibodies against FAP antigen exhibiting specificity towards the F19 epitope have been reported (Schmidt, A., at al., 2001 , Eur. J. Biochem. 268, 1730-1738; Mersmann, M., at al., 2001 , Int. J. Cancer, 92: 240-248). OS4 is another humanized antibody (CDR-grafted) derived from the F19 antibody (Wuest, T., at al., 2001 , J. Biotechnol., 92:159-168). Furthermore, murine anti-FAP antibodies, including chimeric and humanized versions have been evolved (Ostermann, E., at al., 2008, Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res., 14: 4584-4592) (Imlimthan, S. at al., 2021 , Pharmaceuticals, 12: 1023). Therefore, the production of FAP antibodies and suitable derivatives thereof are thereof known to one of skill in the art and incorporated herein by reference.

[0043] As used herein, “antibody” or “antibody unit”, within the scope of it, includes any part of an antibody structure, antigen binding fragment or nanobody or single domain antibody. This unit may bind, reactively associate, or complex with a receptor, antigen or other receptor unit present in the targeted cell population. An antibody can be any protein or proteinaceous molecule that can bind, complex, or otherwise react with a portion of a cell population to be treated or biologically engineered. Thus, the antibodies herein are capable of specifically binding to a FAP antigen.

[0044] The antibody according to the invention may be a monomeric antibody or a multimeric antibody. In particular, the antibody according to the invention is a monomeric antibody. The antibody according to the invention can be monoclonal or polyclonal. Preferably, the antibody according to the invention is monoclonal. In another preferred embodiment, the targeting agent is an anti-FAP antibody or a peptide-binding FAP.

[0045] The term “fragment” as used herein, means a region comprising the antigen binding or variable region of the antibody. Said fragment may be selected, without limitation, from the group consisting of Fv, Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv, single-chain Fv (scFv), di-scFvs or sc(Fv)2, dsFv, Fd, dAb, CDRs, VH, VL, VHH, V-NAR, nanobodies, minibodies, diabodies, and multi-specific antibodies formed from antibodies fragments.

[0046] Antibody-mimic molecules are also contemplated, such as affibodies and designed ankyrin repeat proteins (DARPins). Such antibodies and antibody-mimetic molecules are well known in the art and can be prepared by well-known methods and information. For convenience, antigen-related information well known in the art is labelled as follows, including name, other names, and GenBank accession numbers. Nucleic acid and protein sequences corresponding to FAP antigens can be found in public databases, such as Ensembl. The antibodies target the corresponding FAP antigens including all amino acid sequence variants and homologues, having at least 70%, 80%, 85%, 90% or 95% homology with the sequences identified in the references, or having biological properties and characteristics that are fully identical to the FAP antigen sequences known in the art, (see Fischer, E. at al., 2012, Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res., 18, 6208-6218; Schlothauer, T., at al. 2016, Protein Eng., Des. Sei., 29, 457-466). In some embodiments, the FAP antibody is selected from: ESC11 and ESC14; 28H1 .

[0047] Fap inhibitors (FAPIs)

[0048] The term “inhibit” or “inhibition of’ refers to a reduction in a detectable amount. Thus, FAPIs, or a FAPi, may be considered to have a useful application in endometriosis, where their application may be used in the treatment of endometriosis. In some embodiments, FAP inhibition uses small molecule inhibitors.

[0049] As used herein, "small molecule” or “micro molecule”, includes any low molecular weight molecule (less than 1000 daltons) including but not limited to lipid, monosaccharide, second messenger, other natural products and metabolites, as well as drugs. The compatibility and design of FAP inhibitors (FAPIs) are well known in the art and can be prepared by well-known methods and information, such as discussed in Pure, E., at al., 2018, Oncogene, 37: 4343-4357.

[0050] In some embodiments, the FAPI is selected for the group consisting of, but not limited to, talabostat, PT-100, linagliptin (Tradjenta), FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold as disclosed in Jansen at al., ACS Med Chem Lett. 2013 May 9; 4(5): 491-496, MIP-1232 such as described in Mueller at al. J Biol Chem 1999; 274:24947-52, and FAP inhibitors such as FAPI-02 and FAPI-04 for example such as described in Lindner at al. EJNMMI Radiopharmacy and Chemistry (2019) 4:16 and Lindner at al., Journal of Nuclear Medicine, published on Apr. 6, 2018. In addition to those listed in Imlimthan, S., at al. 2021 , Pharmaceuticals, 14 (10), 1023, such as 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; DOTA.SA.FAPi; DAT A5m. SA. FAPi; 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 instance, FAP inhibitors are described in WG20081522, WO20132661 , WO20245173, WG21005125, WG21005131 , US2020246383, WO19154859, WO19083990, WO191 18932, WO18111989, WO17189569, WO13107820, WO08116054 or WO07085895, the disclosure of which being incorporated herein by reference.

[0051] In a more preferred embodiment, the FAP inhibitor is selected from the group consisting of, but not limited to, 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, DAT A5m. SA. FAPi, DOTA.(SA.FAPi)2, DOTAGA.(SA.FAPi)2, RPS-309, QCP02, FAPI-34, FL-L3, RPS-309, OncoFAP, FAP-2286. In an even more preferred embodiment, the FAP inhibitor comprises an ultra-high-affinity ligand of FAP for targeting application with pan-tumoral potential, such as OncoFAP. OncoFAP will be 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 a most preferred embodiment the FAP inhibitor comprises OncoFAP.

[0052] Further examples are provided in Giesel, FL, at al. 2019. J Nucl Med. ;60:386-92; Loktev A. at al. 2018. J Nucl Med.59:1423-9; Ballal S, 2021. Eur J Nucl Med Mol., I(48):1915-31 ; and Baum RP, at al., 2021. J Nucl Med. ,120.259192.

[0053] Linker

[0054] In some embodiments, the conjugate as described herein additionally comprises a linker between the FAP targeting ligand and the effector.

[0055] The term “linker” or “linker fragment” or “linker unit” as used herein, refers to a chemical moiety or bond that is linked at one end to the FAP ligand, as disclosed herein, and at the other end to the effector, and may be linked to another effector or ligand following attachment of another linker. Linkers may be synthesized by methods known in the art, such as those described in US2005- 0238649A1 for, at least, antibody-drug conjugates. The linker may be a “releasable”, also known as “cleavable”, which would thus facilitate the release of the effector. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (US 5,208,020, identifying at least linkers for use with cytotoxic agents). Examples of “non-releasable”, also known as “non-cleavable” linkers, according to the invention include, without limitations, thioethers. Therefore, in some embodiments, the linker is a releasable linker or a non-releasable linker.

[0056] In some embodiments, the linker is selected from the group consisting of acid-labile linkers, protease-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers. Suitable linkers compatible with OncoFAP may be known from from Backhaus, P. et al. Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga- OncoFAP-DOTAGA.

[0057] Effector

[0058] In some embodiments, the effector is an imaging agent and / or 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 an imaging agent and therapeutic agent.

[0059] Therapeutic Agent

[0060] The term “therapeutic agent” as used herein refers to an agent for use in the treatment or therapy of endometriosis. In some embodiments, the therapeutic agent comprises a photosensitizer or cytotoxic agent. The term “photosensitizer” as used herein refers to molecules, including suitable porphyrin, chlorines and dyes, that can generate reactive oxygen species (ROS) under illumination at certain wavelengths. In some embodiments, the photosensitizer is suitable for use in photodynamic therapy (PDT). PDT involves the combination of light and a photosensitizer (PS), which is activated by absorption of light of a specific wavelength, causing the generation of potentially toxic ROS that induce a cascade of intracellular molecular events resulting in targeted tissue damage (Avci, P., at al., 2014, J. Biomed. Nanotechnol., 10, 1937-1952). In some embodiments, the photosensitizer has an excitation peak at between 600 to 900 nm, such as between 680 to 800 nm. In some embodiments, the photosensitizer is selected from the group consisting of, but not limited to, Allumera, Photofrin, Visudyne, Levulan, Foscan, Metvix, Hexvix, Cysview, Laserphyrin and Spectrum. In some embodiments, the photosensitizer is IRDye 700DX.

[0061] Thus, the possibility of anti-Fap targeted photodynamic therapy (TPT), as disclosed in Dorst, D., et al., 2022, Rheumatology, 61 :2999-3009, Dorst, D at al., 2020, Rheumatology, 59:3952-3960, but herein applied to the treatment and / or diagnosis of endometriosis, is also encompassed by the current invention. Therefore, in some embodiments, the therapeutic agent may also be an imaging agent.

[0062] The term “cytotoxic agent” as used herein refers to a molecule that when entering in contact with a cell, eventually upon internalization into the cell, alters a cell function (e.g. cell growth and / or proliferation and / or differentiation and / or metabolism such as protein and / or DNA synthesis) in a detrimental way or leads to 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, radioisotopes, toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.

[0063] The cytotoxic drug according to the invention may be selected from the group consisting of dolastatins such as dolastin 10, dolastin 15, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin F (MMAF), monomethylauristatin-D (MMAD), monomethyl auristatin E (MMAE), and 5-benzoylvaleric acid-AE ester (AEVB), maytansines such ansamitocin, mertansine (also called emtansine or DM1) and ravtansine (also called soravtansine or DM4), antracyclins such as daunorubicin, epirubicin, pirarubicin, idarubicin, zorubicin, cerubidin, aclarubicin, adriblastin, doxorubicin, mitoxantrone, daunoxome, nemorubicin and PNU-159682, calicheamicins such as calicheamicin beta 1 Br, calicheamicin gamma 1 Br, calicheamicin alpha 21 , calicheamicin alpha 31 , calicheamicin beta 11 , calicheamicin gamma 1 L, calicheamicin delta 11 and ozogamicin, esperamicins such as esperamicin A1 , neocarzinostatins, bleomycin, duocarymycins such as CC- 1065 and duocarmycin A, pyrrolobenzodiazepines such as anthramycin, abbeymycin, chicamycin, DC-81 , mazethramycin, neothramycins A and B, porothramycin prothracarcin, sibanomicin (DC- 102), sibiromycin and tomamycin, pyrrolobenzodiazepine dimers (or PBD), indolino- benzodiazepines, indolino-benzodiazepine dimers, a-amanitins, abraxane, actinomycin, aldesleukin, altretamine, alitretinoin, amsacrine, anastrozole, arsenic, asparaginase, azacitidine, azathioprine, bexarotene, bendamustine, bicalutamide, bortezomib, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, chloramphenicol, ciclosporin, cidofovir, coal tar containing products, colchicine, dacarbazine, dactinomycin, danazol, dasatinib, diethylstilbestrol, dinoprostone, dithranol, dutasteride, dexrazoxane, docetaxel, doxifluridine, erlotinib, estramustine, etoposide, exemestane, finasteride, flutamide, floxuridine, flucytosine, fludarabine, fluorouracil, ganciclovir, gefitinib, gemcitabine, goserelin, hydroxyurea, hydroxycarbamide, ifosfamide, irinotecan, imatinib, lenalidomide, leflunomide, letrozole, leuprorelin acetate, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, menotropins, mifepristone, nafarelin, nelarabin, nitrogen mustard, nitrosoureas, oxaliplatin, ozogamicin, paclitaxel, podophyllyn, pegasparaginase, pemetrexed, pentamidine, pentostatin, procarbazin, raloxifene, ribavarin, raltitrexed, rituximab, romidepsin, sorafenib, streptozocin, sunitinib, sirolimus, temozolomide, temsirolimus, teniposide, thalidomide, thioguanine, thiotepa, topotecan, tacrolimus, taxotere, tafluposide, toremifene, tretinoin, trifluridine, triptorelin, valganciclovir, valrubicin, vinblastine, vidaradine, vincristine, vindesine, vinorelbine, vemurafenib, vismodegib, vorinostat, zidovudine, vedotine, derivatives and combinations thereof. The cytotoxic drug can also be a radionuclide such as Lutetium-177, lodine-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.

[0064] Imaging Agent

[0065] The term “imaging agent” as used herein is an agent that is employed for use in medical imaging techniques. The techniques or modalities include, but are not limited to, X-rays, computed tomography (CT), magnetic resonance imaging (MRI), scintigraphy, fluorescence, luminescent, ultrasound, etc. Therefore, in some embodiments, the imaging agent comprises a fluorescent or radioactive molecule. In some embodiments, an 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.

[0066] The use of an imaging agent has particular application in the use of imaging agent assisted, or guided, surgery. In some embodiments, the imaging agent is a fluorescent molecule for use in fluorescent guided surgery. The term “florescent guided surgery” or “florescent image guided surgery” as used herein refers to a medical imaging technique to detect fluorescently labelled structures during surgery, with 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- methylpyridinium iodide), rhodamine, Cyanine5 (also known as Cy5), Cyanine5.5 (also known as Cy5.5), Cyanine7 (also known as Cy7), sulfoCyanine7 (also known as sulfoCy7), Cyanine7.5 (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-label, any other Cyanine-label, and sulfonated or otherwise modified variants of any of these fluorophores.

[0067] Combinations are also envisioned in the current invention and would be known by one of skill in the art, for example, in some embodiments, the imaging agent is a fluorescent molecule and the effector is a photosensitizer for use in photodynamic therapy (PDT). Such combinations are encompassed in the invention and may be applied in methods of endometriotic lesion removal. Such methods may be alone or in combination with other diagnostic and / or treatments or therapies. For example, in some embodiments, the methods as disclosed herein are used in combination with hormonal suppression focused on reducing estrogen, such as progestins, androgens, gonadotropin-releasing hormone (GnRH) agonists, and aromatase inhibitors.

[0068] The term “radioactive molecule” as used herein also refers to “radioisotopes” and “radionuclides” for use in nuclear medicine, including imaging, diagnosis and therapy, or any combination thereof. In some embodiments, the radioactive molecule is selected from the group consisting of, but not limited to, Technetium-99m, lodine-131 , Chromium-51 , Lutetium-177, lodine-125, lndium-111 , lodine-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, Zicronium-89, or a derivative thereof. A radionuclide for imaging and / or therapy is chosen based on its properties with respect to the radiation / particles emitted (with respect to imaging devices and probes), its half-life (balanced with the pharmacokinetic properties of the ligand), decay products (with respect to radiation exposure / therapeutic efficacy) and radiochemical properties (for efficient labelling).

[0069] In some embodiments, the radioactive molecule is a gamma emitter, more preferably Tc-99m or In- 111 . Suitable application of radiolabelling, compatible linkers, and its application to the current invention would be known to one in the art.

[0070] In some embodiments, the radioactive molecule comprises a PET radioisotope (a positron emitter) or a SPECT radioisotope (gamma emitter). In some embodiments, the radioactive molecule is68Ga,18F,11C,89Zr,64CU,99mTc, or111ln. Therefore, in some embodiments, the PET imaging agent comprises a positron emitting radionuclide, preferably68Ga,18F,11C,89Zr,64CU, preferably18F or68Ga. In a more preferred embodiment, the PET imaging agent comprises68Ga.

[0071] Combinations are also envisioned in the current invention and would be known by one of skill in the art, for example, in some embodiments, the FAP ligand is a FAPI and the effector is a radionuclide (e.g., Lutetium-177), such as the FAP inhibitor FAPI-2286 developed by Clovis Oncology (Zboralski, D., at al., 2022, Eur J Nucl Med Mol Imaging. Sep;49(11):3651-3667). Thus, in some embodiments, the FAP ligand is a FAPI and the effector is a radionuclide. Further examples of radiolabelled FAPI, such as Gallium-68, Fluorine-18, Carbon-11 , Zirconium-89, Copper-64 -FAPI. FAPI, as disclosed herein, may include one or more 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, FAP-2286The synthesis of such radiolabelled-FAPI, would be known to one of skill in the art, such as described in Imlimthan at 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 comprises68Ga. In an alternative embodiment, the FAP ligand comprises OncoFAP and the effector comprises 18p

[0072] The FAP ligand, which may be a FAPI, may also be “linked” or “coupled” to a radionuclide chelator, such as DOTA (FAP-2286) radiotherapeutic tracer. Alternative chelators and approaches are also known by the skilled person, for instance, several other bifunctional chelators, NOTA, DATA5m, DOTA, NODAGA and DOTAGA, are known in the art, with reference to chelator-modified FAPI (Moon, E.S, 2020, EJNMMI radiopharm. chem. 5:19). Therefore, in some embodiments, the effector comprises a radioactive molecule optionally attached to a chelator.

[0073] Chelators are known in the art (see for instance Price and Orvig, DOi: 10.1039 / C3CS60304K), and are useful for associating the ligand with radiolabels. Suitable chelators for a radiolabel are 1 ,4,7, 10-tetraazacyclododecane-tetraacetic acid (DOTA) 1 ,4,7-triazacyclononane-triacetic acid (NOTA), triazacyclononane-phosphinate (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 its hydrolyzed form. Further chelators for radiolabels can be based on 2,2',2",2"'-(Ethane-1 ,2-d iy Id in itrilo)tetraacetic acid (EDTA) or 1 ,4,8,11-Tetraazacyclotetradecane (cyclam) or 1 ,8-diamino-3,6, 10, 13, 16, 19- hexaazabicyclo[6.6.6]icosane (DiamSar). In preferred embodiments, chelators are selected from HYNIC (6-hydrazinonicotinic 15 hydrazide), MAG3 (mercaptoacetyltriglycine), MAS3 (acetylmercaptoacetyltriserine), DTPA (diethylenetriaminepentaacetic acid), chelators for tricarbonyl radiolabels such as Tc-99m-tricarbonyl (for instance L 1 , L8, L9, and L 10 from Banerjee at al., DOi: 10.1021 / jm400823w), DFO (deferoxamine), DFO* (see Patra at al., Chem Commun. 2014;50:11523-5), TAFC (triacetylfusarinine C), FSC (Fusarinine 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, 1 0- tetraazacyclododecane-1 ,4, 7-triyl)triacetic acid), CHX-A"-DTPA (such as [(R)-2-amino-3-(4- isothiocyanatophenyl)propyl]-trans(S,S)-cyclohexane-1 ,2-diamine-pentaacetic acid), TCMC (such as 2-[(4-lsothiocyanatophenyl)methyl]-1 ,4, 7, 1 0-tetraazacyclododecane-1 ,4, 7, 10-tetraacetamide tetrahydrochloride), DO3AM (2-(4,7, 1 0-tris(2-amino-2-oxoethyl)-1 ,4,7, 1 0-tetraazacyclododecan- 1-yl)acetic acid), NET A (({4-[2-(bis-carboxy-methylamino )-5-( 4-nitrophenyl)pentyl]- 7-carbo- xymethyl[1 ,4, 7]triazanonan-1-yl} acetic acid), CB-DO2A (4, 10-bis( carboxymethyl)-1 ,4, 7, 10- tetraazabicyclo[5.5.2]tetradecane, DiamSar, NODA-MPAA (the 1 ,4,7-triazacyclononane-1 ,4- diacetate (NODA) motif with a methylphenylacetic acid (MPAA) backbone, see DOi: 10.1021 / bc200175c), and RESCA (WO2016 / 065435). Highly preferred chelators are DOTA and NOTA, while DOTA is most preferred. DOTA may also be referred to as tetraxetan and comprise the compound formula (CH2CH2NCH2CO2H)4. Preferred Tc-99m-chelators are HYNIC, MAG3, MAS3, DTPA, and chelators for tricarbonyl Tc-99m, more preferably HYNIC, MAG3, MAS3, and DTPA. Preferred Zr-89 chelators are DFO, DFO*, TAFC, and FSC. Preferred Ga-68 chelators are HBED-CC, TAFC, FSC, THP, TRAP, DOTA, 35 NODAGA, and NOTA. Preferred In-111 chelators are DTPA, DOTA, NOTA, DOTAGA, and CHXA"-DTPA. Preferred Pb-chelators are TCMC, DO3AM, and NETA. Preferred Cu chelators are DOTA, CB-DO2A, TETA, and Diamsar. Preferred AIF-18 chelators are NODA-MPAA, NOTA, and RESCA.

[0074] Chelators compatible with OncoFAP may be adapted from Backhaus, P. et al. Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA. Eur J Nucl Med Mol Imaging. 2022 May;49(6):1822-1832. Therefore, in a preferred embodiment the FAP inhibitor comprises OncoFAP and the chelator comprises DOTAGA, or a suitable compatible alternative. In a more preferred embodiment, the FAP inhibitor comprises OncoFAP, the chelator comprises DOTAGA and the effector comprises a PET imaging agent. In an even more preferred embodiment, the FAP inhibitor comprises OncoFAP, the chelator comprises DOTAGA and effector comprises68Ga, such as Ga-68-DOTAGA-OncoFAP.

[0075] Compositions

[0076] In some embodiments, the compositions are used for the treatment of (human) subjects. For that purpose, there is provided a pharmaceutical composition comprising physiological buffers, such as e.g. PBS or HEPES, comprising further stabilizing agents such as e.g. sucrose. Such compositions are compatible with and suitable and intended for use in subsequent intravenous, intramuscular, subcutaneous, intraperitoneal, intrauterine, intravaginal, intraurethral, or other relevant administration or for use in organ-targeted vascular delivery or isolated perfusion.

[0077] Uses & Methods

[0078] The large difference in FAP expression between the endometriotic tissue and the surrounding nonaffected tissue offers the possibility to use FAP expression for the detection of endometriotic lesions. In one aspect, there is provided the conjugate for use as described herein, for use in the treatment of endometriosis lesions.

[0079] In some embodiments, there is provided the FAP inhibitor (FAPI) or labelled FAP inhibitor (FAPI) as described herein for use in the treatment of endometriosis and / or the distinction of endometriotic tissue from the surrounding, non-endometriotic, tissue. Particularly for peritoneal endometriosis this could be a huge improvement over the current diagnostic tools, ultrasound, CT and MRI, which are generally not able to detect these lesions. Therefore, in some embodiments, there is provided the labelled FAP inhibitor (FAPI) as described herein for use in the distinction of peritoneal endometriotic tissue from the surrounding, non-endometriotic, tissue. Frequently, laparoscopic surgery is required to confirm the presence of endometriotic lesions in patients. The use of a labelled FAPI to detect endometriosis is non-invasive and likely to significantly shorten the time required before endometriosis is diagnosed. This could lead to a considerable reduction in patient burden, both physical and psychological.

[0080] Therefore, it is within the scope of the disclosure to have ligands useable for diagnostic and / or preoperative SPECT / PET / CT scanning, intraoperative localization of endometriotic lesions with a relevant probe, such as a gamma probe, or portable SPECT camera (radioguided surgery) and radioligand therapy (e.g. Lu-177 FAP), or for fluorescence imaging or PDT.

[0081] To create a probe visible for the eye of a medical practitioner, and having high penetration depth in human tissue, fluorescence guided surgery (FGS) and radio guided surgery (RGS) can be combined into a single compound according to the invention, wherein the probe contains a fluorescent label as well as a chelating moiety that can carry a radiolabel. Preoperatively the location of the endometriotic lesions can be determined with the help of the radionuclide by PET or SPECT imaging. During the surgical procedure, the medical practitioner can rely on the signal generated by the radionuclide to be guided to the endometriotic lesions, until the fluorescent label becomes visible. The fluorescent label precisely evaluates resection margins once the surgical field is exposed to the surface, which can help the surgeon in removing all pathogenic tissue. Associated methods may be derived from the trial: Onofrio Catalano and Diandrea Galloway. PET / MRI for Evaluation of Endometriosis. ClinicalTrials.gov ID NCT06377553.

[0082] A further application, based on the difference between FAP expression in endometriotic and healthy tissue, would be the eradication of endometriotic lesions by photodynamic therapy which may use the photosensitizer as discussed above. For this approach, it is important that the target cells express FAP and are therefore susceptible to radioactively labelled FAPI, while the surrounding tissue should lack FAP expression and will remain largely unaffected. Therefore, in some embodiments, there is provided the labelled FAP inhibitor (FAPI) as described herein for use in the treatment of endometriosis by photodynamic therapy. In some embodiments, there is provided the labelled FAP inhibitor (FAPI) in combination with photosensitizer as described herein for use in the treatment of endometriosis by photodynamic therapy.

[0083] Due to the radiolabel and fluorescent label linked to a targeting moiety, the compounds according to the invention are suitable for diagnosis. This diagnosis can be prior to treatment, but according to the unique nature of the compounds it can also be during treatment, or (immediately) after treatment. Accordingly, embodiments provide the compound for use according to the invention, wherein the medicament is for imaging, diagnosing, and / or treating endometriosis. Such a method is preferably for achieving a diagnosis via two parameters, such as via a radiolabel and via a fluorescent label. The compounds for use according to the invention, and the compositions for use according to the invention, as described herein, can be very suitably used in a method of imaging, diagnosing, or treating endometriosis in a subject in need thereof, the method comprising the step of administering a compound or composition according to the invention to the subject.

[0084] A "subject" includes an animal, such as a human, monkey, cow, horse, cat, dog, mouse, or rat. The animal can be a mammal such as a non-primate and a primate (e g., monkey and human). In one embodiment, a subject is a human being. In other embodiments the subject is not a human, such as a mouse or a rat.

[0085] In some embodiments, there is provided the conjugate for use as disclosed herein for the treatment and / or diagnosis of one or more endometriosis lesion subgroups selected from: superficial (peritoneal) endometriosis; cystic ovarian endometriosis (endometrioma); and deep endometriosis.

[0086] In some embodiments, there is provided the conjugate for use as disclosed herein for the treatment and / or diagnosis of pain of endometriosis. Example of pain of endometriosis, or endometriotic pain, include but are not limited to, pelvic pain, pain felt during menstrual periods, pain accompanied by swelling of the abdomen, discomfort with bowl movement, general discomfort, uterine pain, and lower abdominal pain.

[0087] In one aspect, there is provided a method of identifying endometriosis lesions comprising the steps of: i) introducing the conjugate described herein, into a subject. ii) visualizing the endometriosis lesions.

[0088] Methods of visualization will be known to one of skill in the art and include, but are not limited to, the implementation of fluorescence camera detection, gamma probe detection and positron probe detection.

[0089] In some embodiments, the method as described herein, additionally comprising the step of: iii) removal of the endometriosis lesions.

[0090] The term “removal” as used herein with references to endometriosis lesions, includes but is not limited to any removal, destruction, or excision by invasive and / or non-invasive means.

[0091] In a particular embodiment, the invention concerns the use of a FAP inhibitor for the treatment of a subject having endometriosis or for use in the treatment of a subject having endometriosis. In some embodiments, the method as disclosed herein is for the identification and / or visualization and / or removal of one or more endometriosis lesions subgroups selected from: superficial endometriosis; cystic ovarian endometriosis (endometrioma); and deep endometriosis.

[0092] In some embodiments, the method as disclosed herein is for the identification and / or visualization and / or removal of endometriosis lesions associated with pain of endometriosis.

[0093] In a further embodiment, the method as disclosed herein is for the identification, visualization and subsequent removal of endometriosis lesions associated with pain of endometriosis.

[0094] Kits

[0095] In one aspect, there is provided a kit comprising the conjugate as described herein, and at least one of: a drug administration vehicle and; an instruction manual.

[0096] The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims.

[0097] Description of the figures

[0098] Figure 1 Peritoneal endometriosis section. A, B and C are whole slide images; D, E and F are zoom-in views of the black box in A. A and D) H&E staining; B and E) FAP immunohistochemistry;

[0099] C) FAP immunohistochemistry negative control; F) CD68 immunohistochemistry.

[0100] Peritoneal endometriosis. A, B and C are whole slide images; D, E and F are zoom-in 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.

[0101] Figure 3 Peritoneal endometriosis. A, B and C are whole slide images; D, E and F are zoom-in 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.

[0102] Figure 4 Ovarian endometriosis. A, B and C are whole slide images; D E and F are zoom-in 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.

[0103] Figure 5 Ovarian endometriosis. A, B and C are whole slide images D, E and F are zoom-in 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. Figure 6. Deep infiltrating endometriosis. A, B and C are whole slide images; D, E and F are zoom- in 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.

[0104] Examples

[0105] Methods

[0106] Collection of samples and ethical approval

[0107] Endometriotic tissue samples were collected from patients who underwent surgery for endometriosis at Radboudumc. Informed consent was obtained prior to the use of the tissue. Tissue fragments from patients aged 18 years and older and with different subtypes of endometriosis were included. Samples were collected from the three endometriosis subtypes; peritoneal endometriosis (PER); ovarian endometriosis or endometrioma (OMA); and deep infiltrating endometriosis (DIE).

[0108] Preparation of samples

[0109] Tissue suspicious for endometriosis was removed during surgery by a gynaecologist experienced in endometriosis surgery. After removal, fragments were fixated using paraformaldehyde (PFA, buffered formaldehyde 4%) and subsequently embedded in paraffin. The presence of endometriosis was confirmed by a pathologist as part of the regular diagnostic procedure in the Netherlands. For FAP expression analysis, eligible sections were first selected based on standard haematoxylin-eosin staining. If endometriosis was detected, based on the presence of endometrial- like stroma and glands and fibrosis, sections were selected for further analysis. Adjacent sections were used for immunohistochemistry. Immunohistochemistry was performed on 17 eligible samples.

[0110] Immunohistochemistry

[0111] For FAP immunohistochemistry staining 4 pm thick sections were mounted on IHC slides. Slides were deparaffinized in xylene and dehydrated in ethanol. Thereafter, antigen retrieval was done for 10 min in 96°C Tris / Borate / EDTA buffer (TBE) with 0.5% (v / v) tween-20, before passive cooling down to room temperature (RT) and washing twice in demi water and thrice in phosphate buffered saline (PBS). Afterwards, endogenous peroxidase activity was blocked by incubation with hydrogen peroxide (3% H2O2 in 10 mM PBS) for 10 min. After washing (2x demi water, 3x PBS), slides were first incubated with avidin block (Vector, SP-2001), washed in PBS twice, and secondly with biotin block, both for 15 min before washing again. Thereafter, slides were pre-incubated for 30 min with normal goat serum (NGS, 20% NGS in PBS). Slides were then incubated for 1 hour with the primary antibody for Fibrocyte Activating Protein alpha (FAP, monoclonal rabbit-anti-human antibody, diluted 1 :500 in PBS+1 % BSA, Abeam, ab207178) at room temperature. After washing in PBS, slides were incubated with the biotinylated secondary antibody (biotinylated Goat anti-rabbit IgG, diluted 1 :200 in PBS+1 %BSA, Vector, BA-1000). For negative controls, slides were incubated without the secondary antibody. After washing in PBS and incubation with Biotin / Streptavidin complex (ABC-Kit Elite, diluted 1 :100 in PBS+1 %BSA, incubated 30 min before use, Vectastain, PK-6100) for 30 min at room temperature slides were stained with 3,3'-diaminobenzidine (Bright DAB, Immunologic, B-500) for 8 min and counterstained with hematoxylin for 2-5 sec before washing in tap water for 10 minutes and washing twice in demi water. After the staining protocol, sections were dehydrated in alcohol and xylene baths and covered with Permount (P46, Fisher, SP15-500).

[0112] Immunohistochemistry for the detection of CD68 was performed according to a standard clinical protocol at a pathology department in the Netherlands. A DAKO OMNIS staining machine was used for the staining with a primary antibody against CD68 (CD68-PG-M1 , DAKO GA613).

[0113] Example 1

[0114] Endometriotic 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 the regular diagnostic procedure in the Netherlands.

[0115] Samples were derived from peritoneal endometriosis (PER) (Figures 1 to 3), ovarian endometriosis or endometrioma (OMA) (Figures 4 and 5) and deep infiltrating endometriosis (DIE) (Figures 6 and 7).

[0116] In the areas where endometriosis was identified based on the HE staining (Figures 1 to 7; A, D), the degree and the pattern of FAP expression using immunohistochemistry was assessed (Figures 1 to 7; B, C, E).

[0117] FAP expression was identified to be the strongest in both the stromal cells directly under the epithelial cells (Figure 3E solid black arrow) and in the myofibroblasts, which were often arranged in a track-like pattern (Figure 3E interrupted black arrow). The epithelial cells, however, stained negative for FAP (Figure 3; E empty black arrow). It was also identified that as the distance to the endometriotic glandular tissue increases, the intensity of FAP staining decreases (Figure 5E solid black arrows).

[0118] In the peripheral parts of endometriotic lesions, the FAP staining is weak, as seen in Figures 5B and E.

[0119] In samples where healthy tissue is included in the biopsy, around the endometriotic lesion, it can be seen that FAP expression is not detectable in this healthy tissue. This finding is most clearly seen in Figures 3B (**; comparison identifier), 5B (**; comparison identifier) and 7B (**; comparison identifier), but is apparent throughout all other samples of the three subtypes of endometriosis. Conclusion

[0120] Strong expression of FAP was identified in endometriotic lesions from all endometriosis subtypes; PER, OMA and DIE. The pattern of expression is comparable between the different subtypes, which all exhibit strong expression in the center of the lesions and a weaker expression towards the periphery. In healthy tissue surrounding the lesions, no FAP expression was detected.

[0121] Example 2

[0122] A suitable FAP binding ligand, such as OncoFAP, is labelled with a radionuclide, such as Ga-68, via a chelator attached to the FAP ligand (such as DOTAGA). 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 as available from Philogen. A suitable amount (activity, for example 100MBq) of the compound is then injected intravenously in a patient with a diagnosis of endometriosis. After incubation of the radiotracer for an hour, the patient is scanned in a PET / CT or PET / MR scanner and the localization of the endometriosis lesions is visualized on the resulting images. Localization of the lesions confirms the diagnosis (for example peritoneal endometriosis) and allows for patient-individual planning of therapy (such as surgical resection). Suitable methods for translational imaging of FAP using Ga-68-DOTAGA-OncoFAP may be adapted from Backhaus, P. et al. Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA. Eur J Nucl Med Mol Imaging. 2022 May;49(6):1822-1832 and / or those investigate and discussed in trail: Onofrio Catalano and Diandrea Galloway. PET / MRI for Evaluation of Endometriosis. ClinicalTrials.gov ID NCT06377553.

[0123] Conclusion

[0124] Using FAP-PET / CT imaging, endometriosis lesions, including peritoneal endometriosis lesions, are detected. In a cohort of patients suffering from endometriosis, PET imaging with68Ga-oncoFAP detected endometriosis lesions, namely peritoneal and other abdominal lesions. Therefore, we see that PET imaging with68Ga-oncoFAP is able to identify endometriosis lesions in subjects suffering from pain of endometriosis.

[0125] Example 3

[0126] Suitable FAP ligands are labelled with a fluorescent dye (for visualization of lesions during operation) or a radioisotope (for detection of lesions by a probe). Such a compound is for example FAP-34 labeled with IRDye800CW, or99mTc-FAP-34. The compound is administered intravenously and within 24h, the patient is operated on.

[0127] Depending on the compound, the surgeon visualizes the fluorescent signal (for example with a fluorescence camera) or detects the radioactive signal with a suitable probe (shielded with a defined field of view). The surgeon is guided to the endometriosis lesions and removes the lesions. Conclusion

[0128] The use of fluorescent or radioactive FAP ligands assists the identification of endometriosis lesions that are otherwise difficult to diagnose by ultrasound and MRI. Moreover, the possibility to distinguish active endometriosis lesions is done by the use of radioactive FAP ligands.

Claims

Claims1 . A conjugate comprising a FAP targeting ligand and an effector, for use in the treatment and / or diagnosis of endometriosis.

2. The conjugate for use according to claim 1 , wherein the FAP targeting ligand is selected from the group consisting of: an antibody; a FAP inhibitor (FAPI); and a peptide.

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

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

5. The conjugate for use according to claim 3 or claim 4, wherein the linker is selected from the group consisting of acid-labile linkers, protease-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers.

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

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

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

9. The conjugate for use according to claim 8, wherein the radioactive molecule comprises a PET radioisotope (a positron emitter) or a SPECT radioisotope (gamma emitter), preferably68Ga,18F,11C,89Zr,64CU,99mTc or111ln.

10. The conjugate for use according to claim 9, wherein the PET imaging agent comprises a positron emitting radionuclide, preferably68Ga,18F,11C,89Zr,64CU.

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

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

13. A method of identifying endometriosis lesions comprising the steps of: i) introducing the conjugate according to any one of claims 6 to 11 into a subject. ii) visualizing the endometriosis lesions.

14. The method according to claim 13, additionally comprising the step of: iii) removal of the endometriosis lesions.

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

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