Treatment for endometriosis and other benign gynecological neoplasms - Patents.com

Abstract

The present invention relates to agents for the treatment and / or diagnosis of endometriosis and other benign gynecological neoplastic diseases or disorders and to agents capable of eliciting a cytotoxic response in benign gynecological neoplastic diseases or disorders.

Description

[Technical field]

[0001] This application claims priority to European patent application EP22167625.7, filed April 11, 2022, which is incorporated herein by reference.

[0002] Field The present invention relates to agents for the treatment and / or diagnosis of endometriosis and other benign gynecological neoplastic diseases or disorders, and to agents capable of eliciting a cytotoxic response in benign gynecological neoplastic diseases or disorders. [Background technology]

[0003] 2. Background of the Invention Benign gynecologic neoplasms share an overdistribution of fibroblasts; examples of such diseases include endometriosis and uterine myoma. Endometriosis has been classically defined as the ectopic presence of endometrial epithelial and stromal cells. However, recent literature has provided accumulating evidence that such a definition is insufficient, with some lesions showing a low distribution of epithelial cells. Similar studies have pointed to the fibrotic nature of the disease as a consistent feature across all stages of the disease. This has led to several calls to redefine endometriosis as a profibrotic disease (P Vigano, et al., Human Reproduction, Volume 33, Issue 3, March 2018, Pages 347-352;Sun-Wei Guo, Patrick G Groothuis, Human Reproduction Update, Volume 24, Issue 5, September-October 2018, Pages 577-598). Attempts to address fibrotic and inflammatory pathways in endometriosis through targeting TNF-a and VEGF, or other pathway interference, have met with limited preclinical and clinical success. Repeated tissue injury and repair induce irreversible differentiation of fibroblasts into myofibroblasts, and such terminal differentiation limits the responsiveness of fibroblasts to transient alterations of their signaling pathways (e.g., anti-TNF-a and TGF-B). A similar picture exists in fibroids.

[0004] FAP is part of a larger family of prolyl-specific serine proteases that have both enzymatic and signaling roles in health and disease. Although the protein has been linked to fibrosis, cancer, and inflammation in various organs, it is rarely expressed in normal tissues, making it an attractive target for treatment. This is because FAP is only expressed in tissues undergoing active injury and repair. FAP is also known to be influenced by estrogen expression in cancer, and estrogen is one of the main drivers of endometriosis. Furthermore, FAP has been shown to be involved in the PTEN / PI3K / ACT and Ras-ERK pathways, which are some of the most common genes with mutations observed in endometriosis.

[0005] Based on the above-mentioned state of the art, the object of the present invention is to provide means and methods for the treatment of diseases or disorders of endometriosis and benign gynecological neoplasms such as fibroids. This object is achieved by the subject matter of the independent claims herein, further advantageous embodiments are described in the dependent claims herein, in the examples, in the figures and in the general description. Summary of the Invention

[0006] The present inventors are the first to describe overexpression of FAP and its use in the treatment of endometriosis and associated adhesions as well as other benign gynecological neoplasms (eg, fibroids).

[0007] A first aspect of the present invention relates to an agent, a. a ligand / targeting moiety capable of specifically binding to FAP; and b. a therapeutic moiety, said therapeutic moiety having the ability to induce cell death directly, or indirectly via immune cell recruitment, of a cell that expresses a FAP on its cell surface; c. Optionally, the ligand and the immune cell engaging moiety are linked (covalently) to each other via a peptide linker; Including, In particular for use in the treatment of benign gynecological neoplastic diseases or disorders.

[0008] A second aspect of the present invention relates to an agent, wherein the agent is a. a target protein binding moiety, wherein said target protein is overexpressed in benign gynecological neoplastic cells; b. a biocidal molecule, a cytotoxic molecule, a radioisotope and / or an immune cell recruiting moiety, said immune cell recruiting moiety having the ability to specifically bind to an immune cell surface molecule, said immune cell recruiting moiety being capable of specifically binding to an immune cell, particularly a CD8 + have the ability to recruit T cells or NK cells); c. a masking moiety capable of masking said immune cell recruiting moiety and / or said target protein binding site from binding to its target. d. a cleavable linker connecting the masking moiety to another component of the agent, the cleavable linker being specifically cleavable (recognizable) by matrix metalloproteinase (MMP) 2, 9, 10, and / or 26; Includes In particular, for use in the treatment of benign gynecological neoplasms.

[0009] A third aspect of the invention relates to an agent according to any one of the preceding aspects.

[0010] A fourth aspect of the present invention relates to a composition comprising: a. a ligand capable of specifically binding to FAP; b. Radioisotope labeling Including, In particular it relates to agents for use in the diagnosis of benign gynecological neoplasms.

[0011] Terms and Definitions For purposes of interpreting this specification, the following definitions shall apply and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that the following definitions conflict with any document incorporated herein by reference, the definitions shall control.

[0012] As used herein, the terms "comprising," "having," "containing," and "including," as well as other similar forms and grammatical equivalents, are intended to be equivalent and open ended in that the item or items following any of these words are not meant to be an exhaustive list of the items or items, nor are they meant to be limited to only the items or items listed. For example, an article "comprising" components A, B, and C can consist of only components A, B, and C (i.e., only components A, B, and C), or it can include not only components A, B, and C, but also one or more other components. Thus, "comprising" and similar forms thereof, as well as their grammatical equivalents, are intended and understood to include the disclosure of an embodiment of "consisting essentially of" or "consisting of."

[0013] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is understood to be encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0014] As used herein, a reference to "about" a value or parameter includes (describes) a variation on the value or parameter itself. For example, a reference to "about X" includes a description of "X."

[0015] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization technology and biochemistry).Standard techniques are used for molecular, genetic and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0017] The term FAP in the present context relates to prolyl endopeptidase FAP or fibroblast activation protein alpha (UniProt ID Q12884).

[0018] The term CD3 in the present context relates to the T cell surface glycoprotein CD3 epsilon chain alone (UniProt ID P07766) and to the heterodimeric forms naturally occurring in humans (CD3 epsilon and CD3 delta (UniProt ID P04234) heterodimer, CD3 epsilon and CD3 gamma heterodimer (UniProt ID P09693), and CD3 epsilon and CD3 zeta heterodimer (UniProt ID P20963)).

[0019] The term CD16a in the present context relates to the low affinity immunoglobulin gamma Fc region receptor III-A (UniProt ID P08637).

[0020] The term CD8 in the present context relates to the T cell surface glycoprotein CD8 alpha (UniProt ID P01732), the T cell surface glycoprotein CD8 beta (UniProt ID P10966) and their heterodimers naturally occurring in humans (CD8 beta and CD8 alpha heterodimers).

[0021] The term Okt3 in the present context relates to a recombinant antibody targeting CD3, as described in US Pat. No. 6,750,325, which is incorporated herein by reference.

[0022] array Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the present invention. In some embodiments, sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segment hybridizes to the complement of the strand under selective hybridization conditions (e.g., under very high stringency hybridization conditions). The nucleic acid can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0023] In the present context, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter that represents the result of sequence comparison, which is determined by comparing two aligned sequences position by position.Methods for aligning sequences for comparison are well known in the art.Sequences for comparison are aligned by Smith and Waterman local homology algorithm, Adv.Appl.Math.2:482 (1981), Needleman and Wunsch global alignment algorithm, J.Mol.Biol.48:443 (1970), Pearson and Lipman similarity search method, Proc.Nat.Acad.Sci.85:2444 (1988), or computerized implementations of these algorithms, including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, for example through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0024] An example of the comparison of amino acid sequences is the BLASTP algorithm using default settings: Expect threshold: 10; Words size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. An example of such a comparison of nucleic acid sequences is the BLASTN algorithm using default settings: Expect threshold: 10; Words size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1.-2; Gap costs: Linear. Unless otherwise indicated, the sequence identity values ​​provided herein refer to values ​​obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)) using the default parameters identified above for protein and nucleic acid comparisons, respectively.

[0025] Reference to identical sequences without specifying a percentage value means 100% identical sequences (ie, the same sequence).

[0026] General Biochemistry: Peptides, Amino Acid Sequences The term polypeptide in the context of this specification relates to a molecule consisting of 50 or more amino acids, the amino acids forming a linear chain linked by peptide bonds. The amino acid sequence of a polypeptide can represent the amino acid sequence of an entire protein (as found physiologically) or a fragment thereof. The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

[0027] The term peptide in the present context relates to a molecule consisting of up to 50 amino acids, in particular of 8 to 30 amino acids, more in particular of 8 to 15 amino acids, wherein the amino acids form a linear chain linked by peptide bonds.

[0028] The sequence of amino acid residues is determined from the amino terminus to the carboxyl terminus. Capital letters at sequence positions refer to L-amino acids in one-letter code (Stryer, Biochemistry, 3 rd ed. p. 21). Lower case letters in amino acid sequence positions refer to the corresponding D- or (2R)-amino acid. Sequences are written from left to right in the amino- to carboxy-terminal direction. According to standard nomenclature, amino acid residue sequences are designated by three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0029] In the present context, the term amino acid linker or peptide linker refers to a polypeptide of variable length that is used to link two polypeptides to generate a single chain polypeptide. An exemplary embodiment of a linker useful for carrying out the invention described herein is an oligopeptide chain of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids. A non-limiting example of an amino acid linker is a monomer or di-, tri- or tetramer of tetraglycine-serine peptide linker.

[0030] General molecular biology: nucleic acid sequence, expression The terms gene expression or expression, or gene product, may refer to either or both of the processes and products of the production of nucleic acids (RNA) or peptides or polypeptides (also called transcription and translation, respectively), or intermediate processes that control the processing of genetic information to yield a polypeptide product. The term gene expression may also be applied to the transcription and processing of RNA gene products, such as regulatory RNA or structural (e.g., ribosomal) RNA. When the expressed polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells. Expression can be assayed at both the transcription and translation levels, in other words, at the level of the mRNA and / or protein product.

[0031] Binding; Binder Ligand Antibody: The term specific binding in the context of the present invention refers to the property of a ligand to bind to its target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of ≦5*10 when bound to its target. -7 mol / L, especially ≦10 -8 mol / L, more particularly ≦10 -9 They have dissociation constants in the mol / L range, but have dissociation constants at least three orders of magnitude higher when interacting with molecules that have similar overall chemical composition to the target but different three-dimensional structures.

[0032] The term aptamer refers to an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers can be created by selecting them from a large pool of random sequences. Nucleic acid aptamers can be generated through repeated rounds of in-vitro selection or SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to molecular targets, such as small molecules, proteins, or nucleic acids, via non-covalent interactions. Aptamers offer molecular recognition properties comparable to antibodies.

[0033] In the context of this specification, the term antibody refers to whole antibodies, including but not limited to immunoglobulin types G (IgG), A (IgA), D (IgD), E (IgE) or M (IgM), antigen-binding fragments or single chains thereof, and related or derived constructs. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region of IgG is composed of C H 1. C H 2. C H Each light chain is composed of three domains: a light chain variable region (referred to herein as V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses so-called nanobodies or single-domain antibodies, which are antibody fragments consisting of a single monomeric variable antibody domain.

[0034] In the present context, the term humanized antibody refers to an antibody originally produced by immune cells of a non-human species, whose protein sequence has been modified to increase similarity with the antibody variants naturally produced in humans. As used herein, the term humanized antibody includes antibodies in which CDR sequences from the germline of other mammalian species, such as llamas, have been grafted onto human framework sequences. Further modifications of the framework region may be made not only within the human framework sequences, but also within the CDR sequences from the germline of other mammalian species.

[0035] The term antibody-like molecule in the context of this specification refers to a molecule that has the ability to specifically bind to other molecules or targets with high affinity (Kd≦10E-6 mol / l). Antibody-like molecules bind to targets in a manner similar to the specific binding of antibodies. The term antibody-like molecule encompasses repeat proteins such as designed ankyrin repeat proteins (Molecular Partners, Zuerich), engineered antibody mimetic proteins that exhibit highly specific and high affinity target protein binding (see US Patent Application Publication No. 2012 / 142611, US Patent Application Publication No. 2016 / 250341, US Patent Application Publication No. 2016 / 075767 and US Patent Application Publication No. 2015 / 368302, all of which are incorporated herein by reference). The term antibody-like molecule further encompasses, but is not limited to, polypeptides derived from armadillo repeat proteins, polypeptides derived from leucine-rich repeat proteins, and polypeptides derived from tetratricopeptide repeat proteins. The term antibody-like molecule further encompasses protein A domains, fibronectin domain FN3, consensus fibronectin domains, lipocalins (see Skerra, Biochim. Biophys. Acta 2000, 1482(1-2):337-50), polypeptides derived from zinc finger proteins (see Kwan et al. Structure 2003, 11(7):803-813), Src homology domain 2 (SH2) or Src homology domain 3 (SH3), PDZ domains, gamma-crystallin, ubiquitin, cysteine ​​knot polypeptides or knottins, cystatins, Sac7d, triple helical coiled coils (also known as alpha bodies), Kunitz domains or Kunitz-type protease inhibitors, and carbohydrate binding module 32-2.

[0036] The term Protein A domain derived polypeptide refers to a molecule that is a derivative of Protein A and has the ability to specifically bind to the Fc and Fab regions of immunoglobulins.

[0037] Armadillo repeat protein refers to a polypeptide that includes at least one armadillo repeat, which is characterized by a pair of alpha helices that form a hairpin structure.

[0038] A single domain antibody refers to a heavy chain only antibody, a VHH, or a nanobody.

[0039] The term single-chain variable fragment (scFv) refers to a recombinant molecule in which the variable regions of an immunoglobulin light chain and an immunoglobulin heavy chain encoding an antigen-binding domain have been engineered into a single polypeptide.

[0040] The term humanized camelid antibody in the context of this specification refers to an antibody consisting only of the heavy chain or the variable region of the heavy chain (VHH domain) whose amino acid sequence has been modified to increase its similarity to antibodies naturally produced in humans, and which therefore exhibits reduced immunogenicity when administered to humans. A general strategy for humanizing camelid antibodies is described in Vincke et al. "General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold", J Biol Chem. 2009 Jan 30;284(5):3273-3284, and in US Patent Application Publication No. 2011 / 165621.

[0041] In the context of this specification, the term fragment crystallizable (Fc) region is used in the sense known in the art of cell biology and immunology, which, as applied to IgG, refers to the C domains covalently linked by disulfide bonds. H 2 domain and C H Refers to a fraction of antibodies that contain two identical heavy chain fragments composed of three domains.

[0042] As used herein, the term pharmaceutical composition refers to the compound of the present invention or a pharma- ceutically acceptable salt thereof, together with at least one pharma- ceutically acceptable carrier. In one embodiment, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral or injectable administration.

[0043] As used herein, the term pharma- ceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, known to those of ordinary skill in the art (see, e.g., Remington. the Science and Practice of Pharmacy, ISBN 0857110624).

[0044] As used herein, the term treating or treatment of any disease or disorder (e.g., endometriosis) refers, in one embodiment, to improving the disease or disorder (e.g., delaying or preventing or alleviating the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. The present methods for assessing the treatment and / or prevention of disease are generally known in the art, unless otherwise noted below.

[0045] The term immune cell in the context of this specification relates to a cell of the immune system that has the ability to kill (directly or indirectly) and / or induce apoptosis of cells of the living body. Examples of immune cells are CD8+ cells or natural killer cells (NK cells).

[0046] The term immune cell surface molecule in the context of this specification relates to molecules, especially proteins, that are located on the cell surface of an immune cell and are accessible to other moieties.

[0047] The term recruiting immune cells in the context of this specification relates to the ability of the molecules of the invention to bring immune cells into intimate contact with endometrial cells, thereby recruiting the immune cells.

[0048] The term bulk protein in the present context relates to any protein that is present in large amounts in blood (more than 1% of blood). Examples of bulk proteins are albumin, globulins, fibronogen.

[0049] The term detectable expression in the context of this specification relates to the amount of protein expressed by a cell. A protein is considered detectable expression if it is detectable on the surface of at least a subset of cells by standard cell biology methods.

[0050] The term shielding in the present context relates to the property of a masking moiety, which can non-covalently interact with a particular portion of an agent, thereby preventing this other portion from binding to the actual target, as long as the masking moiety is covalently attached to the agent.

[0051] The term endometriosis or ectopic endometrium in the context of this specification refers to any disease in which cells similar to those in endometrium (the layer of tissue that normally lines the uterus) grow outside the uterus.The most common site of endometriosis is the ovary, followed by the pouch of Douglas, the posterior broad ligament, and the sacrouterine ligament.Endometriosis in the present invention refers to all subtypes or alternative names, including but not limited to adenomyosis, endometrioma, deep infiltrating endometriosis, superficial peritoneal endometriosis, and abdominal wall endometriosis.

[0052] The terms uterine fibroids, leiomyomas, or myomas are fibroids and uterine myomas that occur in and around the female reproductive tract. Uterine fibroids are often combined with endometriosis. The primary cell population of uterine fibroids is similar to that of endometriosis, but the initial cells are myometrial cells, in contrast to the endometrial epithelial cells in endometriosis.

[0053] The term benign gynecologic neoplasm or neoplastic disease refers to the abnormal growth of non-cancerous cells. Benign gynecologic neoplasm refers to cases where tissues usually found in and around the female reproductive organs grow abnormally in the organ itself or in other organs. For example, endometriosis occurs when a population of cells like the lining of the uterus grow abnormally in other organs, causing pain and other symptoms to the patient. Fibroids or uterine fibroids are other examples of benign gynecologic neoplasms.

[0054] Detailed Description of the Invention A first aspect of the present invention relates to an agent comprising: a. a ligand / targeting moiety capable of specifically binding to FAP; and b. a therapeutic moiety, the therapeutic moiety capable of inducing cell death directly, or indirectly through the recruitment of immune cells, of a cell that expresses a FAP on its cell surface; c. Optionally, the ligand and the immune cell recruiting moiety are linked (covalently) to each other via a peptide linker; and particularly for use in the treatment of a benign gynecological neoplastic disease or disorder.

[0055] In certain embodiments, the therapeutic moiety is capable of inducing cell death in a cell that expresses a FAP on its cell surface. The induction of cell death can be achieved through different mechanisms.

[0056] In certain embodiments, the therapeutic moiety is a biocidal molecule, a cytotoxic molecule, and / or an immune cell recruiting moiety, which has the ability to specifically bind to an immune cell surface molecule (wherein the immune cell recruiting moiety specifically binds to immune cells, particularly CD8 + (has the ability to recruit T cells or NK cells).

[0057] In certain embodiments, the therapeutic moiety is an immune cell engaging moiety, the immune cell engaging moiety having the ability to specifically bind to an immune cell surface molecule.

[0058] In one embodiment, the immune cell surface molecule is a) CD3; b) CD16; c) CD8; is selected from.

[0059] In one embodiment, the immune cell surface molecule is CD3.

[0060] In some embodiments, the immune cell recruiting moiety is selected from the group comprising antibodies, antibody fragments, single chain antigen binding fragments, aptamers, non-immunoglobin scaffolds, small molecules, and antibody-like molecules. In some embodiments, the immune cell recruiting moiety is a single domain antibody. In some embodiments, the immune cell recruiting moiety is selected from the group comprising antibodies, antibody fragments, single chain antigen binding fragments. In some embodiments, the immune cell recruiting moiety is a single domain antibody.

[0061] In some embodiments, the therapeutic moiety is a cytotoxic molecule with a molecular weight <1000 Daltons (Da). In some embodiments, the therapeutic moiety is a cytotoxic molecule with a molecular weight <800 Da. In some embodiments, the therapeutic moiety is a cytotoxic molecule with a molecular weight <500 Da.

[0062] In certain embodiments, the cytotoxic molecule is selected from the group comprising doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine, leurocidin, vindesine, estramustine, cisplatin, cyclophosphamide, leurosine, taxol, desacetylvinblastine, cyclophosphamide, ifosfamide, cytarabine, 6-thioguanine, chlorambucil, carmustine, mitoxantrone, paclitaxel or cytotoxic derivatives thereof.

[0063] In one embodiment, the ligand capable of specifically binding to a FAP is selected from the group comprising antibodies, antibody fragments, single-chain antigen-binding fragments, aptamers, non-immunoglobin scaffolds, antibody-like molecules, single domain antibodies, small molecule pharmaceuticals (1000 Da or less, particularly 800 Da or less, more particularly 500 Da or less) according to the Lipinski Rules of Five criteria, small peptides, and FAP-specific substrates (e.g., (poly)peptide-proline-leucine / asparagine / aminotrifluoromethylcoumarin-(poly)peptide). In one embodiment, the ligand is selected from the group comprising antibodies, antibody fragments, and single-chain antigen-binding fragments. In one embodiment, the ligand is a single domain antibody.

[0064] In some embodiments, the agent comprises a masking moiety attached (covalently linked) to the remainder of the agent via a linker, which has the ability to reduce or eliminate the binding ability of the ligand and / or the immune cell engaging moiety (and prevent the immune cell binding moiety or the ligand from binding to its target). In some embodiments, the linker comprises a protease cleavage site (cleavable linker).

[0065] The masking approach is an addition that may allow for a better therapeutic window.

[0066] In some embodiments, the agent comprises a bulk protein binding site, and the binding site is attached (covalently linked) to the remainder of the agent via a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the bulk protein binding site is a human serum albumin binding site.

[0067] In certain embodiments, the protease cleavage site is specifically cleavable (recognizable) by matrix metalloproteinases 2, 9, 10, and / or 26.

[0068] In some embodiments, two, three, or all of the components of the agent are covalently linked to one another via a peptide linker. In some embodiments, each peptide linker sequence is selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:15, ERK, (GS) n (SEQ ID NO: 18), (G2S) n (SEQ ID NO: 19), (G3S) n (SEQ ID NO: 20), (G4S) n (SEQ ID NO:21), where n is an integer selected from 1 to 10, G is glycine, S is serine, E is glutamic acid, R is arginine, and K is lysine. In certain embodiments, n is 1, 2, 3, or 4.

[0069] In certain embodiments, the benign neoplastic tissue is characterized by detectable expression of the target protein, hi certain embodiments, at least 1% of benign neoplastic cells express the target protein.

[0070] In one embodiment, said ligand capable of specifically binding to a FAP is characterized by the sequence SEQ ID NO:16.

[0071] In one embodiment, the immune cell surface molecule is CD3 and the therapeutic moiety is characterized by the sequence of SEQ ID NO:17.

[0072] A second aspect of the present invention relates to an agent comprising: a. a target protein binding moiety, where the target protein is overexpressed in benign gynecological neoplastic cells; b. Biocidal molecules, cytotoxic molecules, radioisotopes and / or immune cell recruiting moieties, which have the ability to specifically bind to immune cell surface molecules (the immune cell recruiting moieties are capable of binding to immune cells, particularly CD8 + have the ability to recruit T cells or NK cells); c. a masking moiety capable of masking the immune cell engaging moiety and / or the target protein binding moiety from binding to its target; d. a cleavable linker connecting said masking moiety and said other components of said agent, said cleavable linker being specifically cleavable (recognizable) by matrix metalloproteinases (MMPs) 2, 9, 10, and / or 26; and in particular for use in the treatment of benign gynecological neoplasms.

[0073] In one embodiment of the second aspect of the invention, the cleavable linker is specifically cleavable (recognizable) by MMP2 and / or 9.

[0074] In one embodiment of the second aspect, the cleavable linker is characterized by a sequence selected from the group of SEQ ID NOs: 1-4.

[0075] In certain embodiments of the second aspect, the masking moiety is capable of shielding the immune cell engaging moiety from binding to its target.

[0076] In certain embodiments, the benign gynecological neoplastic disease is endometriosis. In certain embodiments, the benign gynecological neoplastic disease is the development of uterine fibroids.

[0077] In certain embodiments, the agent is formulated for subcutaneous or intravenous injection.

[0078] A third aspect of the invention relates to an agent as defined in any one of the preceding aspects.

[0079] A fourth aspect of the present invention is a. A ligand capable of specifically binding to FAP b. Radioisotope labeling In particular, for use in the diagnosis of benign gynecological neoplasms.

[0080] In one embodiment, the radioisotope label is 94m Tc, 186 Re, 203 Pb, 47 Sc, 111 In, 97 Ru, 62 Cu, 88 Y, 121 Sn, 161 Tb, 153 Sm, 166 Ho, 105 It is selected from the group including Rh.

[0081] In certain embodiments, the benign gynecological neoplastic disease is endometriosis. In certain embodiments, the benign gynecological neoplastic disease is the development of uterine fibroids.

[0082] The agent or conjugate thereof for the above may be for use in imaging, detection, diagnosis of benign neoplasms.

[0083] In one embodiment, the diagnosis is an in vivo diagnosis.

[0084] In one embodiment, the diagnosis is radiography. In one embodiment, the diagnosis is selected from the group of X-ray, PET, CT.

[0085] array

[0086] [Table 1]

[0087] [Table 2]

[0088] Medical Treatment Also within the scope of the present invention is a method for treating a benign gynecological neoplasm in a patient in need thereof, comprising administering to said patient an agent according to the above description.

[0089] In certain embodiments, the agent is an antibody, an antibody fragment, an antibody-like molecule, or a polypeptide derived from a Protein A domain.

[0090] In some embodiments, the polypeptide agent is an immunoglobulin consisting of two heavy chains and two light chains.In some embodiments, the polypeptide ligand is a single domain antibody consisting of the variable domains isolated from the heavy or light chain.In some embodiments, the non-agonist polypeptide ligand is a heavy chain antibody consisting of only the heavy chain, such as the antibodies found in camelids.

[0091] In certain embodiments, the agent is an antibody fragment. In certain embodiments, the polypeptide agent is a Fab fragment, i.e., the antigen-binding fragment of an antibody, or a single chain variable fragment, i.e., a fusion protein of the heavy and light chain variable regions of an antibody linked by a peptide linker.

[0092] Pharmaceutical Compositions, Administration / Dosage Forms and Salts According to one compound aspect of the invention, a compound according to the invention is provided as a pharmaceutical composition, pharmaceutical dosage form, or pharmaceutical dosage form.

[0093] In certain embodiments of the present invention, the compounds of the present invention are typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the agent and to present the patient with a simple, easily administered product.

[0094] Similarly, there is provided a dosage form for the prevention or treatment of endometriosis comprising a non-agonist ligand or an antisense molecule according to any of the above aspects or embodiments of the invention.

[0095] The present invention further includes a pharmaceutical composition comprising a compound of the present invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharma- ceutically acceptable carriers, as described herein.

[0096] Certain embodiments of the invention relate to dosage forms for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection dosage forms. Optionally, pharma- ceutically acceptable carriers and / or excipients may be present.

[0097] The dosage regimen for the compounds of the present invention will vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration, the recipient's species, age, sex, health, disease state and weight, the nature and extent of symptoms, type of concurrent treatment, frequency of treatment, route of administration, renal and hepatic function of the patient, and the desired effect. In some embodiments, the compounds of the present invention may be administered in a single dose per day, or the total daily dosage may be administered in divided doses, two, three or four times a day.

[0098] In one embodiment, the pharmaceutical composition or combination of the present invention may be a unit dose of about 1-1000 mg of active ingredient for a subject weighing about 50-70 kg. The therapeutically effective dose of the compound, pharmaceutical composition, or combination thereof depends on the species, weight, age, and individual condition of the subject, the disorder or disease being treated, or its severity. A physician, clinician, or veterinarian of ordinary skill can readily determine the effective amount of each active ingredient required to prevent, treat, and inhibit the progression of a disorder or disease.

[0099] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization, and / or can contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. They can be manufactured by standard processes, such as conventional mixing, granulation, dissolution, or lyophilization processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, L. Lachman et al., Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

[0100] Methods of manufacture and treatment according to the present invention The invention further includes, as an additional aspect, the use of an agent identified herein, or a pharma- ceutically acceptable salt thereof, as described in detail above, for use in a method for the manufacture of a medicament for the treatment or prophylaxis of benign gynecological neoplasms, in particular endometriosis or uterine fibroids.

[0101] Similarly, the invention encompasses a method of treating a patient diagnosed with a disease associated with benign gynecological neoplasms, particularly endometriosis or uterine fibroids, which method involves administering to the patient an effective amount of an agent identified herein, or a pharma- ceutically acceptable salt thereof, as described in detail herein.

[0102] For example, when alternatives of a single separable feature, such as the type of linker or the ligand protein or medical indication, are presented herein as "embodiments," it is understood that such alternatives may be freely combined to form separate embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for the type of linker may be combined with any of the alternative embodiments for the ligand, and these combinations may be combined with any medical indication suggested herein.

[0103] The present application further includes the following:

[0104] item 1. An agent comprising: a. a ligand capable of specifically binding to FAP; and b. a therapeutic moiety, the therapeutic moiety capable of inducing cell death in a cell that expresses a FAP on its cell surface; Including, Agent for use in the treatment of benign gynecological neoplastic disease. 2. The agent for use according to item 1, wherein the therapeutic moiety is an immune cell recruiting moiety, and the immune cell recruiting moiety has the ability to specifically bind to an immune cell surface molecule. 3. The immune cell surface molecule is selected from the group consisting of: a. CD3; b. CD16; c.CD8; is selected from In particular, the agent for use according to item 2, wherein the immune cell surface molecule is CD3. 4. The immune cell recruiting moiety is selected from the group including an antibody, an antibody fragment, a single chain antigen-binding fragment, a single domain antibody, an aptamer, a non-immunoglobin scaffold, and an antibody-like molecule; In particular, said immune cell recruiting moiety is selected from the group comprising antibodies, antibody fragments, single chain antigen binding fragments, More particularly, the agent for use according to any one of items 2 or 3, wherein said immune cell recruiting moiety is a single domain antibody. 5. The therapeutic moiety is a cytotoxic molecule having a molecular weight <1000 Daltons (Da), particularly a molecular weight ≦800 Da, more particularly a molecular weight ≦500 Da; 6. The agent for use according to item 5, wherein said cytotoxic molecule is selected from the group comprising doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine, leurocidin, vindesine, estramustine, cisplatin, cyclophosphamide, leurocidin, taxol, desacetylvinblastine, cyclophosphamide, ifosfamide, cytarabine, 6-thioguanine, chlorambucil, carmustine, mitoxantrone, paclitaxel or cytotoxic derivatives thereof. 7. The ligand is selected from the group including antibodies, antibody fragments, single-chain antigen-binding fragments, single domain antibodies, aptamers, non-immunoglobin scaffolds, and antibody-like molecules, small molecule drugs following the Lipinski Rules of Five set of criteria, peptides, and FAP-specific substrates; In particular, said ligand is selected from the group comprising antibodies, antibody fragments, single chain antigen-binding fragments, More particularly, the agent for use according to any one of the above items, wherein said ligand is a single domain antibody. 8. An agent for use according to any one of the preceding items, wherein the agent comprises a masking moiety attached to the agent via a linker, the masking moiety having the ability to reduce or eliminate the binding ability of the ligand and / or the immune cell recruiting moiety, in particular wherein the linker comprises a protease cleavage site. 9. An agent for use according to any one of the preceding items, wherein the agent comprises a bulk protein binding site, in particular a human serum albumin binding site, which is attached to the agent via a linker, in particular the linker comprises a protease cleavage site. 10. The agent for use according to item 8 or 9, wherein the protease cleavage site is specifically cleavable by matrix metalloproteinases 2, 9, 10, and / or 26. 11. Two, three or all of the components of the agent are covalently linked to each other via a peptide linker, and in particular each peptide linker sequence is selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:15, ERK, (GS) n (SEQ ID NO: 18), (G2S) n (SEQ ID NO: 19), (G3S) n (SEQ ID NO: 20), (G4S) n (SEQ ID NO: 21), wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. 12. The agent for use according to any one of the preceding items, wherein the endometriotic tissue is characterized by detectable expression of FAP, in particular, at least 1% of endometriotic cells express FAP. 13. An agent for use according to any one of the preceding items, wherein the ligand capable of specifically binding to FAP is characterized by the sequence of SEQ ID NO: 16. 14. The agent for use according to any one of items 2 to 4 or 7 to 13 above, wherein the immune cell surface molecule is CD3 and the therapeutic moiety is characterized by the sequence of SEQ ID NO: 17. 15. An agent for use in the treatment of a benign gynecological neoplastic disease, comprising: a. a target protein binding moiety; b. Immune cell recruitment part; c. a masking moiety capable of masking the binding of said immune cell engaging moiety and / or said target protein binding moiety to its target; d. a cleavable linker connecting the masking moiety to another component of the agent, the cleavable linker being specifically cleavable by matrix metalloproteinases (MMPs) 2, 9, 10, and / or 26. A formulation comprising: 16. The agent for use according to item 15, wherein the cleavable linker is specifically cleavable by MMP2 or MMP9. 17. The agent for use according to item 15 or 16, wherein the cleavable linker is characterized by a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4. 18. The agent for use according to any one of items 15 to 17, wherein the masking moiety has the ability to mask the immune cell recruiting moiety from binding to its target. 19. The agent for use according to any one of the above items, wherein the benign gynecological neoplastic disease is endometriosis. 20. The agent for use according to any one of items 1 to 18 above, wherein the benign gynecological neoplastic disease is the development of uterine fibroids. 21. The agent for use according to any one of the preceding items, wherein the agent is formulated for subcutaneous or intravenous injection. 22. An agent as specified in any one of the above items. 23. A drug comprising: A ligand capable of specifically binding to FAP; b. Radioisotope labeling A formulation comprising: 24. The radioisotope label 94m Tc, 186 Re, 203 Pb, 47 Sc, 111In, 97 Ru, 62 Cu, 88 Y, 121 Sn, 161 Tb, 153 Sm, 166 Ho, 105 24. The agent according to item 23, selected from the group including Rh. 25. The agent according to item 23 or 24 for use in diagnosing a benign gynecological neoplastic disease. 26. The agent for use according to item 25, wherein the benign gynecological neoplastic disease is endometriosis or uterine fibroids. 27. The agent for use according to claims 25 to 26, wherein the diagnosis is in vivo diagnosis. 28. The agent for use according to claims 25 to 27, wherein the diagnosis is X-ray imaging, in particular the diagnosis is selected from the group of X-ray, PET, CT.

[0105] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived, which are intended to illustrate the invention but not to limit its scope. [Brief description of the drawings]

[0106] Text description of the illustration image024.gif. [Figure 1] Figure 1 shows single-cell RNA-seq showing unbiased cellular composition of ectopic, orthotopic and control endometrial samples. Fibroblasts are overdistributed in ectopic and orthotopic endometrium of women with endometriosis, much higher than in normal control healthy orthotopic endometrium. Data from Tan et al., 2021 (https: / / doi.org / 10.1101 / 2021.07.28.453839) were reanalyzed for this figure. [Diagram 2] FIG. 2 shows the design applied in this disclosure. [Diagram 3]Figure 3 shows a diagram illustrating an exemplary embodiment of a masking approach to protect immune cell binding domains. The bottom plot shows the masking approach and that the activatable form of the treatment has no binding or cell killing capacity, but can be restored to binding and cell killing capacity upon cleavage and activation. [Figure 4] Figure 4 shows the FAP phenotype panel. -PE569 ovarian endometrioma (OMA). PE569 cultured ovarian endometrioma stromal cells. Gating strategy for PE569 OMA shown. CD90-APC, FAP-PE, L / D-ZombieV. CD44-APC, CD45-BV605, CD105-AF488. L / D-ZombieV. [Diagram 5] Figure 5 shows orthotopic versus ectopic endometrial tissues of FAP antigen density. Qifikit was used for quantitative determination of cell surface FAP antigen by flow cytometry using direct immunofluorescence assay. Antigen density was expressed in Specific Antibody-Binding Capacity (SABC) units. Samples analyzed from lesions (ectopic): PE569_DIE (deep infiltrating endometriosis), PE569_OMA (ovarian endometrioma), PE571_ADH (ovarian endometrioma adhesions), OVCAR-3 (human ovarian cancer cell line). Samples analyzed from orthotopic: PE564, PC573, PC568. Low surface expression was observed in orthotopic endometrium. [Figure 6] FIG. 6 shows that the average orthotopically had 13% FAP-positive cells and the average ectopic had over 83% FAP-positive cells. [Figure 7] Figure 7 shows that expression of FAP and CD90 is evident in stromal cells compared to epithelial cells: MFI and expression levels-phenotypes of PE575 and PE576. [Figure 8] FIG. 8 shows an ex vivo killing assay: patient-derived lesion organoids were co-cultured with immune cells and antibodies for 24 h. [Figure 9] FIG. 9 shows the killing assay CellTiterGlo PC571 using the FAP BITE. [Figure 10]Figure 10 shows the (bright field) microscopic killing assay. Orthotopic endometrial cells - Passage 0 PC571. [Figure 11] Figure 11: (Bright field) microscopic killing assay. Primary cells derived from endometriotic tissue - passage 1 PE571. [Figure 12] Figure 12 shows OVCAR3+ T cells (endometriosis patients) in NOG mice. In vivo study in mice: delayed implant growth for 2 weeks. Mice were implanted with an aggressive tumor cell line expressing the target protein. Treatment was performed 6 times between d7 and d14. T cells were administered twice a week at a dose of 0.1 mg / kg. Tumor volume was measured every 3 days. [Figure 13] FIG. 13 shows the immune response against patient samples, indicating activity of specific constructs against FAP+ endometriotic cells (1:1 mix of epithelial and stromal cells). [Figure 14] FIG. 14 shows flow cytometry data showing a high percentage of FAP+ cells in uterine myoma patient lesions. [Figure 15] FIG. 15. MMP26 serum levels from women with endometriosis, uterine fibroids and healthy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] Working Example As part of a newly launched clinical study (ethics approval number: BASEC2020-02117), to date, 62 women have contributed samples to our study. Samples were collected from women with endometriosis and from women undergoing gynaecological surgery for interventions not related to endometriosis (e.g. uterine fibroids and tubal ligation). For women with endometriosis, the following samples were collected: endometriotic lesions, endometriotic adhesions (if present), and orthotopic endometrium. For the control group, orthotopic endometrium and diseased tissues (e.g. fibroids) if present are collected. Blood is collected for all women and samples are collected and processed according to WERF SOPs (as far as possible). Two additional ethical approvals (BASEC2020-02272) and (BASEC-2021-00885) were obtained for healthy buffy coat and in vivo xenograft studies. The median age of participants to date is 35 years old.

[0108] Example 1: Target Verification We demonstrated for the first time that FAP protein is expressed on the surface of ectopic endometrium as well as endometriosis-associated adhesions in endometriosis patient samples. Depending on the sample, the percentage of FAP+ stromal cells was 67.3% in ovarian endometriosis, 98.1% in deep infiltrating endometriosis, and 85.1% in endometriotic adhesions. In comparison, orthotopic endometrial fibroblasts expressed an average of 13.8% FAP. The epithelial cancer cell line OVCAR3 was 13.60% FAP-positive (see phenotype (Figure 6)). Using a two-tailed t-test, ectopic endometrial stromal cells were found to have a significantly higher percentage of FAP+ cells.

[0109] Using immunohistochemistry, most healthy tissues did not show any FAP expression (see supplementary material in Schuberth et al., 2013, J Transl Med. 11: 187.), and in healthy endometrium, FAP expression was at very low levels in a subset of fibroblasts (see Figures 5-6). Furthermore, we confirm low expression of FAP in the healthy uterus. Differential expression of FAP distinguishes between tissue remodeling associated with the endometrial cycle and active ectopic tissue fibrosis. It also expresses FAP together with epithelial cells during retrograde menstruation. + This highlights the potential origin of ectopic fibroblasts that may carry the phenotype from the orthotopic endometrium to ectopic regions.

[0110] Next, we used flow cytometry (QIFIKIT) to assess the surface copy number of FAP in orthotopic endometrium, ectopic endometrium, and adhesions. The surface copy number of FAP was 20-35 times higher in ectopic endometrium and adhesions of women with endometriosis compared to orthotopic endometrium of women with endometriosis. More specifically, the copy number in orthotopic endometrium was less than 1000, whereas the copy number was between 25000-35000 surface copies in ectopic endometrium and adhesions. To make the numbers easier to understand, healthy hepatocytes that show very low expression immunohistochemically have less than 1000 FAP surface copies. Similar differences in surface density have been noted in cancer immunotherapy, with responders and non-responders showing more than 10000 and less than 2000 copies of the target antigen, respectively.

[0111] The inventors have also shown that uterine fibroids, like endometriosis, are more likely to be FAP positive (Figure 14).

[0112] Example 2: Design of bispecific antibodies for the cytotoxic destruction of endometriotic FAP-positive fibroblasts We designed bispecific antibody constructs to target endometriosis hyperactivated fibroblasts that are FAP positive. These constructs follow the design in Figure 2, where FMC-2 is a bispecific antibody targeting FAP. The design of FMC-2, which we used to generate the data presented here, is based on scFvFAP (-scFvCD3(OKT3)) from the public domain. We generated our own nanobodies (single domain antibodies) against FAP, CD3 and CD16 in alpacas and obtained over 250 positive binders. Our goal is to utilize our unique nanobodies to design 1-2 final lead designs optimized for safety, efficacy, developability and manufacturability.

[0113] The first safety feature is a masking strategy with endometriosis-specific cleavage. The basic principle is to introduce a moiety that prevents one or more antibody domains from binding to the target until the antibody domain fits into the appropriate (endometriotic) microenvironment. Briefly, the masked antibody in circulation has no cytotoxic effect, and upon encountering a defined microenvironment, the linker sensitive to a specific protease is cleaved together with the masking moiety, exposing the binding domain and activating the antibody. Specifically for endometriosis, we designed four different linkers sensitive to enzymes overexpressed in the endometriotic microenvironment. As a first example, we demonstrated the overexpression of MMP26 in patient samples compared to fibroid patients and healthy donors (Figure 15, p-values ​​<0.05 and <0.001, respectively). Another enzyme for which we tested linkers is MMP2, which has been shown to be overexpressed, specifically over 9-fold, in endometriotic fibroblasts compared to control orthotopic fibroblasts (p-value <0.001 under Bonferroni correction, reanalysis of GSE168902 using standard R limma pipeline). MMP2 is also known to contribute to fibrosis and is one of the protein interaction partners of FAP (String-db, v11.5).

[0114] Example 3: Observations Current targeting approaches for FAP are primarily via small molecules, which are not necessarily specific for FAP (due to its 52% homology with another protein) and which are prone to cyclization and inactivation (Fang J et al. 2016;138(4):1013-1023. doi:10.1002 / ijc.29831). Our unique approach is to target the ectodomain of the protein using single domain antibodies, leveraging their ability to access epitopes that are difficult to access with Ig-like antibodies. We are starting with a selection of over 100 strong binders that we have generated in alpaca against FAP. FAP shares up to 62% of its sequence with other known enzymes, and it is hypothesized that previous ineffectiveness observed in cancer may be due to cross-reactivity and non-catalytic activity of FAP. Our nanobody program and selection approach avoids non-specific binding, optimizing antibody safety while ensuring specificity against active fibrosis / adhesion.

[0115] FMC-2 is our bispecific antibody program targeting FAP. The current generation of FMC-2 (scFvFAP-scFvCD3) is >90% FAP + FMC-2 showed high efficacy in destroying patient-derived stromal cells (>90% destruction in 12 hours, 1:1 E:T ratio). Compared to endometrial lesions, >80% of orthotopic endometrial fibroblasts survived 24 hours after treatment with FMC2 under the same conditions (Figure 10). FMC-2 was also highly effective in destroying endometriosis-associated adhesions, removing adhesion cells in less than 6 hours (1:1 E:T ratio). Surgery has been shown to be effective in alleviating pain (despite a high recurrence rate), but is also the main cause of peritoneal adhesions.

[0116] FMC-2 has demonstrated strong efficacy against stromal cells and adhesions derived from endometriosis patients. FMC-2 has great potential to potentially reduce the need for surgery and delay hysterectomy in women with endometriosis-related fibrosis and adhesions. Furthermore, FMC-2 has been shown to be effective in treating FAP. + It may also have potential applications in cardiac fibrosis and peritoneal adhesions associated with general surgery, which occurs in up to 93% of operations.

[0117] Example 4: Materials and Methods Single Purified Cell Suspension Protocol Collagenase I / II Digestion Mix:

[0118] [Table 3]

[0119] Estimate the volume of tissue needed for digestion in ml. Use 3 ml of digestion mix per lesion. Weight of biopsy tissue Shake gently to remove excess blood and mucus. Centrifuge at 300g for 5 minutes. For endometriosis, PBS (Mg 2+ / Ca 2+ Wash once with (without). Centrifuge at 300 x g for 5 minutes. · Cut the first fragment without medium and transfer it to a retrospective tube to preserve the tissue for fixation, freezing, etc. Transfer the biopsy to a petri dish containing 3mL of digestion medium. For fibroids use 6mL. Add digestion medium over the tissue and digest: Cut the fresh tissue into 1mm pieces using forceps and scissors in digestion medium. 3 Finely chop into small pieces. Using a 25 ml stripper, transfer the pieces and the digestion medium into a gentleMACS flask (C tube). Rinse the mincing dish with 1-5 mL of Digestion Medium and add to the gentleMACS Flask. Incubate for 40 minutes at 37°C in the gentleMACS program. Do not open the flask after the gentleMACS program has finished until centrifugation. Centrifuge the samples at room temperature for 5 minutes at 300 x g. Gently remove the digestion medium with a pipette or stripper. Mg 2+ / Ca 2+ Wash the fibroids with PBS containing no ethanol (until the supernatant becomes transparent). Centrifuge at 300g for 5 minutes and remove the supernatant. · Resuspend in 1-3 ml of tryple and Rock Inhibitor (10 μl / ml) and transfer to a new 15 ml tube. Incubate at 37°C for 15 minutes in a 37°C shaker. Stop the digestion by adding 14 ml of PBS. PBS(Mg 2+ / Ca 2+ Wash once with (without). Centrifuge at 300g for 5 minutes. Remove the supernatant. 5 ml PBS (Mg 2+ / Ca 2+ (none). Filter the digestion medium through a 70 mm cell strainer. o Collect the flow-through fraction; o The backwash fraction is collected in 5 ml of culture medium and plated in a dish. Centrifuge at 300g for 5 minutes. 1-10ml of PBS (Mg 2+ / Ca 2+ Resuspend in 0.5% CO (no trypan blue) and count viable cells (trypan blue). Centrifuge at 300g for 5 minutes. Add 1-3 ml of ACK lysis buffer. Incubate at RT for 8 min. Fill the tube with PBS (15 ml) to stop the reaction. Centrifuge at 300g for 5 minutes. Mg 2+ / Ca 2+ Wash once with PBS without. · Pellets can be reconstituted or frozen.

[0120] Organoid Media material · DMEM F-12(+L-Glutamine,+Hepes,+Phenol Red);Gibco TM ;#11330032 B27 supplement 10ml; Gibco TM ;#12587-010(Stock 50X;-20C) N-2 Supplement (100X) - 5mL; Gibco TM ;#17502048 (Stock 100X;-20C) A-83-01; Sigma Aldrich; #SML0788-5MG (Stock 5000X-2500μM; -20C) SB -202190;Sigma Aldrich;#S7067-5MG (Stock 2500X-25000μM;-20C) Recombinant human EGF, CF; Bio-Techne AG; #236-EG-01M (stock 100 μg / mL; -80C) Recombinant human Noggin; Peprotech; #120-10C-20uG (stock 200μg / mL; -80C) Recombinant human FGF basic (146aa); Bio-Techne AG; #233-FB-025 (stock 4μg / mL; -80c) Penicillin-Streptomycin; Sigma Aldrich; #P4333-100ML (Stock 100X; -20°C) Insulin-Trans-Cell-G, 100X; Gibco TM ;#41400045(Stock 100X;4C) GlutaMAX Supplement - 100mL; Gibco TM ;#35050061(Stock 100X;4C) Human FGF-10; Pepro Tech EC Ltd; #100-26-100uG (Stock 25mg / mL; -20C) · B-Estradiol; Sigma Aldrich; #E8876-1G (stock 10mg / mL; 4c) N-Acetyl-L-Cysteine ​​- SIGMA - GRADE; Sigma Aldrich; #A7250 - 50mg (Stock 100μg / mL) Y-27632 dihydrochloride (100MG); AbMole BioScience; #M1817 (Stock 1mM; -20C) RSPO1 conditioned medium; homemade (5%; -20C) NICOTINAMIDE;Sigma Aldrich;#72340-100G (Stock 1M;-20C) Recombinant human HGF; Peprotech; #100-39-100UG (stock 100μg / mL; -20℃).

[0121] procedure Thaw reagents on ice Prepare medium on ice according to Boretto et al., 2019: Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening. Use the medium within one week. Filter the medium (0.22μm) before use For 50mL use: DMEM F-12: 41.1 mL B27 supplement 1ml N-2 supplement 500 μl A-83-01: 10μl SB-202190: 20μl Recombinant human EGF, CF: 25 μl Recombinant human Noggin: 20μl Recombinant human FGF basic (146aa): 25μl Penicillin-streptomycin: 500 μl Insulin-Trans-Cell-G: 500 μl GlutaMAX supplement: 500μl Human FGF-10: 20 μl B-estradiol: 20μl N-acetyl-L-cysteine: 102 μl Y-27632 dihydrochloride: 500 μl RSPO1 conditioned medium: 5mL Nicotinamide: 100μl Recombinant human HGF: 50 μl

[0122] Organoid Harvesting Procedure Cultrex TM Organoid Harvesting Solution Materials (follow manufacturer's guidelines) · Cultrex TM Organoid Harvesting Solution;#3700-100-01(100mL) · Cold PBS1X · Tryple Express;#12604-013 Y-27632 dihydrochloride (100MG); AbMole BioScience; #M1817 (Stock 1mM) · Cultrex Reduced Growth Factor Basement Membrane Extract,Type 2,Pathclear;Biotechne;# 3533-005-02

[0123] procedure Aspirate the cell culture medium while still on ice and gently wash each well with 10 volumes of cold (2–8°C) PBS, being careful not to disrupt the basement membrane matrix containing the organoids. Aspirate the PBS and add 10 volumes of cold (2-8°C) Cultrex to each well. TMAdd Organoid Harvesting Solution. Incubate the plate at 2-8°C or on ice for 30-90 minutes with moderate shaking. This incubation is complete when the basement membrane matrix dome is no longer visible at the bottom of the well and the organoids are visible floating on the bottom of the well. Note: Removing the dome with a cell scraper or pipette may hasten this process. Once the matrix has depolymerized, transfer the contents of the wells to a tube on ice. For single wells, transfer to a microfuge tube, but for multi-dome wells, you will need a 15mL or 50mL conical tube. Pellet the organoids by centrifugation in a swinging bucket rotor at 500 x g for 5 minutes at 2–8°C. Aspirate the supernatant. Wash the organoids with 10 volumes of cold (2-8°C) PBS and pellet the organoids by repeated centrifugation at 500 x g for 5 minutes at 2-8°C in a swinging bucket rotor. Aspirate the PBS. Add 10μl of Rock inhibitor to 1mL of tryple and incubate at 37 for 15 minutes. · Add 10 volumes of PBS to neutralize the Tryple with Rock inhibitor. Centrifuge the tube at 500 x g for 5 minutes. Aspirate the PBS. The isolated organoids can be: o For further organoid culture, suspend them in basement membrane matrix. o Resuspend in freezing medium for cryopreservation. o Killing assay

[0124] Phenotypic analysis of endometrial cells isolated from tissue Phenotyping of endometrial cells orthotopic and ectopic using established panels. Panel 1 is used to assess the ratio of epithelial to stromal fractions and Panel 2 is used to further characterize the stromal fraction. Concurrent with phenotyping, quantification of FAP is performed using Qifikit.

[0125] material Orthotopic and ectopic cells · OVCAR3 cells PBS (Gibco, Phosphate Buffered Saline, without CaCl2 or MgCl2, Ref 10010-015, Lot 2375270) · FBS(Gibco, Cat 10500064,Lot 229262H) NaN3 (Sigma-Aldrich, Sodium Azide, S2002-100g, Lot STBJ9889) · VersaComp Antibody Capture Bead Kit (Beckmancoulter, ref B22804, Lot 4132061K) ArC Amine Reactive Compensation Bead Kit (Invitrogen, ref. A10628, lot 2409708) · Fc Block(Human TruStain, Biolegend, Cat 422302, Lot B328706) · Antibodies

[0126] [Table 4]

[0127] procedure · Harvest cells with Trypsin / Accutase, count and plate into 96-well U-bottom plates in PBS at 100μl / well (50-100k / well). Prepare FACS buffer: PBS + 2% FBS + 0.1% NaN 3 . Add 100 μl FB per well, resuspend and spin at 300 x g for 5 minutes at 4°C. Repeat wash with 180 μl FB. 50 μl BD Human Fc Block (1:100, 1-10×10 6 Add 100 ml of PBS (for cells) and incubate at RT for 15 min. During incubation, prepare the phenotypic panel. Make sure to add the L / D stain last. Add 50μl of direct conjugate antibody dilution to each well. Add 1 drop of VersaComp Compensation Capture Beads to each well for compensation (use ArC reactive beads for L / D). Make sure to vortex for at least 30 seconds before adding the beads. Add single color antibody conjugate to corresponding wells at the antibody concentration used for your application. Incubate in the dark at 4°C for 40 minutes. Add 150μl FB (PBS for compensation beads), spin at 300xg for 5 minutes and discard the supernatant. Wash once with 180 μl FB. · Resuspend cells in 200μl PBS + 2mM EDTA. Store plates at 4°C protected from light until reading on the Celesta. Transfer plate contents to labeled FACS tubes prior to reading. · Before running on the flow cytometer, remember to add one drop of the corresponding negative beads (Component B) to each compensation tube and vortex. Panel 1: CD90-AF647, FAP-PE, L / D-Bv421 Panel 2: CD45-BV605, CD105-AF488, CD44-APC, L / D-BV421

[0128] FAP quantification by Qifikit (following the manufacturer's recommendations) Qifikit contains a series of beads with a diameter of 10 μm, coated with different but well-defined amounts of mouse Mab molecules. The beads mimic cells with different antigen densities, labeled with an isotype IgG, primary mouse Mab. The cells are labeled with a primary mouse monoclonal antibody against the antigen of interest. In a separate test tube, the cells are labeled with an irrelevant mouse monoclonal antibody (control). The cells, setup beads and calibration beads from the kit are then labeled in parallel with a fluorescein-conjugated anti-mouse secondary antibody.

[0129] The primary antibody used to label the cells is used at saturating concentrations. Saturating conditions are determined by performing titration studies for each Mab studied with a FITC-conjugated anti-mouse secondary antibody. The primary antibody can be of any mouse IgG isotype. Under these conditions, the number of bound primary antibody molecules corresponds to the number of antigen sites present on the cell surface. The secondary antibody is also used at saturating concentrations. As a result, the fluorescence correlates with the number of bound primary antibody molecules on the cells and on the beads.

[0130] material Qifikit (Dako, K0078), setup beads (vial 1), calibration beads (vial 2), FITC conjugate (vial 3). · FAP-unlabeled primary antibody (FAP Monoclonal Antibdoy, eBioscience, Invitrogen, BMS168), 1mg / ml · Unconjugated IgG1 primary antibody (BioLegend, Cat No 401402), 0.5mg / ml FACS buffer (PBS+FBS+NaN3) · 100k for FAP Quant & Full Panels and 80k for all FMOs.

[0131] procedure Mix 100μl of cell suspension with 10μl of unconjugated FAP primary mouse monoclonal antibody (1:200). For a negative control, replace the unconjugated antibody with an irrelevant IgG1 mouse monoclonal antibody of the same isotype and adjusted to the same concentration. Make sure to use the primary antibody at a saturating concentration. Incubate at 4°C for 45 minutes. Add 150μl of FACS buffer and mix gently to keep the cells in suspension. Centrifuge at 300 x g for 5 minutes. Aspirate and discard the supernatant, leaving approximately 50 μl of liquid in the wells. Repeat washing with 180μl FACS buffer. Prepare setup and calibration beads: Dispense 100 μl (50 μl may be sufficient) of vortexed beads from vial 1 and vial 2 into two separate wells. Resuspend well in 150 μl PBS-BSA and centrifuge at 300 x g for 5 min. Aspirate the supernatant. Add 100 μl of FITC conjugate, vial 3, diluted 1:50 in PBS (add to cell suspension as well as the setup and calibration beads). Mix gently to bring cells into suspension. Incubate in the dark at 4°C for 45 minutes. Add 150 μl of FACS buffer and centrifuge at 300×g for 5 minutes. Aspirate and discard the supernatant. Repeat washing with 180 μl FACS buffer. Resuspend the pellet in 200 μl of FACS buffer. Store the tubes at 4°C for no more than 2 hours before analysis.

[0132] Data Acquisition Set up the flow cytometer using standard operating procedures. Set the analysis window first. Since the autofluorescence of Qifikit beads tends to be higher than that of lymphocytes, it may be necessary to pre-adjust the PMT of the corresponding fluorescence detector so that both the population of negative cells and the setup beads are visible on the scale. Acquire data from setup beads. Fluorescence analysis is restricted to debris-free bead singlets as defined by a dot plot of forward scatter versus side scatter. Acquire data for calibration beads and samples without chaining analysis windows. →Data from beads and cell preparations must be collected simultaneously. The PMT voltages of the corresponding fluorescence detectors must be the same, but the scattering settings may differ for cell to Qifikit bead data acquisition. Samples are analysed in the following order: → Vial 1: Setup beads. This sample is used to set the analysis window. The setup beads contain a mixture of brand and high level beads. → Vial 2: Calibration beads: Used to create a calibration curve (MFI against ABC). →Analyze the cells using a flow cytometer and calculate ABD based on the calibration curve equation.

[0133] CellTiter-Glo® Killing Assay Prepare CellTiter-Glo® Reagent according to manufacturer's guidelines. material T cell donor: Expanded T cells from PE551 donor Target cells P1 PC571, stromal + epithelial fraction mixed 1:1 · Cell culture medium used for cell seeding: DMEM / F12 medium (Gibco, Ref 11330-032)+10%FBS+1%P / S Trypsin 0.25%, Phenol Red (product number: 25200056) Assay medium: RPMI without phenol red (10% FBS and 1X L-Glut) Clear bottom 96-well black plate (product number: 353219) BITEs: FAP BITE, CellTiter Glo Viability Assay Kit · procedure P0 cells were detached with Trypsin for the stromal fraction and Accutase for the epithelial fraction for approximately 5 and 15 minutes, respectively. Cells were harvested, centrifuged at 300×g for 5 minutes, and the two cell fractions were mixed and used for the next experiment. For this assay, 7000 cells / well (in 100 μl) were seeded in DMEM / F12+10%FBS+1%P / S medium and left to adhere overnight. The next day, the cell culture medium was replaced with T cell culture medium (only 50 μl of cell culture medium was added) and T cells (28000 / well) (target cell:T cell ratio 1:4) and / or Bites were added to each well in a total volume of 25 μl (final concentration 1200 pg / mL) according to the layout below. As a control condition for cell death, Lysis Buffer was added to some wells, which was added 30 minutes before adding CellTiter Glo Reagent. 4 μL of Lysis Buffer was added to each well as needed. CellTiter Glo was determined after 24 hours: an aliquot of CellTiter Glo reagent was thawed and protected from light. At each time point (in this case 24 hours after addition of T cells + / - Bites), 100μL of Celltiter Glo reagent was added per well (104μL for wells with Lysis buffer) and the plate was incubated for 20 minutes in a 37C incubator protected from light. After the 20 minute incubation, the luminescence signal was measured on a Tecan plate reader. Record the light emission. The percent cytotoxicity is calculated as follows: o [T-(TEAb-E)] / [(T-(Tdead-E)]×100 o T = target luminescence signal o E = effector luminescence signal o Tdead = luminescence signal of lysed target cells o TEAb = target + effector + antibody luminescence signal

[0134] Killing assay - microscopy (bright field) tracking material · Target cells PC5711 donor (passage 0), 25k epithelial cells + 25k stromal cells (in a 24-well plate) PE571 donor (passage 1): 50k cells total (in a 96-well plate) T cell donor: Expanded T cells from PE551 donor · BITEs: FAP BITE Assay medium: RPMI without phenol red (10% FBS and 1X L-Glut)

[0135] procedure Use a 1:1 T cell:target cell ratio. o 24-well plate: 1:1. Seed 300 μL of 50kT cells per well (total volume of Tcell+BiTE per well is 400 μL). o 96-well plate: 10k T cells seeded per well, total volume 100 μL (total volume per well is 50 μL Tcell+BiTE) · Images of each well were taken at different time points: 0 hours, 2 hours, 4 hours, 21 hours and 24 hours.

[0136] Flow cytometric killing assay Target cells (OVCAR3 or PE559-2) were stained with CellTrace Yellow (5 μM) for 30 min. They were then washed and co-cultured with T cells at a ratio of 4:1 (E:T) for 24 h. After incubation, cells were washed, fixed, and acquired on a Fortessa.

[0137] Bispecific T Cell Engager (BiTe) Staining Protocol material · The desired BiTe Biotinylated Mouse Anti-Fab (https: / / www.jacksonimmuno.com Cat 115-066-006) Streptavidin PE (Biolegend Cat: 405203) Staining buffer

[0138] procedure Dispense 100k target cells into each well (96u bottom) and centrifuge at 500g for 5 minutes (don't forget to add an extra well for a secondary control for each cell type). Remove the supernatant and wash the cells with 200 μl staining buffer and repeat the same centrifugation step. Remove the supernatant and resuspend the cells in 100 μl staining buffer. Add 5 μl (or amount depending on your titration) of BiTe to the appropriate wells (you can also prepare a mix and resuspend the cells with it). Incubate the cells at 4°C for 1 hour. Centrifuge at 300g for 5 minutes, remove the supernatant, wash with 200μl staining buffer, and repeat the centrifugation and removal of the supernatant. · From this point on, a secondary control condition will be included in all stainings. Prepare a mixture of mouse anti-Fab and staining buffer (1:200); for example, 5 wells (consider a little more) 550μl staining buffer + 2.75mouse anti-Fab. Resuspend the cells in 100μl of this mixture. Incubate the cells at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes, remove the supernatant, wash with 200μl staining buffer, and repeat the centrifugation and removal of the supernatant. Prepare a mixture of Streptavidin-PE and staining buffer (1:200); for example, for 5 wells (consider a little more) 550 μl staining buffer + 2.75 μl Streptavidin-PE. Resuspend the cells in 100 μl of this mixture. Incubate the cells at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes, remove the supernatant, wash with 200μl staining buffer, and repeat the centrifugation and removal of the supernatant. · Resuspend the cells in 150μl staining buffer and transfer each well to a flow tube. Harvest the cells.

[0139] Masked BITE assay Masked BITEs were resuspended in staining buffer in the presence / absence (+ / -) of HAS (100 μg / ml). T cells were added and incubated for 1 h at 4°C. Protease digestion: 2 μg BiTe in 50 μl was incubated with protease and PBS or HBSS containing Ca and Mg for 3 hours at 37C. 25k T cells were added directly to the mixture (1 hour, 4°C). Follow the BITE staining protocol.

Claims

1. The agent is, a. Ligands having the ability to specifically bind to FAP; and b. A therapeutic portion having the ability to induce cell death in cells, wherein the cells express FAP on their cell surface; including, A drug for use in the treatment of benign gynecological neoplasms.

2. The agent for use according to claim 1, wherein the therapeutic portion is an immune cell mobilization portion, and the immune cell mobilization portion has the ability to specifically bind to immune cell surface molecules.

3. The aforementioned immune cell surface molecules are as follows: a. CD3; b. CD16; c. CD8; Selected from a group consisting of, In particular, the agent for use according to claim 2, wherein the immune cell surface molecule is CD3.

4. The immune cell mobilization portion is selected from the group including antibodies, antibody fragments, single-chain antigen-binding fragments, single-domain antibodies, aptamers, non-immunoglobin scaffolds, and antibody-like molecules. In particular, the immune cell mobilization portion is selected from the group including antibodies, antibody fragments, and single-chain antigen-binding fragments. More particularly, the agent for use according to any one of claims 2 or 3, wherein the immune cell mobilization portion is a single-domain antibody.

5. The therapeutic portion is a cytotoxic molecule with a molecular weight < 1000 Datons (Da), particularly ≤ 800 Da, and more particularly ≤ 500 Da. In particular, the agent for use according to claim 1, wherein the cytotoxic molecule is selected from the group comprising doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porphyromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine, leulosidine, vindesine, estramustine, cisplatin, cyclophosphamide, leulosine, taxol, desacetylvinblastine, cyclophosphamide, ifosfamide, cytarabine, 6-thioguanine, chlorambucil, carmustine, mitoxantrone, paclitaxel, or cytotoxic derivatives thereof.

6. The ligand is selected from the group comprising antibodies, antibody fragments, single-chain antigen-binding fragments, single-domain antibodies, aptamers, non-immunoglobin scaffolds, and antibody-like molecules, small molecule drugs that comply with a set of criteria of Lipinski Rules of Five, peptides, and FAP-specific substrates. In particular, the ligand is selected from the group including antibodies, antibody fragments, and single-chain antigen-binding fragments. More particularly, the agent for use according to any one of claims 1 to 3, wherein the ligand is a single-domain antibody.

7. The agent for use according to any one of claims 1 to 3, wherein the agent comprises a masking portion bound to the agent via a linker, the masking portion having the ability to reduce or remove the binding ability of the ligand and / or the immune cell mobilization portion, and in particular the linker comprises a protease cleavage site.

8. The agent for use according to any one of claims 1 to 3, wherein the agent comprises a bulk protein binding site, particularly a human serum albumin binding site, the binding site being bound to the agent via a linker, the linker particularly comprising a protease cleavage site, and the protease cleavage site particularly being specifically cleavable by matrix metalloproteinases 2, 9, 10, and / or 26.

9. The agent for use according to any one of claims 1 to 3, wherein the endometrial tissue is characterized by detectable expression of FAP, in particular by at least 1% of the endometrial cells expressing FAP.

10. A drug for use in the treatment of benign gynecological neoplasms, a. Target protein binding site; b. Immune cell recruitment part; c. A masking portion having the ability to block the binding of the immune cell mobilization portion and / or the target protein binding portion to its target; d. A cleavable linker connecting the masking portion to other components of the agent, wherein the cleavable linker is specifically cleavable by matrix metalloproteinases (MMPs) 2, 9, 10, and / or 26, and in particular, the cleavable linker is specifically cleavable by MMP2 or MMP9. A drug containing [a specific ingredient].

11. The agent for use according to claim 10, wherein the cleavable linker is characterized by an sequence selected from the group of sequence numbers 1 to 4.

12. A drug for use in the diagnosis of benign gynecological neoplasms, a. Ligands that have the ability to specifically bind to FAP; b. Radioisotope labeling: Includes , the radioactive isotope label is ９４ｍ Tc, １８６ Re, ２０３ Pb, ４７ Sc, １１１ In, ９７ Ru, ６２ Cu, ８８ Y, １２１ Sn, １６１ Tb, １５３ Sm, １６６ Ho, １０５ An agent selected from the group comprising Rh.

13. The agent for use according to claim 12, wherein the diagnosis is an in vivo diagnosis.

14. The agent for use according to claim 12 or 13, wherein the diagnosis is an X-ray, and in particular the diagnosis is selected from the group consisting of X-ray, PET, and CT.

15. The agent for use according to any one of claims 1 to 3 or 10 to 13, wherein the benign gynecological neoplasm is endometriosis.

16. The agent for use according to any one of claims 1 to 3 or 10 to 13, wherein the benign gynecological neoplasm is the development of uterine fibroids.

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