Compositions targeting molecules expressed on endothelial cells (ECS) of endometrium and endometriotic lesions and methods of use thereof

Abstract

Provided herein is a targeting molecule, wherein the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) in which expression of the surface molecule is upregulated.

Description

Attorney Docket No. 117823-37820COMPOSITIONS TARGETING MOLECULES EXPRESSED ON ENDOTHELIAL CELLS (ECS) OF ENDOMETRIUM AND ENDOMETRIOTIC LESIONS AND METHODS OF USE THEREOFRELATED APPLICATIONS

[0001] The instant application claims priority to U.S. Provisional Application No. 63 / 700,000, filed on September 27, 2024. The entire contents of the foregoing application are expressly incorporated by reference herein.GOVERNMENT SUPPORT

[0002] This invention was made with government support under All 55865 and AI175379 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.FIELD OF THE INVENTION

[0003] Described herein are compositions and methods related to targeting and treating diseases or disorders of the uterus with targeting molecules that bind upregulated surface molecules on endometrial endothelial cells (EECs).BACKGROUND OF THE INVENTION

[0004] Endometriosis is an incurable chronic gynecological disorder characterized by the presence and growth of endometrium-like tissue outside of uterus. The tissue growth is referred to as ectopic or EM lesions. Although the precise pathogenesis of endometriosis is unknown, the commonly accepted Sampson’s theory suggests that EM lesions originate by cells detached from endometrium and lodged into abdominal cavity via fallopian tubes by retrograde menstruation. However, 75-90% of women experience retrograde menstruation, but not all of these women develop endometriosis, which is mostly seen in individuals with hormonal or immunological issues (Chauhan S. et al. Endometriosis: A Review of Clinical Diagnosis, Treatment, and Pathogenesis. Cureus. 2022 Sep 6;14(9):e28864). Common symptoms include dysmenorrhea, pelvic pain, cramping, pain during intercourse, bowel movements and urination, heavy bleeding, and infertility.1MEl\57624905.v3Attorney Docket No. 117823-37820

[0005] The diagnosis of endometriosis is difficult. The median time elapsed from the onset of the symptoms to the definitive diagnosis is seven years, and the delay is even longer in younger women (M.S. Arruda, et al. Time elapsed from onset of symptoms to diagnosis of endometriosis in a cohort study of Brazilian women, Human Reproduction, Volume 18, Issue 4, April 2003, Pages 756-759). Ultrasound can show large lesions and scarring, which is another sign of endometriosis (Leonard! M., et al. How to perform an ultrasound to diagnose endometriosis. Australas J Ultrasound Med. 2018 Apr 22;21(2):61-69). Currently, the most definitive procedure to diagnose endometriosis is surgically invasive laparoscopy (Mak J, Leonard! M., et al. 'Seeing is believing': arguing for diagnostic laparoscopy as a diagnostic test for endometriosis. Reprod Fertil. 2022 Jun 10;3(3):C23-C28). During laparoscopy, detectable EM lesions are surgically removed, but since the lesions may vary in size, depth of penetration, location and appearance, it is nearly impossible to completely remove affected tissue, therefore the symptoms persist. Moreover, rare cases of thoracic (Yao J. et al., Endometriosis of the lung: A case report and review of literature. World J Clin Cases. 2023 Jun 26;11(18):4326-4333) and cerebral endometriosis (Sarma D. et al., Cerebellar endometriosis. Am J Roentgenol. 2004; 182(6): 1543-6) have been described, where laparoscopy cannot be performed.

[0006] Non-invasive endometriosis therapy mostly consists of symptomatic treatment, which usually starts with nonsteroidal anti-inflammatory drugs (NSAIDs) or steroids with androgenic action. Other treatments may include Gonadotropin-releasing hormone (GnRH) or vascular endothelial growth factor (VEGF) inhibitors, which can cause serious side effects. As a result of insufficient removal of endometriotic lesions during laparoscopy and high- failure rate of non-surgical therapies, ~30 out of 100,000 women diagnosed with endometriosis undergo a hysterectomy, a procedure that eliminates the anatomic source of endometrial cells but does not remove already existing lesions outside the uterus, thus the operation cannot be considered a radical method of treatment, which eliminates the disease.

[0007] Accordingly, there is a need for improved compositions and methods related to diagnosing and treating endometriosis. The present disclosure meets such needs.2MEl\57624905.v3Attorney Docket No. 117823-37820SUMMARY OF THE INVENTION

[0008] In some aspects, provided herein is a targeting molecule, wherein the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) in which expression of the surface molecule is upregulated.

[0009] In some embodiments, the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein-coupled receptor 37 (GPR37), Germ cell-specific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C).

[0010] In some embodiments, the expression of the surface molecule is upregulated in normal endometrium. In some embodiments, the expression of the surface molecule is upregulated in normal endometrium as compared to expression in non-endometrial tissue. In some embodiments, the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in normal endometrium as compared to expression in non-endometrial tissue.

[0011] In some embodiments, the expression of the surface molecule is upregulated in endometriotic (EM) lesions. In some embodiments, the expression of the surface molecule is upregulated in EM lesions as compared to expression in non-endometrial tissue. In some embodiments, the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in EM lesions as compared to expression in non-endometrial tissue. In some embodiments, the expression of the surface molecule is upregulated in EM lesions as compared to expression in normal endometrium. In some embodiments, the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3- fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in EM lesions as compared to expression in normal endometrium.

[0012] In some embodiments, the surface molecule is not expressed in normal endometrium. In some embodiments, the surface molecule is not expressed in normal endometrium in the proliferative stage of the estrous cycle. In some embodiments, the surface molecule is EVA1C.3MEl\57624905.v3Attorney Docket No. 117823-37820

[0013] In some embodiments, the surface molecule is weakly expressed in normal endometrium. In some embodiments, the surface molecule is weakly expressed in normal endometrium in the secretory stage of the estrous cycle. In some embodiments, the surface molecule is EVA1C.

[0014] In some embodiments, the targeting molecule is an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a multispecific antibody, or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is 1) an Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2; 2) a diabody, ScFv, SMIP, single chain antibody, affibody, avimer, or nanobody; 3) a fusion of one or more heavy chains and one or more nanobodies; or 4) a single domain antibody.

[0015] In some embodiments, the antigen-binding fragment is a nanobody. In some embodiments, the nanobody specifically binds to EVA1C. In some embodiments, the nanobody comprises an amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of GTISTWRDELVAAITQGGTTYYAVYYCAAVGTRKTTGEPYDYAY (SEQ ID NO: 1); GSISSEYDELVAAISGGGTTYYAVYYCAAPGYHRSSYYY (SEQ ID NO: 2); GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3); GTISARRTEFVAGINIGGITYYAVYYCAAPGGPDREHNY (SEQ ID NO: 4);GTISAVQDELVAAINIGTTTYYAVYYCAAVYESKGYYALSHYY (SEQ ID NO: 5); and GTIFWYGDELVAAISNGGTTYYAVYYCAVVVIASIRGHPPHYY (SEQ ID NO: 6). In some embodiments, the nanobody comprises an amino acid sequence selected from the group consisting of: GTISTWRDELVAAITQGGTTYYAVYYCAAVGTRKTTGEPYDYAY (SEQ ID NO: 1); GSISSEYDELVAAISGGGTTYYAVYYCAAPGYHRSSYYY (SEQ ID NO: 2); GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3); GTISARRTEFVAGINIGGITYYAVYYCAAPGGPDREHNY (SEQ ID NO: 4); GTISAVQDELVAAINIGTTTYYAVYYCAAVYESKGYYALSHYY (SEQ ID NO: 5); and GTIFWYGDELVAAISNGGTTYYAVYYCAVVVIASIRGHPPHYY (SEQ ID NO: 6). In some embodiments, the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).4MEl\57624905.v3Attorney Docket No. 117823-37820

[0016] In some aspects, provided herein is a composition comprising: 1) the targeting molecule of any of the preceding embodiments, and 2) an agent. In some embodiments, the agent is selected from the group consisting of radioisotopes, small molecules, biologies, nanoparticles, or a second targeting molecule. In some embodiments, the second targeting molecule specifically binds to an antigen on the surface of an NK cell or a nanoparticle.

[0017] In some aspects provided herein, is an engineered immune cell comprising: 1) a first polynucleotide encoding a first targeting molecule that specifically binds to a surface molecule on an endometrial endothelial cell (EEC); and 2) a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell. In some embodiments, the first targeting molecule is the targeting molecule of any one of the preceding embodiments. In some embodiments, the immune cell is a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell.

[0018] In some aspects, provided herein is a pharmaceutical composition comprising: 1) the targeting molecule of any one of the preceding embodiments, the composition of any one of the preceding embodiments, or the engineered immune cell of any one of the preceding embodiments; and 2) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a lipid formulation.

[0019] In some aspects, provided herein is a method of treating a disease or disorder of the uterus in a subject in need thereof, comprising administering to the subject the composition of any one of the preceding embodiments, the engineered immune cell of any one of the preceding embodiments, or the pharmaceutical composition of any one of the preceding embodiments. In some embodiments, the disease or disorder of the uterus is endometriosis.

[0020] In some aspects, provided herein is a method of treating endometriosis in a subject in need thereof, wherein the endometriosis is characterized by endometriotic (EM) lesions, comprising administering to the subject a composition comprising a targeting molecule which binds to the surface molecule on an endometrial endothelial cell (EEC) and an agent to treat the endometriotic (EM) lesions.

[0021] In some aspects, provided herein is a medical imaging method comprising (i) administering to a subject having endometriosis characterized by endometriotic (EM) lesions a composition comprising a targeting molecule which binds to a surface molecule on an5MEl\57624905.v3Attorney Docket No. 117823-37820 endometrial endothelial cell (EEC) and an agent to treat the EM lesions, wherein the targeting molecule is associated with a detectable marker, and (ii) detecting the detectable marker in the body of the subject.

[0022] In some aspects, provided herein is a medical imaging method comprising (i) administering to a subject a composition comprising a targeting molecule which binds to a surface molecule on an endometrial endothelial cell (EEC), wherein the targeting molecule is associated with a detectable marker, and (ii) detecting the detectable marker in the body of the subject.

[0023] In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes.

[0024] In some aspects, provided herein is a method of diagnosing or prognosing a disease or disorder of the uterus in a subject, comprising determining the expression in vivo of a surface molecule on an endometrial endothelial cell (EEC) located within an endometriotic (EM) lesion, wherein upregulation of expression of the surface molecule on the EEC, as compared to a control level, is indicative of the presence or progression of the disease or disorder of the uterus.

[0025] In some embodiments, the targeting molecule is the targeting molecule of any one of the preceding embodiments.

[0026] In some embodiments, the composition is the composition of any one of the preceding embodiments or the pharmaceutical composition of the preceding embodiments.

[0027] In some embodiments, the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein-coupled receptor 37 (GPR37), Germ cell-specific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C).

[0028] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the disclosure will become apparent to one of skill in6MEl\57624905.v3Attorney Docket No. 117823-37820 the art. These and other embodiments of the disclosure are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows bulk RNAseq analysis of endothelial cells (ECs) sorted from 20 organs (uterus is underlined). Data is shown for venular (black bars), non-venular (grey bars) and lymphatic (white bars) ECs. Abbreviations: ADRA2B - Alpha-2B adrenergic receptor, P2RX2 - Purinergic Receptor P2X2, GPR37 - G protein-coupled receptor 37, RXFP1 - Relaxin / insulin-like family peptide receptor 1, GSG1 - Germ cell-specific gene 1; PLN - peripheral lymph nodes, MLN - mesenteric lymph node, PP - Peyer’s patch, VAT - visceral adipose tissue, SAT - subcutaneous adipose tissue, SI - small intestine, BM - bone marrow, Chp - choroid plexus, Pia - pia mater, NTMTS - nasoturbinate / maxiloturbinate / septum, MS- maxillary sinus, NALT - nasal-associated lymphoid tissue.

[0030] FIGS. 2A-2B provide violin plots showing the computational analysis results of single-cell RNAseq of human biopsies. In FIG. 2A, the results are presented by biopsy. In FIG. 2B, each of three patients with no endometriosis (C01-C03) and eleven patients with endometriosis (E01-E11) demonstrated expression of RXFP1 on ECs of endometrium or endometriotic (EM) lesions. Abbreviations: ADRA2B - Alpha-2B adrenergic receptor, Ctrl - normal endometrium, EuE - eutopic endometrium in endometriosis patients, EcP - ectopic peritoneal lesions, EcP A - ectopic peritoneum adjacent lesions, EcO - ectopic ovarian lesions, GPR37 - G protein-coupled receptor 37, GSG1 - Germ cell-specific gene 1, RXFP1- Relaxin / insulin-like family peptide receptor 1.

[0031] FIGS. 3A-3B show representative fluorescence-activated cell sorting (FACS) dotplots demonstrating PCR hormone-binding receptor Relaxin R1 (RXFP1) expression on the surface of ECs in normal uterus and EM lesions (FIG. 3A) and other tissues (FIG. 3B). Single cell suspensions were prepared from the identified tissues, and blood ECs were identified as live CD45-CD31+gp38- population. Gates for RXFP1 Expression in small intestine (SI), bladder, and ovary were set according to staining of isotype control performed for each individual organ.

[0032] FIGS. 4A-4B show identification of RXFP1 expression on ECs of murine normal endometrium (FIG. 4A) or EM lesions (FIG. 4B). Frozen tissue sections were co-stained7MEl\57624905.v3Attorney Docket No. 117823-37820 with anti-CD31 to identify ECs and anti-RXFPl. Channels for CD31 and RXFP1 are shown as gray scale. RXFP1+ vessels are identified by white arrows. RXFP1+ cells outside the vessels represent endometrial stromal and granular epithelial cells

[0033] FIGS. 5A-5B show identification of RXFP1 expression on ECs of human normal endometrium (FIG. 5A) or EM lesions (FIG. 5B). Paraffin-embedded tissues were sectioned and co-stained with anti-CD31 to identify ECs and anti-RXFPl. Channels for CD31 and RXFP1 are shown as gray scale. RXFP1+ vessels are identified by white arrows. RXFP1+ cells outside the vessels represent endometrial stromal and granular epithelial cells.

[0034] FIG. 6 provides a volcano plot showing computational analysis results of singlecell RNAseq of human biopsies. Over-expressed genes encoding for transmembrane proteins are identified by name. Results reflect a comparison between expression on ECs of eutopic endometrium in endometriotic patients (EuE) and ectopic peritoneal endometriosis lesions (EcP).

[0035] FIGS. 7A-7B show identification of enhancer of ventral-axon guidance defects homolog C (EVA1C) expression on ECs of murine normal endometrium (FIG. 7A) or EM lesions (FIG. 7B). Frozen tissue sections were co-stained with anti-CD31 to identify ECs and anti-EVAlC. Channels for CD31 and EVA1C are shown as gray scale. EVA1C+ vessels are identified by white arrows. FIGS. 7C-7D show identification of EVA1C expression on ECs of eutopic human endometrium (FIG. 7C) and EM lesions (FIG. 7D).

[0036] FIGS. 8A-8C show identification of RXFP1 in murine EM lesion. As shown in FIG. 8A, mice were injected into the peritoneal cavity with fragments of uterine tissue resulting in formation of EM-like lesions. Tissues were harvested on day 28 and prepared for immunohistochemistry (IHC). Tissue sections were stained with fluorescent antibodies recognizing different structural elements: CD31, a marker of ECs (top left, FIG. 8C); RXFP1 (bottom left, FIG. 8C); smooth muscle actin (SMA) (bottom right, white, FIG. 8C); and CD326 (top right, epithelium (epi), FIG. 8C). FIG. 8B provides an IHC image of murine EM lesions stained for CD31, CD326, and smooth muscle actin. As shown in FIG. 8C, channels for CD31 and RXFP1 are shown as gray scale (left). Stained vessels are identified by arrow heads. Right: pseudo image of the four merged channels. Vessels expressing both CD31 and RXFP1 are indicated by the arrows. Most lesions are surrounded by smooth muscle cells, as evidenced by SMA staining.8MEl\57624905.v3Attorney Docket No. 117823-37820

[0037] FIGS. 9 shows that EVA1C is expressed on ECs in experimental murine EM, but not on ECs in normal mouse endometrium. Experimental EM lesions were induced and prepared for IHC as described in FIG. 8A. Sections of normal uterus tissue were analyzed in parallel.

[0038] FIG. 10 shows that EVA1C is expressed on ECs in human EM, but not in normal endometrium. Biopsies of normal human endometrium (left) and a peritoneal EM lesion (right) were analyzed by IHC. Microvascular structures in both micrographs are identified by arrows.

[0039] FIGS. 11A-11F show the discovery of transcriptional and proteomic landscapes of inflammatory diseases using the lOx Genomics Xenium and MACSima™ platform. FIG. HA shows a Xenium image of Hidradenitis suppurativa (HS) lesions in skin. FIG. 11B shows interleukin 2 receptor subunit alpha (IL2RA) expression in HS skin. FIG. 11C shows uniform manifold approximation and projection (UMAP) identification of cell populations in the sample shown in FIG. 11 A. FIG. HD shows spatial distribution of cell populations in HS skin. FIG. HE shows regulatory T cells markers associated with skin disease pathology. FIG. HF shows inflammatory disease tissue images produced by MACSima™.

[0040] FIG. 12 provides a molecular imaging strategy to detect EM. A. EM can manifest anywhere in the peritoneal cavity, but most commonly on the uterus or ovaries. B. Lesions often form blood filled cysts (‘chocolate cysts’) and are surrounded by a dense network of small blood vessels in which ECs express RXFP1 and EVA1C. These surface molecules are absent from ECs in other tissues (except RXFP1 is found on ECs in normal endometrium). C. NAbs that specifically recognize RXFP1 or EVA1C can be characterized and modified with a chelator (e.g. deferoxamine (DFO) or methyltetrazine) to carry a radiotracer (e.g.18F- fhiorodeoxyglucose or89Zr). These imaging reagents can be tested by intravenous (IV) injection. D. Radio-NAbs are predicted to accumulate on ECs in EM lesions where they emit gamma radiation detectable by PET imaging. Instead of radioisotopes, NAbs may also be used to target other payloads to EM lesions. Abbreviations: PET - positron emission tomography, IVIS - in vivo imaging system, Nabs - nanobodies, NIR - near-infrared, VHH - (or nanobody) the antigen binding fragment of heavy chain only antibodies.

[0041] FIG. 13 provides a synthetic homing receptor (SHORE) strategy to target natural killer (NK) cells to EM lesions. SHOREs are modified NAbs that are displayed at high9MEl\57624905.v3Attorney Docket No. 117823-37820 density on the surface of a therapeutic lymphocyte to confer the ability to adhere to a specific endothelial ligand in physiologically perfused microvessels. Preliminary findings from a SHORE strategy to target T cells to solid tumors are encouraging. For the present project, SHOREs may either be transfected into NK cells as a chimeric transmembrane protein (similar to a chimeric antigen receptor or CAR, not depicted) or could be constructed as bispecific ‘universal’ SHOREs, whereby a NAb domain recognizes an EC antigen (e.g. RXFP1 or EVA1C), while the second binding domain binds a highly expressed common surface moiety on NK cells (e.g. CD45 or a carbohydrate). Several lectins that bind to NK and T cells at a very high density without causing apparent activation have been identified by the present inventors, and a series of NAb-lectin fusion constructs to array SHOREs on a lymphocyte surface are being constructed.

[0042] FIGS. 14A-14B demonstrate generation of anti-EVAlC NAbs using a yeast display library screening study. FIG. 14A provides a schematic drawing of the yeast display library screening strategy. Recombinant EVA1C ectodomain tagged with a FLAG peptide (DYKDDDDK) was used as a bait by incubating with NAb-expressing yeast cells (identified by staining for an HA tag) followed by immuno-magnetic bead selection. Bait binding was detected by subsequent staining with a fluorescent anti-FLAG 2ndstage monoclonal antibody. FIG. 14B provides flow cytometry data reflecting incremental enrichment of EVA1C-FLAG binders in the total yeast population with successive selection steps on human (selections 1 to 4) followed by mouse (selections 5 to 6) recombinant EVA1C proteins.

[0043] FIG. 15 provides flow cytometry data reflecting the screening of sorted subclones for binding to murine (upper panel) and human (lower panel) EVA1C-FLAG soluble proteins. Binding of NAb-expressing yeast (determined by an HA tag) to EVA1C protein (determined by a FLAG tag) is shown for individual subclones A3, B4, C3, C7, and C12.

[0044] FIGS. 16A-16C provide FACS histograms showing binding of various NAb subclones A3, A5, B4, C3, C7, and C12 to EVA1C KO 293T cells (FIG. 16A), human EVA1C transfected 293T cells (FIG. 16B), and mouse EVA1C transfected 293T cells (FIG. 16C). Anti-GFP NAb was used as a control.

[0045] FIGS. 17A-17B provide ELISA results showing binding of NAb subclones A3, A5, B4, C3, and C7 to plate-bound human EVA1C-FLAG protein. ELISA plates were coated with human EVA1C-FLAG protein or negative control protein (both at 1 ug / ml), incubated10MEl\57624905.v3Attorney Docket No. 117823-37820 with various concentrations of NAbs (ranging from 5 uM to 1 nM), and absorbance at 450 nm wavelength was assessed by spectrophotometry. FIG. 17A shows the absorbance at 450 nm wavelength (raw data) as a function of NAb concentration (NB concentration, nM). FIG. 17B shows the percent of maximum absorbance as a function of NAb concentration (NB concentration, nM).

[0046] FIGS. 18A-18B provide immunofluorescence images reflecting the identification of EVA1C expression on ECs of murine endometriotic lesions by anti-EVAlC NAb clone B4. Frozen tissue sections were co-stained with anti-CD31 to identify ECs and either control anti-GFP NAb (FIG. 18A) or anti-EVAlC NAb clone B4 (FIG. 18B). NAb staining was followed by staining with anti-VHH polyclonal antibody. Channels for CD31 and NAb staining are shown in gray scale. CD31+ vessels co-stained with B4 NAb are identified by white arrows.

[0047] FIGS. 19A-19C provide a micrograph of normal Human endometrium (Proliferative stage) shown in grey scale. Section was stained with anti-CD31 to demarcate the vessels and co-stained with anti-EVAlC antibody followed by a 2nd stage antibody. FIG. 19A shows the absence of co-localization of anti-CD31 and anti-EVAlC staining. FIG. 19B shows no expression of EVA1C on stromal cells. FIG. 19C shows single color staining of blood vessels by anti-CD31.

[0048] FIGS. 20A-20C provides a micrograph of normal Human endometrium (Secretory stage) shown in grey scale. Section was stained with anti-CD31 to demarcate the vessels and co-stained with anti-EVAlC antibody followed by a 2nd stage antibody. FIG. 20A shows co-localization of anti-CD31 and anti-EVAlC expression depicted by white arrow. FIG. 20B shows low to moderate expression of EVA1C on stromal cells. In addition, weak expression of EVA1C on EC in one microvessel (indicated by white arrow) is shown. FIG. 20C shows single color staining of blood vessels by anti-CD31.

[0049] FIG. 21 provides a schematic of an exemplary workflow for discovering novel targets in human endometriosis lesions.11MEl\57624905.v3Attorney Docket No. 117823-37820DETAILED DESCRIPTION OF THE INVENTIONDEFINITIONS

[0050] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0051] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural (i.e., one or more), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising, “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited.

[0052] The term “about” or “approximately” means within 5%, or more preferably within 1%, of a given value or range.

[0053] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.12MEl\57624905.v3Attorney Docket No. 117823-37820

[0054] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0055] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0056] The expressions “at least about A, B, and C” and “at least about A, B, or C” may be construed to mean at least about A, at least about B, or at least about C. The expressions “at most about A, B, and C” and “at most about A, B, or C” may be construed to mean at most about A, at most about B, or at most about C.

[0057] The expression “about A to B and C to D” may be construed to mean between about A and about B and between about C and about D. The expression “about A to B or C to D” may be construed to mean between about A and about B or between about C and about D.

[0058] The term “exemplary” as used herein means “serving as an example, instance, or illustration.” Any embodiment described herein as “exemplary” is not to be construed as preferred or advantageous over other embodiments.

[0059] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0060] As used herein, the term “encode” or “encoding” refers to a property of sequences of nucleic acids, such as a vector, a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of other molecules such as proteins.13MEl\57624905.v3Attorney Docket No. 117823-37820

[0061] The terms “increased,” “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0062] As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” may therefore be used in some embodiments herein to capture potential lack of completeness inherent in many biological and chemical phenomena.

[0063] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0064] As used herein, a homologue of any polypeptide or nucleic acid sequence contemplated herein includes sequences having a certain homology with the wildtype amino acid and nucleic sequence. A homologous sequence may include a sequence, e.g. an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%< 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the subject sequence. The term “percent identical” or “percent identity” in the context of amino acid or nucleotide sequences refers to the percent of residues in two sequences that are the same when aligned for maximum correspondence. In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence. Sequence identity may be measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group,14MEl\57624905.v3Attorney Docket No. 117823-37820University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0065] As used herein, the terms “endometriotic lesions” or “EM lesions” refer to ectopic growths of endometrial-like tissue located outside the uterine cavity. In some embodiments, EM lesions occur in or on various anatomical sites including, but not limited to, the pelvic area, peritoneum, ovaries, fallopian tubes, bladder, urinary tract, ligaments in the pelvis, intestines, rectum, brain, liver, lungs, or eyes. In some embodiments, EM lesions are classified into distinct types based on location and depth, including: (1) superficial peritoneal lesions, which are typically small, flat or raised patches on the peritoneum; (2) endometriomas, which are cystic lesions commonly found on the ovaries; and (3) deep infiltrating endometriosis (DIE), characterized by the presence of lesions 5 mm or more in depth under the peritoneal surface. EM lesions may exhibit bleeding, inflammation, fibrosis, and neovascularization, and are associated with symptoms such as dysmenorrhea, pelvic pain, cramping, pain during intercourse, bowel movements and urination, heavy bleeding, and infertility.

[0066] It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

[0067] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety for all purposes, as if each individual publication, patent, and patent application were specifically and individually incorporated by reference.OVERVIEW

[0068] Endometriosis (EM), a gynecological condition affecting women of child-bearing age, is characterized by the ectopic growth of endometrial mucosa outside the uterus. EM causes infertility, chronic pain, and other severe complications, imposing a significant burden15MEl\57624905.v3Attorney Docket No. 117823-37820 on both patients and healthcare systems. Although the exact prevalence of EM is unknown, at least 10% of women aged 15 to 49 years are thought to be affected (-200 million globally, including -6-10 million in the US). Despite the high prevalence, EM is challenging to diagnose and treat. To date, the only definitive clinical test to diagnose EM is laparoscopy, a surgical procedure that requires anesthesia and carries a risk of bleeding and infection. Treatments are palliative and limited to the use of NSAIDs, hormonal therapies or surgical interventions, often culminating in hysterectomy. Because EM lesions can vary widely in size, penetration depth, distribution and appearance, laparoscopic surgery rarely results in complete removal of diseased tissue, so symptoms frequently persist. Moreover, EM lesions can also manifest outside the abdominal cavity, e.g., in the lung or central nervous system, which are not accessible by laparoscopy.

[0069] Accordingly, the present disclosure provides improved compositions and methods related to diagnosing and treating diseases and disorders of the uterus (e.g., endometriosis). Provided herein are unique surface molecules (e.g., proteins) that are upregulated in uterine microvascular endothelial cells (ECs) and not in ECs of non-endometrial tissues. Further, provided herein are surface molecules (e.g., proteins) that are upregulated in ECs of EM lesions in comparison with ECs of normal uterine endometrium. These differentially- expressed surface molecules allow for uterine targeted treatment, via a targeting molecule, such as an antibody or antigen-binding fragment thereof (e.g., nanobody) and other modes of targeted delivery of therapeutics. In another aspect, the present targeting molecules enable highly specific diagnostic imaging.

[0070] Herein, it was discovered that microvascular ECs in uterine endometrium surprisingly express several surface molecules that are not found on ECs of non-endometrial tissues. These proteins include, for example, G-protein coupled receptor (GPCR) Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), orphan GPCR GPR37, Germ cell-specific gene 1 (GSG1) protein, and Relaxin / insulin-like family peptide receptor 1 (RXFP1). Moreover, RXFP1 is unexpectedly upregulated in human normal endometrium and EM lesions. Additionally, it was discovered that enhancer of ventral-axon guidance defects homolog C (EVA1C) is surprisingly upregulated on ECs of EM lesions in comparison with normal uterine endometrium. Finally, it was discovered that EVA1C expression in normal endometrium is dependent on the estrous cycle. Interestingly, EVA1C is not expressed in16MEl\57624905.v3Attorney Docket No. 117823-37820 normal endometrium at the proliferative stage of the estrous cycle, but is weakly expressed on EECs of normal endometrium in the secretory stage of the estrous cycle.

[0071] In some aspects, provided herein is a targeting molecule (e.g., an antibody or antigen-binding fragment thereof), wherein the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) in which expression of the surface molecule is upregulated in endometriotic (EM) lesions. In some embodiments, the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein-coupled receptor 37 (GPR37), Germ cell-specific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C).

[0072] In some aspects, provided herein is a composition comprising: 1) a targeting molecule of the present disclosure (e.g., an antibody or antigen-binding fragment thereof) that binds to a surface molecule (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C) on an endometrial endothelial cell (EEC); and 2) an agent (e.g., radioisotopes, small molecules, biologies, nanoparticles, or a second targeting molecule).

[0073] In some aspects, provided herein is an engineered immune cell comprising: 1) a first polynucleotide encoding a first targeting molecule of the present disclosure (e.g., an antibody or antigen-binding fragment thereof) that specifically binds to a surface molecule (e g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C) on an endometrial endothelial cell (EEC); and / or a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell (e.g., a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell).

[0074] In some aspects, provided herein is a pharmaceutical composition (e.g., a lipid formulation) comprising: 1) a targeting molecule, composition, or engineered immune cell of the present disclosure; and 2) a pharmaceutically acceptable carrier.

[0075] In some aspects, provided herein is a method of treating a disease or disorder of the uterus (e.g., endometriosis) in a subject in need thereof, comprising administering to the subject a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure.

[0076] In some aspects, provided herein is a method of treating endometriosis in a subject in need thereof, wherein the endometriosis is characterized by endometriotic (EM) lesions,17MEl\57624905.v3Attorney Docket No. 117823-37820 comprising administering to the subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure to treat the endometriotic (EM) lesions. In some embodiments, the method comprises administering to the subject a composition comprising a targeting molecule (e.g., an antibody or antigenbinding fragment thereof) which binds to the surface molecule (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C) on an endometrial endothelial cell (EEC) and an agent (e.g., therapeutic payload) to treat the endometriotic (EM) lesions.

[0077] In some aspects, provided herein is a medical imaging method comprising: (i) administering to a subject having endometriosis characterized by endometriotic (EM) lesions a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure, wherein the targeting molecule, composition, engineering immune cell, or pharmaceutical composition is associated with a detectable marker (e.g., fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radioisotopes, metals, metals chelates or metallic cations, chromophores, or enzymes), and (ii) detecting the detectable marker in the body of the subject. In some embodiments, the method comprises administering to the subject a composition comprising a targeting molecule which binds to a surface molecule on an endometrial endothelial cell (EEC) and an agent (e.g., therapeutic payload) to treat the EM lesions, wherein the targeting molecule is associated with a detectable marker.

[0078] In some aspects, provided herein is a medical imaging method comprising: (i) administering to a subject a composition comprising a targeting molecule which binds to a surface molecule on an endometrial endothelial cell (EEC), wherein the targeting molecule is associated with a detectable marker (e.g., fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores, or enzymes), and (ii) detecting the detectable marker in the body of the subject.

[0079] In some aspects, provided herein is a method of diagnosing or prognosing a disease or disorder of the uterus (e.g., endometriosis) in a subject, comprising determining the expression in vivo of a surface molecule (e.g., EVA1C) on an endometrial endothelial cell (EEC) located within an EM lesion, wherein upregulation of expression of the surface molecule on the EEC, as compared to a control level (e.g., expression of the surface molecule18MEl\57624905.v3Attorney Docket No. 117823-37820 on an EEC from normal endometrium of an individual without endometriosis), is indicative of the presence or progression of the disease or disorder of the uterus.TARGETING MOLECULES

[0080] Provided herein is a targeting molecule, wherein the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) in which expression of the surface molecule is upregulated.

[0081] As used herein, a “targeting molecule” refers to any molecule that binds to a component associated with an organ, tissue, cell, extracellular matrix, and / or subcellular locale. In some embodiments, such a component is referred to as a “target” or a “marker”.

[0082] A targeting molecule may be a nucleic acid, polypeptide, glycoprotein, carbohydrate, lipid, small molecule, etc. For example, a targeting molecule can be a nucleic- acid targeting molecule (e.g., an aptamer, Spiegelmer®, etc.) that binds to a cell type specific marker. In general, an aptamer is an oligonucleotide (e.g., DNA, RNA, or an analog or derivative thereof) that binds to a particular target, such as a polypeptide. In some embodiments, a targeting molecule may be a naturally occurring or synthetic ligand for a cell surface receptor, e.g., a growth factor, hormone, LDL, transferrin, etc. A targeting molecule can be an antibody, which term is intended to include antibody fragments, characteristic portions of antibodies, single chain antibodies, etc. Synthetic binding proteins such as Affibodies®, Nanobodies™, AdNectins™, Avimers™, etc., can be used. In a particular embodiment, the targeting molecule is a nanobody. Peptide targeting molecule can be identified, e.g., using procedures such as phage display. This widely used technique has been used to identify cell specific ligands for a variety of different cell types.

[0083] In accordance with the present disclosure, a targeting molecule recognizes one or more “targets” or “markers” associated with a particular organ, tissue, cell, and / or subcellular locale. In some embodiments, a target may be a marker that is exclusively or primarily associated with one or a few cell types, with one or a few diseases, and / or with one or a few developmental stages.

[0084] In some embodiments, the target or marker is a surface molecule expressed on endometrial endothelial cells (EECs). In some embodiments, the target or marker is a surface molecule expressed on endometrial endothelial cells (EECs) of endometriotic (EM) lesions.19MEl\57624905.v3Attorney Docket No. 117823-37820In some embodiments, the target or marker is a surface molecule expressed on endometrial endothelial cells (EECs) of normal endometrium (e.g., endometrium of an individual without a disease or disorder of the uterus, e.g., endometriosis). In some embodiments, the target or marker is a surface molecule expressed on endometrial endothelial cells (EECs) of eutopic endometrium (e.g., endometrial tissue located in the proper place in the uterus of an individual, e.g., of a healthy individual or an individual with endometriosis). In some embodiments, the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein- coupled receptor 37 (GPR37), Germ cell-specific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C).

[0085] In some embodiments, the target or marker is a surface molecule expressed on endometrial endothelial cells (EECs) of endometriotic (EM) lesions, eutopic endometrium, and normal endometrium. In some embodiments, the target or marker is RXFP1.

[0086] In some embodiments, the target or marker is a surface molecule expressed on endometrial endothelial cells (EECs) of endometriotic (EM) lesions. In some embodiments, the target or marker is a surface molecule that is not expressed on EECs of eutopic endometrium. In some embodiments, the target or marker is a surface molecule that is not expressed on EECs of normal endometrium, e.g., normal endometrium in the proliferative stage of the estrous cycle (e.g., the follicular stage). In some embodiments, the target or marker is a surface molecule that is weakly expressed on EECs of normal endometrium, e.g., normal endometrium in the secretory stage of the estrous cycle (e.g., the luteal stage). In some embodiments, the target or marker is a surface molecule that is minimally to moderately expressed on stromal cells of normal endometrium, e.g., normal endometrium in the secretory stage of the estrous cycle (e.g., the luteal stage). In some embodiments, the target or marker is EVA1C.

[0087] In some embodiments, the target is a surface molecule on an endometrial endothelial cell (EEC). In some embodiments, expression of the surface molecule is upregulated on the EEC of an endometriotic (EM) lesion. In some embodiments, the EM lesion is selected from the group consisting of an ectopic peritoneal lesion, ectopic peritoneum adjacent lesion, and ectopic ovarian lesion. In some embodiments, expression of20MEl\57624905.v3Attorney Docket No. 117823-37820 the surface molecule is upregulated on the EEC of the eutopic endometrium (e.g., endometrial tissue located in the proper place in the uterus of an individual, e.g., of a healthy individual or an individual with endometriosis). In some embodiments, expression of the surface molecule is upregulated on the EEC of normal endometrium (e.g., the endometrium of an individual without a disease or disorder of the uterus, e.g., endometriosis). In some embodiments, the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein- coupled receptor 37 (GPR37), Germ cell-specific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C).

[0088] In some embodiments, expression of the surface molecule is upregulated on EECs as compared to expression on non-EECs (e.g., endothelial cells of non-endometrial tissue, e.g., peripheral lymph node (PLN), mesenteric lymph node (MLN), Peyer’s patch (PP), thymus, visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), small intestine (SI), colon, bone, bone marrow (BM), brain, dura, choroid plexus (Chp), pia mater (Pia), parenchyma, nasoturbinate / maxiloturbinate / septum (NTMTS), maxillary sinus (MS), nasal- associated lymphoid tissue (NALT), and skin). In some embodiments, the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein-coupled receptor 37 (GPR37), Germ cell-specific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C). In some embodiments, the surface molecule is RXFP1. In some embodiments, the surface molecule is EVA1C.

[0089] In some embodiments, the surface molecule (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C) is expressed at levels at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5- fold greater in EECs than in a reference population of cells (e.g., non-EECs or ECCs of normal endometrium) which may consist, for example, of a mixture containing an approximately equal amount of cells (e.g., approximately equal numbers of cells, approximately equal volume of cells, approximately equal mass of cells, etc.). In some embodiments, the surface molecule is present at levels at least 1.5 fold, at least 2 fold, at least21MEl\57624905.v3Attorney Docket No. 117823-378203 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 500 fold, at least 1000 fold, at least 5000 fold, or at least 10,000 fold greater than its average expression in a reference population. Detection or measurement of the surface molecule may make it possible to distinguish the cell type or types of interest from cells of many, most, or all other types.

[0090] In some embodiments, the expression of the surface molecule is upregulated in normal endometrium (e.g., the endometrium of an individual without a disease or disorder of the uterus, e.g., endometriosis). In some embodiments, the expression of the surface molecule is upregulated in normal endometrium as compared to expression in non-endometrial tissue (e.g., peripheral lymph node (PLN), mesenteric lymph node (MLN), Peyer’s patch (PP), thymus, visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), small intestine (SI), colon, bone, bone marrow (BM), brain, dura, choroid plexus (Chp), pia mater (Pia), parenchyma, nasoturbinate / maxiloturbinate / septum (NTMTS), maxillary sinus (MS), nasal- associated lymphoid tissue (NALT), and skin). In some embodiments, the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, and RXFP1. In some embodiments, the expression of the surface molecule is upregulated at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 500 fold, at least 1000 fold, at least 5000 fold, or at least 10,000 fold more in normal endometrium as compared to expression in non-endometrial tissue. In some embodiments, the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in normal endometrium as compared to expression in non-endometrial tissue. Suitable techniques for measuring the expression of the surface molecule in vivo (e.g., measurement of radiotracers using positron emission tomography (PET)) or in vitro (e.g., e.g., ELISA, western blotting, mass spectrometry, immunofluorescence, flow cytometry, etc.) will be clear to the skilled person.

[0091] In some embodiments, the expression of the surface molecule is upregulated in endometriotic (EM) lesions. In some embodiments, the expression of the surface molecule is upregulated in EM lesions as compared to expression in non-endometrial tissue (e.g., peripheral lymph node (PLN), mesenteric lymph node (MLN), Peyer’s patch (PP), thymus,22MEl\57624905.v3Attorney Docket No. 117823-37820 visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), small intestine (SI), colon, bone, bone marrow (BM), brain, dura, choroid plexus (Chp), pia mater (Pia), parenchyma, nasoturbinate / maxiloturbinate / septum (NTMTS), maxillary sinus (MS), nasal-associated lymphoid tissue (NALT), and skin). In some embodiments, the surface molecule is selected from RXFP1 and EVA1C. In some embodiments, the expression of the surface molecule is upregulated at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 500 fold, at least 1000 fold, at least 5000 fold, or at least 10,000 fold more in EM lesions as compared to expression in non-endometrial tissue. In some embodiments, the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in EM lesions as compared to expression in non-endometrial tissue. Suitable techniques for measuring the expression of the surface molecule in vivo (e.g., measurement of radiotracers using positron emission tomography (PET)) or in vitro (e.g., e.g., ELISA, western blotting, mass spectrometry, immunofluorescence, flow cytometry, etc.) will be clear to the skilled person.

[0092] In some embodiments, the expression of the surface molecule is upregulated in endometriotic (EM) lesions. In some embodiments, the expression of the surface molecule is upregulated in EM lesions as compared to expression in eutopic endometrium (e.g., endometrial tissue located in the proper place in the uterus of an individual, e.g., of a healthy individual or an individual with endometriosis). In some embodiments, the surface molecule is EVA1C. In some embodiments, the expression of the surface molecule is upregulated at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 500 fold, at least 1000 fold, at least 5000 fold, or at least 10,000 fold more in EM lesions as compared to expression in eutopic endometrium. In some embodiments, the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5- fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in EM lesions as compared to expression in eutopic endometrium. Suitable techniques for measuring the expression of the surface molecule in vivo (e.g., measurement of radiotracers using positron emission tomography (PET)) or in vitro (e.g., e.g., ELISA, western blotting,23MEl\57624905.v3Attorney Docket No. 117823-37820 mass spectrometry, immunofluorescence, flow cytometry, etc.) will be clear to the skilled person.

[0093] In some embodiments, the expression of the surface molecule is upregulated in endometriotic (EM) lesions. In some embodiments, the expression of the surface molecule is upregulated in EM lesions as compared to expression in normal endometrium (e.g., the endometrium of an individual without a disease or disorder of the uterus, e.g., endometriosis). In some embodiments, the surface molecule is EVA1C. In some embodiments, the expression of the surface molecule is upregulated at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 500 fold, at least 1000 fold, at least 5000 fold, or at least 10,000 fold more in EM lesions as compared to expression in normal endometrium. In some embodiments, the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in EM lesions as compared to expression in normal endometrium. Suitable techniques for measuring the expression of the surface molecule in vivo (e.g., measurement of radiotracers using positron emission tomography (PET)) or in vitro (e.g., e.g., ELISA, western blotting, mass spectrometry, immunofluorescence, flow cytometry, etc.) will be clear to the skilled person.

[0094] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecules are coupled (e.g., covalently associated) with an agent (e.g., therapeutic payload). In some embodiments, the agent is capable of inducing cell death of EECs of EM lesions or trigger local intra-vascular coagulation to shut down blood flow. In some embodiments, the agent is a small molecule, saccharide, oligosaccharide, polysaccharide, biologic (e.g., peptide, protein, peptide analog and derivatives, siRNAs, shRNAs, antisense RNAs, ribozymes, aptamers), peptidomimetic, dendrimers, nanoparticle, a second targeting molecule (e.g., a second targeting molecule that specifically binds to an antigen on the surface of an natural killer (NK) cell or a nanoparticle), or any combination thereof. In some embodiments, the agent is a pain reliever such as a nonsteroidal anti-inflammatory drug (e.g., aspirin, ibuprofen, or naproxen). In some embodiments, the agent is a hormonal therapy (e.g., gonadotropin-releasing hormone (Gn-24MEl\57624905.v3Attorney Docket No. 117823-37820RH) agonists and antagonists). In some embodiments, the agent is a vascular endothelial growth factor (VEGF) inhibitor.

[0095] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecule is coupled (e.g., covalently associated) with an agent (e.g., therapeutic payload). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC), wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C. In some embodiments, the agent is a small molecule, saccharide, oligosaccharide, polysaccharide, biologic (e.g., peptide, protein, peptide analog and derivatives, siRNAs, shRNAs, antisense RNAs, ribozymes, aptamers), peptidomimetic, dendrimers, nanoparticle, a second targeting molecule (e.g., a second targeting molecule that specifically binds to an antigen on the surface of an natural killer (NK) cell or a nanoparticle), or any combination thereof. In some embodiments, the agent is a pain reliever such as a nonsteroidal anti-inflammatory drug (e.g., aspirin, ibuprofen, or naproxen). In some embodiments, the agent is a hormonal therapy (e.g., gonadotropin-releasing hormone (Gn- RH) agonists and antagonists). In some embodiments, the agent is a vascular endothelial growth factor (VEGF) inhibitor.

[0096] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecule is coupled (e.g., covalently associated) with an agent (e.g., therapeutic payload). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of eutopic endometrium (e.g., endometrial tissue located in the proper place in the uterus of an individual, e.g., of a healthy individual or an individual with endometriosis), wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, and RXFP1. In some embodiments, the agent is a small molecule, saccharide, oligosaccharide, polysaccharide, biologic (e.g., peptide, protein, peptide analog and derivatives, siRNAs, shRNAs, antisense RNAs, ribozymes, aptamers), peptidomimetic, dendrimers, nanoparticle, a second targeting molecule (e.g., a second targeting molecule that specifically binds to an antigen on the surface of an natural killer (NK) cell or a nanoparticle), or any combination thereof. In some embodiments, the agent is a pain reliever such as a nonsteroidal antiinflammatory drug (e.g., aspirin, ibuprofen, or naproxen). In some embodiments, the agent is25MEl\57624905.v3Attorney Docket No. 117823-37820 a hormonal therapy (e.g., gonadotropin-releasing hormone (Gn-RH) agonists and antagonists). In some embodiments, the agent is a vascular endothelial growth factor (VEGF) inhibitor.

[0097] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecule is coupled (e.g., covalently associated) with an agent (e.g., therapeutic payload). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of normal endometrium, wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, and RXFP1. In some embodiments, the agent is a small molecule, saccharide, oligosaccharide, polysaccharide, biologic (e.g., peptide, protein, peptide analog and derivatives, siRNAs, shRNAs, antisense RNAs, ribozymes, aptamers), peptidomimetic, dendrimers, nanoparticle, a second targeting molecule (e.g., a second targeting molecule that specifically binds to an antigen on the surface of an natural killer (NK) cell or a nanoparticle), or any combination thereof. In some embodiments, the agent is a pain reliever such as a nonsteroidal anti-inflammatory drug (e.g., aspirin, ibuprofen, or naproxen). In some embodiments, the agent is a hormonal therapy (e.g., gonadotropin-releasing hormone (Gn- RH) agonists and antagonists). In some embodiments, the agent is a vascular endothelial growth factor (VEGF) inhibitor.

[0098] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecule is coupled (e.g., covalently associated) with an agent (e.g., therapeutic payload). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of an EM lesion, wherein the surface molecule is EVA1C and RXFP1. In some embodiments, the agent is a small molecule, saccharide, oligosaccharide, polysaccharide, biologic (e.g., peptide, protein, peptide analog and derivatives, siRNAs, shRNAs, antisense RNAs, ribozymes, aptamers), peptidomimetic, dendrimers, nanoparticle, a second targeting molecule (e.g., a second targeting molecule that specifically binds to an antigen on the surface of an natural killer (NK) cell or a nanoparticle), or any combination thereof. In some embodiments, the agent is a pain reliever such as a nonsteroidal anti-inflammatory drug (e.g., aspirin, ibuprofen, or naproxen). In some embodiments, the agent is a hormonal therapy (e.g., gonadotropin-26MEl\57624905.v3Attorney Docket No. 117823-37820 releasing hormone (Gn-RH) agonists and antagonists). In some embodiments, the agent is a vascular endothelial growth factor (VEGF) inhibitor.

[0099] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecule is coupled (e.g., covalently associated) with an agent (e.g., therapeutic payload). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of an EM lesion but not an EEC of the eutopic endometrium or normal endometrium, wherein the surface molecule is EVA1C. In some embodiments, the agent is a small molecule, saccharide, oligosaccharide, polysaccharide, biologic (e.g., peptide, protein, peptide analog and derivatives, siRNAs, shRNAs, antisense RNAs, ribozymes, aptamers), peptidomimetic, dendrimers, nanoparticle, a second targeting molecule (e.g., a second targeting molecule that specifically binds to an antigen on the surface of an natural killer (NK) cell or a nanoparticle), or any combination thereof. In some embodiments, the agent is a pain reliever such as a nonsteroidal anti-inflammatory drug (e.g., aspirin, ibuprofen, or naproxen). In some embodiments, the agent is a hormonal therapy (e.g., gonadotropin-releasing hormone (Gn- RH) agonists and antagonists). In some embodiments, the agent is a vascular endothelial growth factor (VEGF) inhibitor. In some embodiments, the targeting molecule is a nanobody, wherein the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

[0100] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecules are coupled (e.g., covalently associated) with an detectable marker to allow for in vivo imaging of EECs (e.g., EECs of eutopic endometrium and / or EM lesions). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC), wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes.

[0101] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecules are coupled (e.g.,27MEl\57624905.v3Attorney Docket No. 117823-37820 covalently associated) with an detectable marker to allow for in vivo imaging of EECs of eutopic endometrium (e.g., endometrial tissue located in the proper place in the uterus of an individual, e.g., of a healthy individual or an individual with endometriosis). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of eutopic endometrium, wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, and RXFP1. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes.

[0102] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecules are coupled (e.g., covalently associated) with an detectable marker to allow for in vivo imaging of EECs of normal endometrium. In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of normal endometrium, wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, and RXFP1. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radioisotopes, metals, metals chelates or metallic cations, chromophores and enzymes.

[0103] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecules are coupled (e.g., covalently associated) with an detectable marker to allow for in vivo imaging of EECs of an EM lesion. In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of an EM lesion, wherein the surface molecule is EVA1C and RXFP1. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes.

[0104] In some aspects, provided herein is a composition comprising a targeting molecule provided herein, and an agent. In some embodiments, targeting molecules are coupled (e.g., covalently associated) with an detectable marker to allow for in vivo, selective imaging of EECs of an EM lesion (e.g., in vivo imaging of EECs of an EM lesion and not eutopic or28MEl\57624905.v3Attorney Docket No. 117823-37820 normal endometrium). In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of an EM lesion, wherein the surface molecule is EVA1C. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes. In some embodiments, the targeting molecule is a nanobody, wherein the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

[0105] In some embodiments, covalent association is mediated by a linker.

[0106] In some embodiments, targeting molecules are not covalently associated with an agent (e.g., therapeutic payload). For example, targeting molecules may be associated with the surface of, encapsulated within, surrounded by, and / or distributed throughout the lipid formulation or polymeric matrix of an lipid nanoparticle, nanosphere, nanocarrier, microsphere, or microparticle. For example, in some embodiments, a targeting molecule can be encapsulated within, surrounded by, and / or dispersed throughout the liposomal membrane and / or polymeric matrix of a lipid nanoparticle, a nanosphere, a nanocarrier, a microsphere or a microparticle. Alternatively or additionally, a targeting molecule can be associated with a lipid nanoparticle or a nanocarrier by charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof.

[0107] The nanoparticles, nanospheres, nanocarrier, microparticles or microspheres may comprise one or more of polysaccharides, proteins, lipids, chitosan, alginate, pectin, xanthan gum, and cellulose. The nanoparticles, nanospheres or nanocarriers may be liposomes, polymeric micelles, dendrimers. Exemplary dendrimers include those comprising poly-L- lysine, polyamidoamine (PAMAM), polypropylene imine (PPI), liquid crystalline, core-shell, chiral, peptide, glycodendrimers and PAMAMOS dendrimers. Alternatively, the nanoparticles, nanospheres, nanocarrier, microparticles or microspheres may comprise an inorganic compound such as silver, gold, iron oxide, silica, zinc oxide, titanium oxide, platinum, selenium, gadolinium, palladium, or cerium dioxide.29MEl\57624905.v3Attorney Docket No. 117823-37820

[0108] In some embodiments, the targeting molecule is covalently linked to a lipid nanoparticle, nanosphere, nanocarrier, microsphere or microparticle. For example, the targeting molecule is linked to a nanoparticle, nanosphere, nanocarrier, microsphere or microparticle by a peptide linker. Exemplary peptide linkers include the dipeptide Vai Cit (VC), the tripeptide AAN, or longer peptide such as (GGGGS)(n=l, 2, 3, or 4) (SEQ ID NO: 7), (Gly)8 (SEQ ID NO: 8), (Gly)6 (SEQ ID NO: 9), (EAAAK)3(SEQ ID NO: 10), (EAAAK)(n=l-3) (SEQ ID NO: 11), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 12), PAPAP (SEQ ID NO: 13), AEAAAKEAAAKA (SEQ ID NO: 14), (Ala-Pro)n (10 - 34 aa) (SEQ ID NO: 15) . Other types of linkers include GPI-anchors and cross-linked polymers.

[0109] Alternatively, the targeting molecule is linked to a nanoparticle, nanosphere, nanocarrier, microsphere or microparticle by a cleavable linker such as an acid-labile linker, a protease cleavable linker, an enzyme cleavable linker, or a reducible disulfide linkage. Exemplary cleavable linkers include those comprising an ester bond such as a glutaryl linker, those comprising an amide bond and those comprising a carbamate bond. An exemplary acid-labile linker are hydrozone linkers.

[0110] In addition, the targeting molecule is linked to a nanoparticle, nanosphere, nanocarrier, microsphere or microparticle by an uncleavable such as an amide bond and a succinimidyl thioester linker or an amide bond and triazole linker or an oxime linker or a triazole linker.[OHl] In some embodiments, a targeting molecule in accordance with the present disclosure may be a protein or peptide. In certain embodiments, peptides range from about 5 to about 100, from about 5 to about 50, from about 10 to about 75, from about 15 to about 50, or from about 20 to about 25 amino acids in size. In some embodiments, a peptide sequence can be based on the sequence of a protein. In some embodiments, a peptide sequence can be a random arrangement of amino acids.

[0112] The terms “polypeptide” and “peptide” are used interchangeably herein, with “peptide” typically referring to a polypeptide having a length of less than about 100 amino acids. Polypeptides may contain L-amino acids, D-amino acids; or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, lipidation, phosphorylation, glycosylation, acylation, famesylation, sulfation, etc.30MEl\57624905.v3Attorney Docket No. 117823-37820

[0113] As used herein, the terms “fragment,” “derivative,” and “analog” refer to a polypeptide that substantially retains the same biological function or activity of a protein, e.g., antibody. Polypeptide fragments, derivatives or analogs of the disclosure may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably non-conservative amino acid residues) substituted. Such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing a mature polypeptide and another compound (such as a compound that increases the half-life of the polypeptide, for example, polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope of one of ordinary skill in the art.

[0114] Exemplary proteins that may be used as targeting molecules in accordance with the present disclosure include, but are not limited to, antibodies, receptors, cytokines, peptide hormones, glycoproteins, glycopeptides, proteoglycans, proteins derived from combinatorial libraries (e.g., Avimers™, Affibodies®, etc.), and characteristic portions thereof. Synthetic binding proteins such as Nanobodies™, AdNectins™, etc., can be used. In some embodiments, protein targeting molecules can be a nanobody.

[0115] In some embodiments, the nanobody specifically binds to EVA1C. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to GTISTWRDELVAAITQGGTTYYAVYYCAAVGTRKTTGEPYDYAY (SEQ ID NO: 1). In some embodiments, the nanobody comprises an amino acid sequence having at least 90% identity to GTISTWRDELVAAITQGGTTYYAVYYCAAVGTRKTTGEPYDYAY (SEQ ID NO: 1). In some embodiments, the nanobody comprises the amino acid sequence of GTISTWRDELVAAITQGGTTYYAVYYCAAVGTRKTTGEPYDYAY (SEQ ID NO: 1).

[0116] In some embodiments, the nanobody specifically binds to EVA1C. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,31MEl\57624905.v3Attorney Docket No. 117823-3782098% or 99% identity to GSISSEYDELVAAISGGGTTYYAVYYCAAPGYHRSSYYY (SEQ ID NO: 2). In some embodiments, the nanobody comprises an amino acid sequence having at least 90% identity to GSISSEYDELVAAISGGGTTYYAVYYCAAPGYHRSSYYY (SEQ ID NO: 2). In some embodiments, the nanobody comprises the amino acid sequence of GSISSEYDELVAAISGGGTTYYAVYYCAAPGYHRSSYYY (SEQ ID NO: 2).

[0117] In some embodiments, the nanobody specifically binds to EVA1C. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3). In some embodiments, the nanobody comprises an amino acid sequence having at least 90% identity to GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3). In some embodiments, the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

[0118] In some embodiments, the nanobody specifically binds to EVA1C. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to GTISARRTEFVAGINIGGITYYAVYYCAAPGGPDREHNY (SEQ ID NO: 4). In some embodiments, the nanobody comprises an amino acid sequence having at least 90% identity to GTISARRTEFVAGINIGGITYYAVYYCAAPGGPDREHNY (SEQ ID NO: 4). In some embodiments, the nanobody comprises the amino acid sequence of GTISARRTEFVAGINIGGITYYAVYYCAAPGGPDREHNY (SEQ ID NO: 4).

[0119] In some embodiments, the nanobody specifically binds to EVA1C. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to GTISAVQDELVAAINIGTTTYYAVYYCAAVYESKGYYALSHYY (SEQ ID NO: 5). In some embodiments, the nanobody comprises an amino acid sequence having at least 90% identity to GTISAVQDELVAAINIGTTTYYAVYYCAAVYESKGYYALSHYY (SEQ ID32MEl\57624905.v3Attorney Docket No. 117823-37820NO: 5). In some embodiments, the nanobody comprises the amino acid sequence of GTISAVQDELVAAINIGTTTYYAVYYCAAVYESKGYYALSHYY (SEQ ID NO: 5).

[0120] In some embodiments, the nanobody specifically binds to EVA1C. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to GTIFWYGDELVAAISNGGTTYYAVYYCAVVVIASIRGHPPHYY (SEQ ID NO: 6). In some embodiments, the nanobody comprises an amino acid sequence having at least 90% identity to GTIFWYGDELVAAISNGGTTYYAVYYCAVVVIASIRGHPPHYY (SEQ ID NO: 6). In some embodiments, the nanobody comprises the amino acid sequence of GTIFWYGDELVAAISNGGTTYYAVYYCAVVVIASIRGHPPHYY (SEQ ID NO: 6).

[0121] One of ordinary skill in the art will appreciate that any protein and / or peptide that specifically binds to a desired target as described herein, can be used in accordance with the present disclosure.

[0122] In some embodiments, a targeting molecule may be an antibody and / or characteristic portion thereof. The term “antibody” refers to any immunoglobulin, whether natural or wholly or partially synthetically produced and to derivatives thereof and characteristic portions thereof. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.

[0123] As used herein, an antibody fragment (i.e. characteristic portion of an antibody) refers to any derivative of an antibody which is less than full-length. In some embodiments, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of such antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragments also include, but are not limited, to Fc fragments.

[0124] An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and / or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively or additionally, an antibody fragment may comprise multiple chains which are linked together, for example, by disulfide linkages. An antibody fragment may33MEl\57624905.v3Attorney Docket No. 117823-37820 optionally comprise a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0125] In some embodiments, antibodies may include chimeric (e.g. “humanized”) and single chain (recombinant) antibodies. In some embodiments, antibodies may have reduced effector functions and / or bispecific molecules. In some embodiments, antibodies may include fragments produced by a Fab expression library.

[0126] Nanobodies are recombinant antibody fragments consisting of one variable heavy chain. In some embodiments, the variable heavy chain of a nanobody comprises a CDR1, CDR2, and CDR3. The CDR1 and CDR2 segments can be short in comparison to the CDR3 segment, which is longer than the typical CDR3 in a conventional antibody or scFv molecule.

[0127] In some embodiments, the nanobodies can comprise multiple (two or more) VH segments, such as a dimer. Peptide linker can be between VH segments. Each VH segment in a multimer nanobody can be the same VH sequence binding to the same antigen, or different VH sequence binding to different antigens, or different VH sequences binding the same antigen at non-overlapping epitopes. In some embodiments, the nanobodies can comprise multiple segments of VH segments as described above and scFv molecules.

[0128] In some embodiments, the nanobodies can be covalently linked to a drug (e.g., chemotherapeutic drug), imaging probe, or displayed on the surface of nanoparticles, viruses, or CAR T cells.

[0129] In some embodiment, the antibody or antigen-binding fragment thereof is covalently linked to one or more detectable markers (e.g., imaging probe or detectable labels) or other signal-generating groups or moieties, depending on the intended use of the labeled nanobody. Suitable markers and techniques for attaching, using and detecting them will be clear to the skilled person and, for example, include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes, metals, metals chelates or metallic cations or other metals or metallic cations that are particularly34MEl\57624905.v3Attorney Docket No. 117823-37820 suited for use in in vivo, in vitro or in situ diagnosis and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- VI- phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person and, for example, include moieties that can be detected using NMR or ESR spectroscopy.

[0130] Single-chain Fvs (scFvs) are recombinant antibody fragments consisting of only the variable light chain (VL) and variable heavy chain (VH) covalently connected to one another by a polypeptide linker. Either VL or VH may comprise the NH2 -terminal domain. The polypeptide linker may be of variable length and composition so long as the two variable domains are bridged without significant steric interference. Typically, linkers primarily comprise stretches of glycine and serine residues with some glutamic acid or lysine residues interspersed for solubility.

[0131] Diabodies are dimeric scFvs. Diabodies typically have shorter peptide linkers than most scFvs, and they often show a preference for associating as dimers.

[0132] An Fv fragment is an antibody fragment which consists of one VH and one VL domain held together by noncovalent interactions. The term “dsFv” as used herein refers to an Fv with an engineered intermolecular disulfide bond to stabilize the VH-VL pair.

[0133] An F(ab')2 fragment is an antibody fragment essentially equivalent to that obtained from immunoglobulins by digestion with an enzyme pepsin at pH 4.0-4.5. The fragment may be recombinantly produced.

[0134] A Fab' fragment is an antibody fragment essentially equivalent to that obtained by reduction of the disulfide bridge or bridges joining the two heavy chain pieces in the F(ab')2 fragment. The Fab' fragment may be recombinantly produced.

[0135] A Fab fragment is an antibody fragment essentially equivalent to that obtained by digestion of immunoglobulins with an enzyme (e.g., papain). The Fab fragment may be recombinantly produced. The heavy chain segment of the Fab fragment is the Fd piece.

[0136] In some aspects, provided herein is an engineered immune cell comprising: 1) a first polynucleotide encoding a first targeting molecule that specifically binds to a surface35MEl\57624905.v3Attorney Docket No. 117823-37820 molecule on an endometrial endothelial cell (EEC); and 2) a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell. Examples of the immune cell include, but not limited to, a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell. In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC), wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C.

[0137] In some aspects, provided herein is an engineered immune cell comprising: 1) a first polynucleotide encoding a first targeting molecule that specifically binds to a surface molecule on an endometrial endothelial cell (EEC); and 2) a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell. In some embodiments, the immune cell is selected from the group consisting of a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell. In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of eutopic endometrium (e.g., endometrial tissue located in the proper place in the uterus of an individual, e.g., of a healthy individual or an individual with endometriosis), wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, and RXFP1.

[0138] In some aspects, provided herein is an engineered immune cell comprising: 1) a first polynucleotide encoding a first targeting molecule that specifically binds to a surface molecule on an endometrial endothelial cell (EEC); and 2) a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell. In some embodiments, the immune cell is selected from the group consisting of a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell. In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of normal endometrium, wherein the surface molecule is selected from GPCR, ADRA2B, P2RX2, GPR37, GSG1, and RXFP1.

[0139] In some aspects, provided herein is an engineered immune cell comprising: 1) a first polynucleotide encoding a first targeting molecule that specifically binds to a surface molecule on an endometrial endothelial cell (EEC); and 2) a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell. In some embodiments, the immune cell is selected from the group consisting of a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell. In some embodiments,36MEl\57624905.v3Attorney Docket No. 117823-37820 the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of an EM lesion, wherein the surface molecule is EVA1C and RXFP1.

[0140] In some aspects, provided herein is an engineered immune cell comprising: 1) a first polynucleotide encoding a first targeting molecule that specifically binds to a surface molecule on an endometrial endothelial cell (EEC); and 2) a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell. In some embodiments, the immune cell is selected from the group consisting of a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell. In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) of an EM lesion but not an EEC of the eutopic endometrium or normal endometrium, wherein the surface molecule is EVA1C. In some embodiments, the targeting molecule is a nanobody, wherein the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).METHODS OF TREATMENT AND DIAGNOSIS

[0141] As used herein, the term “therapeutically effective amount” means an amount of a therapeutic, prophylactic, and / or diagnostic agent (e.g., inventive vaccine nanocarrier) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, alleviate, ameliorate, relieve, alleviate symptoms of, prevent, delay onset of, inhibit progression of, reduce severity of, and / or reduce incidence of the disease, disorder, and / or condition.

[0142] As used herein, the term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, prophylactic, and / or diagnostic effect and / elicits a desired biological and / or pharmacological effect.

[0143] As used herein, the term “treating” refers to a partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition (e.g., dysmenorrhea, pelvic pain, cramping, pain during intercourse, bowel movements and urination, heavy bleeding, and infertility associated with endometriosis). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a37MEl\57624905.v3Attorney Docket No. 117823-37820 disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment comprises delivery of a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure to a subject in need thereof.

[0144] In some embodiments, the disease or disorder is a disease or disorder of the uterus. In some embodiments, the disease or disorder is endometriosis, adenomyosis, uterine fibroids, polycystic ovary syndrome (PCOS), pelvic inflammatory disease (PID), and / or ovarian cysts. In some embodiments, the disease or disorder is endometriosis.

[0145] In some aspects, provided herein is a method of treating a disease or disorder of the uterus in a subject in need thereof, comprising administering to the subject a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure. In some aspects, provided herein is the use of a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease or disorder of the uterus in a subject in need thereof. In some aspects, provided herein is a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure for use in a method of treating a disease or disorder of the uterus in a subject in need thereof. In some embodiments, the subject is administered a targeting molecule that binds to a surface molecule on an endometrial endothelial cell (EEC) (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C). In some embodiments, the targeting molecule further comprises an agent (e.g., therapeutic payload).

[0146] In some aspects, provided herein is a method of treating a disease or disorder of the uterus in a subject in need thereof, comprising administering to the subject a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure. In some aspects, provided herein is the use of a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease or disorder of the uterus in a subject in need thereof. In some aspects, provided herein is a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure for use in a method of treating a disease or disorder of the uterus in a subject in need thereof. In some embodiments, the subject is administered a targeting molecule that38MEl\57624905.v3Attorney Docket No. 117823-37820 binds to a surface molecule on an endometrial endothelial cell (EEC), wherein the targeting molecule is RXFP1 or EVA1C. In some embodiments, the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) in which expression of the surface molecule is upregulated in endometriotic (EM) lesions. In some embodiments, the targeting molecule further comprises an agent (e.g., therapeutic payload). In some embodiments, the agent is capable of inducing cell death of EECs of EM lesions or trigger local intra-vascular coagulation to shut down blood flow. In some embodiments, the disease or disorder of the uterus is endometriosis.

[0147] In some aspects, provided herein is a method of treating endometriosis in a subject in need thereof, wherein the endometriosis is characterized by endometriotic (EM) lesions, comprising administering to the subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure to treat the endometriotic (EM) lesions. In some aspects, provided herein is the use of a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating endometriosis in a subject in need thereof, wherein the endometriosis is characterized by endometriotic (EM) lesions. In some aspects, provided herein is a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure for use in a method of treating endometriosis in a subject in need thereof. In some embodiments, the subject is administered a composition comprising a targeting molecule (e.g., an antibody or antigen-binding fragment thereof) which binds to the surface molecule and an agent (e.g., therapeutic payload) to treat the endometriotic (EM) lesions. In some embodiments, expression of the surface molecule is upregulated in endometriotic (EM) lesions (e.g., as compared to expression in non- endometrial tissue). In some embodiments, the surface molecule is RXFP1.

[0148] In some aspects, provided herein is a method of treating endometriosis in a subject in need thereof, wherein the endometriosis is characterized by endometriotic (EM) lesions, comprising administering to the subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure to treat the endometriotic (EM) lesions. In some aspects, provided herein is the use of a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating endometriosis in a subject39MEl\57624905.v3Attorney Docket No. 117823-37820 in need thereof, wherein the endometriosis is characterized by endometriotic (EM) lesions. In some aspects, provided herein is a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure for use in a method of treating endometriosis in a subject in need thereof. In some embodiments, the subject is administered a composition comprising a targeting molecule (e.g., an antibody or antigen-binding fragment thereof) which binds to the surface molecule on an endometrial endothelial cell (EEC) and an agent (e.g., therapeutic payload) to treat the endometriotic (EM) lesions. In some embodiments, expression of the surface molecule is upregulated in endometriotic (EM) lesions (e.g., as compared to expression in non-endometrial tissue, eutopic endometrium, and / or normal endometrium). In some embodiments, the surface molecule is EVA1C. In some embodiments, the subject is administered a targeting molecule (or a composition or pharmaceutical composition provided herein comprising said targeting molecule), wherein the targeting molecule is a nanobody. In some embodiments, the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

[0149] The term “diagnosis” as used herein refers to methods by which the skilled artisan can estimate and / or determine whether or not a patient is suffering from a given disease or condition. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, e.g., a biomarker, the presence, absence, amount, or change in amount of which is indicative of the presence, severity, or absence of the condition.

[0150] As used herein the term “prognosis” shall be taken to mean an indicator of the predicted progression of the disease (including but not limited to symptom recurrence, fertility outlook, and quality of life).

[0151] As used herein, the term “identifying” or grammatical variations thereof refer to determining the presence of a diagnostic indicators, e.g., uterine endothelial cells expressing one or more surface molecules of the present disclosure (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C), wherein the one or more surface molecules are upregulated when compared to a non-diseased control.

[0152] The term “control sample,” as used herein, refers to any clinically relevant control sample, including, for example, a sample from a healthy subject not afflicted with the disease or condition being assayed (e.g., endometriosis), a sample from a subject having a less severe40MEl\57624905.v3Attorney Docket No. 117823-37820 or slower progressing disease or condition (e.g., endometriosis) than the subject to be assessed, a sample from a subject having some other type of disease or disorder of the uterus, and the like. A control sample may include a sample derived from one or more subjects. A control sample may also be a sample made at an earlier timepoint from the subject to be assessed. For example, the control sample could be a sample taken from the subject to be assessed before the onset of the disease or condition being assayed (e.g., endometriosis), at an earlier stage of disease, or before the administration of treatment or of a portion of treatment. The control sample may also be a sample from an animal model, or from a tissue or cell lines derived from the animal model, of the disease or condition being assayed (e.g., endometriosis). For example, the expression level of a molecule, such as GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C, in a control sample that consists of a group of measurements may be determined based on any appropriate statistical measure, such as, for example, measures of central tendency including average, median, or modal values.

[0153] The term “control level” refers to an accepted or pre-determined expression level of a molecule (e g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C), which is used to compare with the expression level of a molecule (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, and EVA1C) from a subject (e.g., in a sample derived from the subject or measured in the subject in vivo). In one embodiment, the control level of a molecule is based on the expression level of the molecule from a subject(s) (e.g., in a sample derived from the subject(s) or measured in the subject(s) in vivo) having slow disease progression. In another embodiment, the control level of the molecule is based on the expression level from a subject(s) having rapid disease progression. In another embodiment, the control level of the molecule is based on an unaffected, i.e., non-diseased, subject(s), i.e., a subject who does not have a disease or disorder (e.g., endometriosis). In yet another embodiment, the control level of the molecule is based on the expression level of the molecule from a subject(s) prior to the administration of a therapy for the disease or disorder (e.g., endometriosis). In yet another embodiment, the control level of the molecule is based on the expression level of the molecule from a subject(s) after the administration of a therapy for the disease or disorder (e.g., endometriosis). In one embodiment, the control level of the molecule is based on the level from an animal model of a disease or disorder, (e.g.,41MEl\57624905.v3Attorney Docket No. 117823-37820 endometriosis), a cell, or a cell line derived from the animal model of a disease or disorder, (e.g., endometriosis).

[0154] In some aspects, provided herein is a medical imaging method comprising: (i) administering to a subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure, wherein the targeting molecule, composition, engineering immune cell, or pharmaceutical composition is associated with a detectable marker, and (ii) detecting the detectable marker in the body of the subject. In some embodiments, the method comprises administering to the subject a composition comprising a targeting molecule which binds to a surface molecule (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C) on an endometrial endothelial cell (EEC), wherein the targeting molecule is associated with a detectable marker. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes.

[0155] In some aspects, provided herein is a medical imaging method comprising: (i) administering to a subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure, wherein the targeting molecule, composition, engineering immune cell, or pharmaceutical composition is associated with a detectable marker, and (ii) detecting the detectable marker in the body of the subject. In some embodiments, the subject has or is suspected of having endometriosis characterized by endometriotic (EM) lesions. In some embodiments, the method comprises administering to the subject a composition comprising a targeting molecule which binds to the surface molecule RXFP1 on an endometrial endothelial cell (EEC), wherein the targeting molecule is associated with a detectable marker. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes. In some embodiments, the composition further comprises an agent (e.g., therapeutic payload) to treat the EM lesions. In some embodiments, expression of RXFP1 is upregulated in endometriotic (EM) lesions (e.g., as compared to expression in non- endometrial tissue).42MEl\57624905.v3Attorney Docket No. 117823-37820

[0156] In some aspects, provided herein is a medical imaging method comprising: (i) administering to a subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure, wherein the targeting molecule, composition, engineering immune cell, or pharmaceutical composition is associated with a detectable marker, and (ii) detecting the detectable marker in the body of the subject. In some embodiments, the subject has or is suspected of having endometriosis characterized by endometriotic (EM) lesions. In some embodiments, the method comprises administering to the subject a composition comprising a targeting molecule which binds to the surface molecule EVA1C on an endometrial endothelial cell (EEC), wherein the targeting molecule is associated with a detectable marker. In some embodiments, the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes. In some embodiments, the composition further comprises an agent (e.g., therapeutic payload) to treat the EM lesions. In some embodiments, expression of EVA1C is upregulated in endometriotic (EM) lesions (e.g., as compared to expression in non-endom etrial tissue, eutopic endometrium, and normal endometrium). In some embodiments, the subject is administered a targeting molecule (or a composition or pharmaceutical composition comprising said targeting molecule), wherein the targeting molecule is a nanobody. In some embodiments, the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

[0157] In some aspects, provided herein is a method of diagnosing or prognosing a disease or disorder of the uterus in a subject, comprising determining the expression of a surface molecule (e g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C) on an endometrial endothelial cell (EEC) of the subject. In some embodiments, determining the expression of a surface molecule on an endometrial endothelial cell (EEC) is performed in vivo. In some embodiments, prior to determining the expression of a surface molecule on an endometrial endothelial cell (EEC) of the subject, the method comprises administering to a subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure, wherein the targeting molecule, composition, engineering immune cell, or pharmaceutical composition is associated with a detectable43MEl\57624905.v3Attorney Docket No. 117823-37820 marker. In some embodiments, determining the expression of a surface molecule on an endometrial endothelial cell (EEC) comprising measuring the detectable marker. In some embodiments, upregulation of expression of the surface molecule on the EEC, as compared to a control level (e.g., expression of the surface molecule on an EEC from normal endometrium of an individual without endometriosis), is indicative of the presence or progression of the disease or disorder of the uterus. In some embodiments, upregulation of expression of the surface molecule on the EEC, as compared to a control level (e.g., expression of the surface molecule on an EEC from normal endometrium of an individual without endometriosis), is indicative of the presence or progression of EM lesions. In some embodiments, the disease or disorder of the uterus is endometriosis.

[0158] In some aspects, provided herein is a method of diagnosing or prognosing a disease or disorder of the uterus in a subject, comprising determining the expression of the surface molecule EVA1C on an endometrial endothelial cell (EEC) of the subject. In some embodiments, determining the expression of EVA1C on an endometrial endothelial cell (EEC) is performed in vivo. In some embodiments, prior to determining the expression of EVA1C on an endometrial endothelial cell (EEC) of the subject, the method comprises administering to a subject a targeting molecule, composition, engineering immune cell, or pharmaceutical composition of the present disclosure, wherein the targeting molecule, composition, engineering immune cell, or pharmaceutical composition is associated with a detectable marker. In some embodiments, determining the expression of EVA1C on an endometrial endothelial cell (EEC) comprising measuring the detectable marker. In some embodiments, upregulation of expression of EVA1C on the EEC, as compared to a control level (e.g., expression of EVA1C on an EEC from normal endometrium of an individual without endometriosis), is indicative of the presence or progression of the disease or disorder of the uterus. In some embodiments, upregulation of expression of EVA1C on the EEC, as compared to a control level (e.g., expression of EVA1C on an EEC from normal endometrium of an individual without endometriosis), is indicative of the presence or progression of EM lesions. In some embodiments, the disease or disorder of the uterus is endometriosis. In some embodiments, the subject is administered a targeting molecule (or a composition or pharmaceutical composition comprising said targeting molecule), wherein the targeting molecule is a nanobody. In some embodiments, the nanobody comprises the amino44MEl\57624905.v3Attorney Docket No. 117823-37820 acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

[0159] Suitable detection techniques for detectable markers will be clear to the skilled person (e.g., detection of radiotracers using positron emission tomography (PET)).

[0160] In some aspects, provided herein is a method of diagnosing or prognosing a disease or disorder of the uterus in a subject, comprising determining the expression of a surface molecule on an endometrial endothelial cell (EEC) of the subject. In some embodiments, prior to determining the expression of the surface molecule, the method comprises obtaining a biological sample comprising EECs from the subject. In some embodiments, the biological sample comprises an EM lesion. In some embodiments, after determining the expression of the surface molecule, the method further comprises comparing the expression of the surface molecule to expression of the surface molecule from a control sample (e.g., non-endometrial tissue or normal endometrium of an individual without the disease or disorder of the uterus). In some embodiments, upregulation of expression of the surface molecule in the sample, as compared to a control sample, is indicative of the presence or progression of the disease or disorder of the uterus. In some embodiments, the disease or disorder of the uterus is endometriosis. In some embodiments, the surface molecule is selected from the group consisting of GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C.

[0161] In some aspects, provided herein is a method of diagnosing or prognosing a disease or disorder of the uterus in a subject, comprising determining the expression of EVA1C on an endometrial endothelial cell (EEC) of the subject. In some embodiments, prior to determining the expression of EVA1C, the method comprises obtaining a biological sample comprising EECs from the subject. In some embodiments, the biological sample comprises an EM lesion. In some embodiments, after determining the expression of EVA1C, the method further comprises comparing the expression of EVA1C to expression of the surface molecule from a control sample (e.g., non-endometrial tissue or normal endometrium of an individual without the disease or disorder of the uterus). In some embodiments, upregulation of expression of EVA1C in the sample, as compared to a control sample, is indicative of the presence or progression of the disease or disorder of the uterus. In some embodiments, the disease or disorder of the uterus is endometriosis.45MEl\57624905.v3Attorney Docket No. 117823-37820

[0162] Suitable techniques for measuring expression of a surface molecule in a sample will be clear to the skilled person (e.g., ELISA, western blotting, mass spectrometry, immunofluorescence, flow cytometry, etc.).

[0163] In some embodiments, the methods of use provided herein include identifying if a subject has EECs that express one or more surface molecules of the present disclosure (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVAlC). In some embodiments, the method comprises administering a targeting molecule coupled to a diagnostic agent and determine a subject has EECs that express one or more surface molecules of the present disclosure. In some embodiments, the method comprises determining the one or more surface molecules are upregulated in comparison to a control cell (e.g., a non-endometrial endothelial cell or an EEC from normal endometrium of an individual without endometriosis). In some embodiments, the surface molecule is RXFP1. In some embodiments, the surface molecule is EVA1C.

[0164] In some embodiments, the methods of use provided herein include determining if a treatment of a disease or disorder of the uterus (e.g., endometriosis) in a subject is effective. In some embodiments, the targeting molecule is used to determine the presence of uterine vascular endothelial cells expressing the one or more surface molecules (e.g., GPCR, ADRA2B, P2RX2, GPR37, GSG1, RXFP1, or EVA1C) before treatment and after treatment and compared to a control cell (e.g., a non-endometrial endothelial cell or an EEC from normal endometrium of an individual without endometriosis). In some embodiments, after treatment, the presence of uterine endothelial cells expressing one or more surface molecules is decreased in comparison to prior administration of the treatment indicating the treatment is effective. In some embodiments, after treatment the presence of uterine endothelial cells expressing one or more surface molecules is increased or stays the same in comparison to prior administration of the treatment indicating the treatment is not effective.

[0165] In some embodiments, the methods of use provided herein include determining if a treatment of a disease or disorder of the uterus (e.g., endometriosis) in a subject is effective. In some embodiments, the targeting molecule is used to determine the presence of uterine vascular endothelial cells expressing the EVA1C before treatment and after treatment and compared to a control cell (e.g., a non-endometrial endothelial cell or an EEC from normal endometrium of an individual without endometriosis). In some embodiments, after treatment,46MEl\57624905.v3Attorney Docket No. 117823-37820 the presence of uterine endothelial cells expressing EVA1C is decreased in comparison to prior administration of the treatment indicating the treatment is effective. In some embodiments, after treatment the presence of uterine endothelial cells expressing EVA1C is increased or stays the same in comparison to prior administration of the treatment indicating the treatment is not effective. In some embodiments, the targeting molecule is a nanobody. In some embodiments, the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

[0166] In certain circumstances it will be desirable to deliver the targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure in suitably formulated compositions disclosed herein either by pipette, retro-orbital injection, subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricular (ICV), intravenous injection into the cistema magna (ICM), intracerebro-ventricularly, intramuscularly, intrathecally, intraspinally, orally, intraperitoneally, by oral or nasal inhalation, or by direct application or injection to one or more cells, tissues, or organs.

[0167] As used herein, the term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. The term further refers to a coding sequence for a desired expression product of a polynucleotide sequence such as a polypeptide, peptide, protein or interfering RNA including short interfering RNA (siRNA), miRNA or small hairpin RNA (shRNA). The sequences can also include degenerate codons of a reference sequence or sequences that may be introduced to provide codon preference in a specific organism or cell type. As used herein, the term “heterologous gene” refers to a gene provided to the target cell by an exogenous source, such as a viral vector, e.g., rAAV. In some embodiments, the gene encodes a polypeptide or a nucleic acid molecule, such as microRNA (miRNA), artificial microRNA (amiRNA), and short hairpin RNA (shRNA).FORMULATIONS

[0168] The targeting molecule, composition, or engineered immune cell of the present disclosure (referred to herein as physiologically active components) can be formulated with a47MEl\57624905.v3Attorney Docket No. 117823-37820 carrier that is suitable for administration to a cell, tissue slice, animal (e.g., mouse, nonhuman primate), or human. Physiologically active components within pharmaceutical compositions described herein can be prepared in neutral forms, as freebases, or as pharmacologically acceptable salts.

[0169] Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0170] Carriers of physiologically active components can include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, and the like. The use of such carriers for physiologically active components is well known in the art. Except insofar as any conventional media or agent is incompatible with the physiologically active components, it can be used with pharmaceutical compositions as described herein.

[0171] The phrase “pharmaceutically acceptable carriers” refer to carriers that do not produce an allergic or similar untoward reaction when administered to a human, and in some embodiments, when administered intravenously e.g., at the retro-orbital plexus).

[0172] In some embodiments, pharmaceutical compositions can be formulated for intravenous, intraocular, intravitreal, parenteral, subcutaneous, intracerebro-ventricular, intramuscular, intracerebroventricular, intravenous injection into the cisterna magna (ICM), intrathecal, intraspinal, oral, intraperitoneal, oral or nasal inhalation, or by direct injection in or application to one or more cells, tissues, or organs.

[0173] Pharmaceutical compositions may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles.

[0174] As used herein, the term “lipid nanoparticle” refers to a vesicle formed by one or more lipid components. Lipid nanoparticles are typically used as carriers for nucleic acid delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical48MEl\57624905.v3Attorney Docket No. 117823-37820 ingredient (API). Generally, lipid nanoparticle compositions for such delivery are composed of synthetic ionizable or cationic lipids, phospholipids (especially compounds having a phosphatidylcholine group), cholesterol, and a polyethylene glycol (PEG) lipid; however, these compositions may also include other lipids. The sum composition of lipids typically dictates the surface characteristics and thus the protein (opsonization) content in biological systems thus driving biodistribution and cell uptake properties.

[0175] As used herein, the “liposome” refers to lipid molecules assembled in a spherical configuration encapsulating an interior aqueous volume that is segregated from an aqueous exterior. Liposomes are vesicles that possess at least one lipid bilayer. Liposomes are typical used as carriers for drug / therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient. Liposome compositions for such delivery are typically composed of phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids.

[0176] As used herein, the term “ionizable lipid” refers to lipids having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. Ionizable lipids are also referred to as cationic lipids herein.

[0177] As used herein, the term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid.

[0178] As used herein, the term “conjugated lipid” refers to a lipid molecule conjugated with a non-lipid molecule, such as a PEG, polyoxazoline, polyamide, or polymer (e g., cationic polymer).

[0179] As used herein, the term “excipient” refers to pharmacologically inactive ingredients that are included in a formulation with the API, e.g., ceDNA and / or lipid49MEl\57624905.v3Attorney Docket No. 117823-37820 nanoparticles to bulk up and / or stabilize the formulation when producing a dosage form. General categories of excipients include, for example, bulking agents, fillers, diluents, antiadherents, binders, coatings, disintegrants, flavours, colors, lubricants, glidants, sorbents, preservatives, sweeteners, and products used for facilitating drug absorption or solubility or for other pharmacokinetic considerations.

[0180] The formation and use of liposomes is generally known to those of skill in the art. Liposomes have been developed with improved serum stability and circulation half-times (see, for instance, U.S. Pat. No. 5,741 ,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (see, for instance U.S. Pat. Nos. 5,567,434; 5,552, 157; 5,565,213; 5,738,868; and 5,795,587).

[0181] The disclosure also provides for pharmaceutically acceptable nanocapsule formulations of the physiologically active components. Nanocapsules can generally entrap compounds in a stable and reproducible way (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12): 11 13-1 128, 1998; Quintanar-Guerrero et al, Pharm Res. 15(7): 1056- 1062, 1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1): 107-1 19, 1998; Douglas et al, Crit Rev Ther Drug Carrier Syst 3 (3):233- 261, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles can be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure. Such particles can be easily made, as described in Couvreur et al., J Pharm Sci 69(2): 199-202, 1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlen et al., EurJ Pharm Biopharm, 45(2): 149-155, 1998; Zambau x et al., J Control Release 50(l-3):31- 40, 1998; and U.S. Pat. No. 5,145,684.

[0182] Injectable pharmaceutical compositions can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468). For delivery via injection, the form is sterile and fluid to the extent that it can be delivered by syringe. In some embodiments, it is stable under the conditions of manufacture and storage, and optionally contains one or more preservative compounds against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like),50MEl\57624905.v3Attorney Docket No. 117823-37820 suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and / or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the preparation will include an isotonic agent(s), for example, sugar(s) or sodium chloride. Prolonged absorption of the injectable pharmaceutical compositions can be accomplished by including in the pharmaceutical compositions of agents that delay absorption, for example, aluminum monostearate and gelatin. Injectable pharmaceutical compositions can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.

[0183] Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. As indicated, under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0184] Sterile pharmaceutical compositions can be prepared by incorporating the physiologically active component in an appropriate amount of a solvent with other optional ingredients (e.g., as enumerated above), followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized physiologically active components into a sterile vehicle that contains the basic dispersion medium and the required other ingredients (e.g., from those enumerated above). In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation can be vacuumdrying and freeze-drying techniques which yield a powder of the physiologically active components plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0185] Oral pharmaceutical compositions may be in liquid form, for example, as solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).51MEl\57624905.v3Attorney Docket No. 117823-37820The pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Tablets may be coated by methods well-known in the art.

[0186] Inhalable pharmaceutical compositions can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, tri chlorofluoromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0187] Pharmaceutical compositions can also include microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al, Prog Retin Eye Res, 17(l):33-58, 1998), transdermal matrices (U.S. Pat. No. 5,770,219 and U.S. Pat. No. 5,783,208) and feedback- controlled delivery (U.S. Pat. No. 5,697,899).

[0188] Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.

[0189] Typically, pharmaceutical compositions can include at least 0.1 % of the physiologically active components or more, although the percentage of the physiologically active components may, of course, be varied and may conveniently be between 1 or 2% and 70% or 80% or more or 0.5-99% of the weight or volume of the total composition. Naturally, the amount of physiologically active components in each physiologically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of pharmaceutical compositions and dosages may be desirable.52MEl\57624905.v3Attorney Docket No. 117823-37820

[0190] In some embodiments, for administration to humans, pharmaceutical compositions should meet sterility, pyrogenicity, and the general safety and purity standards as required by United States Food and Drug Administration (FDA) or other applicable regulatory agencies in other countries.KITS

[0191] A variety of kits and components can be prepared for use in the methods described herein, depending upon the intended use of the kit. Accordingly, in another aspect, provided herein is a kit comprising a targeting molecule, composition, engineered immune cell, or pharmaceutical composition of the present disclosure. A kit is any manufacture (e.g., a package or container) comprising a targeting molecule, composition, engineered immune cell, or pharmaceutical composition described herein. The manufacture can be promoted, distributed, or sold as a unit for performing the methods described herein.

[0192] The kits described herein can optionally comprise additional components and reagents. As will be appreciated by one of skill in the art, components of the kit can be provided in any desired form, e.g., in a lyophilized form, a liquid form, a solid form, or a concentrated. In some embodiments of the various aspects described herein, the kit comprises ampoules, syringes, or the like.

[0193] In some embodiments, the kit comprises informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein. The informational material of the kits is not limited in its form. In some embodiments, the informational material can include information about production of the reagents, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for using or administering the components of the kit.

[0194] It is noted that the components of a kit can provided singularly or in any combination as a kit. Such a kit includes the components described herein and packaging materials thereof.

[0195] In some embodiments, the compositions in a kit can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit. For example, the reagents described herein can be supplied in more than one53MEl\57624905.v3Attorney Docket No. 117823-37820 container, e.g., it can be supplied in a container having sufficient reagent for a predetermined number of applications, e.g., 1, 2, 3 or greater. One or more components as described herein can be provided in any form, e.g., liquid, dried or lyophilized form. Liquids or components for suspension or solution of the reagents can be provided in sterile form and should not contain microorganisms or other contaminants. When the components described herein are provided in a liquid solution, the liquid solution preferably is an aqueous solution.

[0196] The kit will typically be provided with its various elements included in one package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a Styrofoam box. The enclosure can be configured so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time.

[0197] In order that the present disclosure may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present disclosure in any manner.EXAMPLESEXAMPLE 1Summary

[0198] Herein, several protein molecules, which are expressed on the surface of endothelial cells (ECs) in endometrium and endometriotic (EM) lesions, were discovered. ECs form the inner lining of the body’s blood vessels, and molecules expressed on the surface of ECs are accessible via blood stream. The data showed that microvascular ECs in uterine endometrium express several surface molecules that are not found on ECs in other tissues. These proteins include G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), orphan GPCR GPR37, Germ cellspecific gene 1 (GSG1) protein, and Relaxin / insulin-like family peptide receptor 1 (RXFP1).

[0199] In addition, a computational analysis of a public single-cell RNAseq dataset of human biopsies (Tan, Y. et al. Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol 24, 1306-1318 (2022)) revealed that mRNA for RXFP1 is also robustly expressed in human normal endometrium and EM lesions. Further analysis of the54MEl\57624905.v3Attorney Docket No. 117823-37820 same dataset identified several proteins that are upregulated on ECs of EM lesions in comparison with normal uterine endometrium. Of these proteins, enhancer of ventral-axon guidance defects homolog C (EVA1C) is the most promising candidate because of its transmembrane expression and unique confinement to EM lesions ECs. These differentially expressed proteins are useful as targets for the detection and / or treatment of uterine and endometrial diseases and disorders.

[0200] Using antibodies (Abs), the protein surface expression of some of above-described molecules was confirmed on ECs of endometrium and EM lesions in the absence of their expression on ECs in other analyzed tissues. This allows for the direction of specifically engineered antibody-like proteins called anchorbodies (AxB), that carry functional payloads (such as radioisotopes, small molecules, biologies, nanoparticles) to proteins uniquely expressed on luminal surface of blood vessels of EM lesions. Delivery of radioisotopes and various biological materials will facilitate better diagnostics and potential treatment of endometriosis.Background

[0201] Endometriosis is an incurable chronic gynecological disorder characterized by the presence and growth of endometrium-like tissue outside of uterus. The tissue growth is referred to as ectopic or EM lesions. Although the precise pathogenesis of endometriosis is unknown, the commonly accepted Sampson’s theory suggests that EM lesions originate by cells detached from endometrium and lodged into abdominal cavity via fallopian tubes by retrograde menstruation. However, 75-90% of women experience retrograde menstruation, but not all of these women develop endometriosis, its mostly seen in individuals with hormonal or immunological issues (Chauhan S. et al. Endometriosis: A Review of Clinical Diagnosis, Treatment, and Pathogenesis. Cureus. 2022 Sep 6;14(9):e28864). Common symptoms include dysmenorrhea, pelvic pain, cramping, pain during intercourse, bowel movements and urination, heavy bleeding, and infertility.

[0202] The diagnosis of endometriosis is difficult. The median time elapsed from the onset of the symptoms to the definitive diagnosis is seven years, and the delay is even longer in younger women (M.S. Arruda, et al. Time elapsed from onset of symptoms to diagnosis of endometriosis in a cohort study of Brazilian women, Human Reproduction, Volume 18, Issue55MEl\57624905.v3Attorney Docket No. 117823-378204, April 2003, Pages 756-759). Ultrasound can show large lesions and scarring, which is another sign of endometriosis (Leonard! M., et al. How to perform an ultrasound to diagnose endometriosis. Australas J Ultrasound Med. 2018 Apr 22;21(2):61-69). Currently, the most definitive procedure to diagnose endometriosis is surgically invasive laparoscopy (Mak J, Leonard! M., et al. 'Seeing is believing': arguing for diagnostic laparoscopy as a diagnostic test for endometriosis. Reprod Fertil. 2022 Jun 10;3(3):C23-C28). During laparoscopy, detectable EM lesions are surgically removed, but since the lesions may vary in size, depth of penetration, location and appearance, it is nearly impossible to completely remove affected tissue, therefore the symptoms persist. Moreover, rare cases of thoracic (Yao J. et al., Endometriosis of the lung: A case report and review of literature. World J Clin Cases. 2023 Jun 26;11(18):4326-4333) and cerebral endometriosis (Sarma D. et al., Cerebellar endometriosis. Am J Roentgenol. 2004; 182(6): 1543-6) have been described, where laparoscopy cannot be performed.

[0203] Non-invasive endometriosis therapy mostly consists of symptomatic treatment, which usually starts with nonsteroidal anti-inflammatory drugs (NSAIDs) or steroids with androgenic action. Other treatments may include Gonadotropin-releasing hormone (GnRH) or vascular endothelial growth factor (VEGF) inhibitors, which can cause serious side effects. As a result of insufficient removal of endometriotic lesions during laparoscopy and high- failure rate of non-surgical therapies, ~30 out of 100,000 women diagnosed with endometriosis undergo a hysterectomy, a procedure that eliminates the anatomic source of endometrial cells but does not remove already existing lesions outside the uterus, thus the operation cannot be considered a radical method of treatment, which eliminates the disease.Data

[0204] The results provided herein comprises evidences that several molecular proteins are expressed on the surface of mouse (FIG. 1) and human (FIG. 2) uterine endometrium ECs on mRNA level. FIG. 1 demonstrates the results of bulk RNAseq analysis of mouse ECs sorted from 20 various tissues. The top five differentially expressed EC surface markers in the mouse uterus were RXFP1, ADRA2B, P2RX2, GPR37, and GSG1.

[0205] To determine if the detected proteins are also expressed on human ECs, a computational analysis of a public single-cell RNAseq dataset of human biopsy material was56MEl\57624905.v3Attorney Docket No. 117823-37820 performed (Tan, Y., et al. Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol 24, 1306-1318 (2022)) (FIG. 2). The results indicate, that from the previously identified candidates, RXFP1 was the most robustly expressed on ECs in normal human endometrium and in EM lesions from all analyzed patients.

[0206] To verify that RNAseq data translate into surface protein expression of RXFP1 on ECs, Ab against extracellular domain of RXFP1 was used to visualize the presence of the protein on ECs in normal endometrium and EM lesions by fluorescence-activated cell sorting (FACS) (FIGS. 3A-3B) and by confocal microscopy (FIGS. 4A-4B). To address the expression of molecules on ECs in EM lesions, endometriosis was induced in young female mice as described previously (Fattori V. et al. Nonsurgical mouse model of endometriosis- associated pain that responds to clinically active drugs. Pain. 2020 Jun; 161(6): 1321-1331). Briefly, recipient mice were injected intraperitoneally with small pieces of uterine tissue obtained from donor female mice. Peritoneal EM lesions were harvested on day 28 post injection and prepared for FACS or immunohistochemistry (IHC) (FIG. 8A).

[0207] FACS data indicate that RXFP1 was sufficiently expressed on CD45- CD31+gp38- blood ECs in both normal endometrium and peritoneal EM lesions (-43-45%) (FIG. 3A), but not in other tissues analyzed (FIG. 3B).

[0208] In addition, protein expression of RXFP1 was verified on mouse ECs in normal endometrium (FIG. 4A) and in peritoneal EM lesions (FIG. 4B) by confocal imaging.

[0209] In accordance to mouse data, RXFP1 expression was confirmed on ECs in normal human endometrium and in EM lesions (FIGS. 5A-5B).

[0210] Further evaluation of differential gene expression in human EM lesions versus normal endometrium (Tan, Y., et al. Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol 24, 1306-1318 (2022)), uncovered another EC surface molecule, EVA1C, which expression was upregulated on ECs of EM lesions when compared to normal endometrium (FIG. 6). According to the atlas, all other transmembrane hits except EVA1C were either poorly expressed on ECs across analyzed murine tissues or expressed on tissues other than normal endometrium (not shown).57MEl\57624905.v3Attorney Docket No. 117823-37820

[0211] To ensure that EVA1C was expressed on ECs of EM lesions on a protein level, confocal imaging of mouse normal uterus and peritoneal EM lesions was performed (FIGS. 7A-7B). Notably, EVA1C staining was absent in mouse normal endometrium. Further, EVA1C expression was confirmed on ECs in eutopic human endometrium and in EM lesions (FIG. 7C and FIG. 7D, respectively).

[0212] FIG. 21 provides a schematic of an exemplary workflow for discovering novel protein targets in human endometriosis lesions.Utility

[0213] Herein, several proteins, which are expressed on the surface of EC in endometrium and EM lesions, are identified. This identification allows for the use of the proteins as “anchors” on ECs for delivering various materials to endometrium and EM lesions via the blood stream. The precise material (such as radioisotopes, small molecules, and biologicals) are conjugated to antigen-like proteins called “anchorbodies” (AxBs), specific for the identified proteins. Among the EC-expressed proteins, EVA1C is prioritized, since it is more selective for EM lesions. However, AxBs specific for RXFP1, which is robustly expressed on EC of both normal endometrium and EM lesions, also are developed. RXFP1- or EVAlC-specific AxBs are generated using the following approach:1) Screening of an established yeast display library of cameloid nanobodies (NBs) for clones against ectodomains of RXFP1 or EVA1C, followed by isolation of the specific clones.2) Generation of AxBs by fusing human and murine IgG heavy chains with high affinity NBs. Since IgG-NBs fusions are much bigger in size than free NBs, they are expected to have a longer circulation half-life. Moreover, due to their small size (~10- 12kDa), free NBs might escape the circulation and bind off-target to extravascular expressed RXFP1 and EVAlC.Commercial Application

[0214] The disclosure herein provides is a novel, non-invasive approach to the diagnosis and treatment of endometriosis. The initial focus is on developing a molecular diagnostic that allows for the detection of EM lesions by non-invasive positron emission tomography (PET)58MEl\57624905.v3Attorney Docket No. 117823-37820 imaging utilizing AxBs specific for EC-expressed proteins in normal endometrium and EM lesions and functionalized by radioisotopes payload. This strategy may shorten the time between the onset of the disease and diagnosis, eliminate the need for costly and invasive diagnostic laparoscopy and allow to monitor the disease progression and response to therapy. In addition, the intra-operative use of fluorescently labeled AxBs could potentially improve the efficiency of diagnostic laparoscopy by enabling better detection of disseminated EM lesions under epi-fluorescent light.

[0215] The disclosure herein also provides a therapeutic AxBs specific for EC-expressed proteins exclusively in EM lesions (such as EVA1C). These AxBs could deliver drugs that kill EM tissue or trigger local intra-vascular coagulation to shut down blood flow. The ability to image EM lesions pre- and post-treatment provides an objective biomarker to monitor effects of these therapeutic interventions.EXAMPLE 2

[0216] Endometriosis, a debilitating gynecological condition that is thought to affect as many as -10% of women of child-bearing age, presents significant diagnostic and therapeutic challenges due to associated chronic pain, infertility, and complex presentation and pathophysiology. The only definitive diagnostic method available to date is laparoscopic surgery, and current treatments are generally limited to pain management, hormonal therapy or surgery, with high associated healthcare costs. This proposal aims to address the critical unmet need for improved diagnostics and treatment of endometriosis by focusing on two novel endothelial cell (EC) biomarkers, RXFP1 and EVA1C). These novel molecular targets were identified in preliminary work using a comprehensive transcriptome atlas of microvascular ECs and validated at the protein level in both human and mouse endometriosis samples.

[0217] Preliminary data show that ECs in endometriosis lesions upregulate RXFP1 and EVA1C on their luminal surface, whereby RXFP1 is also expressed in eutopic endometrium, whereas EVA1C is uniquely expressed in ectopic lesions. These findings suggest that targeting of endometriotic EC targets (EMECT) by engineered nanobodies (NAbs) could have therapeutic and / or diagnostic utility. Thus, this collaborative research seeks to develop and characterize anti-EMECT NAbs to explore their potential use as non-invasive diagnostics59MEl\57624905.v3Attorney Docket No. 117823-37820 and as delivery vehicles to target NK cells to endometriosis lesions as a novel means of cellular therapy. In the process, a panel of in vivo experimental mouse models combined with high-resolution spatial transcriptomics and proteomics can be utilized to refine the understanding of endometriosis pathology and to validate the impact of NK cells on the molecular, cellular and tissue level. This integrative strategy may ultimately enhance diagnostic precision, offer novel therapeutic targets, and eventually transform the clinical management of endometriosis.

[0218] The goals of this collaborative project are achieved in three specific aims: 1) to develop nanobodies targeting EMECT for non-invasive diagnosis and monitoring (HMS); 2) to explore the hypothesized protective role of NK cells in the disease and the potential to develop EMECT targeted NK cell therapies (HMS); and 3) to map the immunological landscape of human and murine endometriosis samples using cutting-edge spatial transcriptomics and proteomics (SNUH-SNUCM).Background

[0219] Endometriosis (EM), a gynecological condition affecting women of child-bearing age, is characterized by the ectopic growth of endometrial mucosa outside the uterus1. EM causes infertility, chronic pain, and other severe complications, imposing a significant burden on both patients and healthcare systems. Although the exact prevalence of EM is unknown, at least 10% of women aged 15 to 49 years are thought to be affected (-200 million globally, including -6-10 million in the US). Despite the high prevalence, EM is challenging to diagnose and treat. Patients spend a median time of 7 years after symptom onset, often visiting multiple providers and undergoing many inconclusive tests, before diagnosis. To date, the only definitive clinical test to diagnose EM is laparoscopy, a surgical procedure that requires anesthesia and carries a risk of bleeding and infection2. Treatments are palliative and limited to the use of NSAIDs, hormonal therapies or surgical interventions, often culminating in hysterectomy3. Because EM lesions can vary widely in size, penetration depth, distribution and appearance, laparoscopic surgery rarely results in complete removal of diseased tissue, so symptoms frequently persist. Moreover, EM lesions can also manifest outside the abdominal cavity, e.g., in the lung or central nervous system, which are not accessible by laparoscopy.60MEl\57624905.v3Attorney Docket No. 117823-37820Consequently, the economic impact of EM is profound, with healthcare costs in the US alone estimated at $100 billion annually4.

[0220] Considering the high unmet need for EM diagnosis and treatment, identifying tissue-specific markers is crucial for developing targeted therapies that enable non-invasive strategies to improve diagnostic precision and minimize off-target effects. A few clinical studies investigate non-invasive diagnostic tests for EM by analyzing either peripheral or menstrual blood5,6or uterine brush biopsies (herabiotech.com), however, these tests cannot identify the number, size or anatomic location of EM lesions. Medical imaging methods could theoretically provide this information, but EM lesions cannot be readily distinguished from other pathological changes (e.g. tumors, inflammation, cysts, etc.). This problem could potentially be addressed by novel molecular imaging strategies, but no reliable specific biomarkers for EM are known to exist.

[0221] The preliminary research provided herein has identified two novel markers, RXFP1 and EVA1C, which are upregulated in microvessels in EM lesions but not in surrounding non-uterine tissues. This discovery was made possible by a comprehensive RNA-Seq atlas generated from purified venular and non-venular microvascular ECs7across 20 different normal tissues in mice. Analysis of uterine ECs revealed a set of genes encoding surface molecules, including RXFP1, unique to uterine ECs and shared only by EM ECs. Furthermore, by mining public human single-cell RNA-Seq (scRNA-Seq) datasets of EM patients8, a second surface molecule, EVA1C, was identified that is preferentially expressed on ECs in EM lesions. These were validated findings at the protein level through immunohistochemistry, confirming expression in human endometrial and EM biopsies and experimentally induced EM lesions in mice.

[0222] A yeast display library of camelid NAbs9is screened to isolate clones specific for EVA1C, and an analogous screen for NAbs against RXFP1 is performed. After IV injection, NAbs are expected to accumulate rapidly within microvessels in EM lesions (RXFP1 and EVA1C) and / or normal endometrium (RXFP1 only). These NAbs could be modified with a radioisotope to allow PET imaging of EM lesions, whereby EVA1C would be strongly preferred as it would minimize radiation exposure to eutopic endometrial tissue. NAbs could also be engineered as synthetic homing receptors (SHOREs) expressed at high density on the surface of therapeutic lymphocytes, particularly natural killer (NK) cells which have both61MEl\57624905.v3Attorney Docket No. 117823-37820 been implicated in exerting a protective role in EM10. However, the role of NK cells has not been rigorously tested, and their utility as targets in EM therapy remains largely conjecture.

[0223] To gain a more detailed understanding of the immunological landscape of EM lesions, spatial multi-omics platforms at the single-cell level are applied to human and murine EM samples. This approach enables the mapping of the transcriptional and proteomic landscapes of ECs and immune cells, particularly focusing on the status of NK cells and microvascular ECs within human and murine EM lesions. By integrating omics data with functional studies in mouse models, the aim is to uncover the impact of NK cells on EM lesions and define cellular and molecular mechanisms to identify candidate therapeutic targets. Furthermore, considering that the initial validation cohort predominantly consisted of Caucasian patients, it is planned to evaluate EM biopsies from SNUH patients to assess the applicability of these studies to Asian populations.Objectives and Aims

[0224] The disclosure herein allows for (1) the identification of EM-specific EC targets (EMECTs) and (2) clarification of the role of NK cells in EM, enabling novel non-invasive diagnostic and therapeutic modalities for EM. To achieve this, there are three specific aims.1. The disclosure allows for the development of NAb panels targeting RXFP1 and EVA1C for non-invasive imaging of EM lesions.2. The disclosure allows for the defining of the role of NK cells in EM and evaluation of the potential for targeted NK cell therapy.3. The disclosure allows for the characterization of the immunological landscape of EM lesions using high resolution spatial transcriptomics and proteomics.Data

[0225] Herein, a molecular atlas of RNA-Seq profiles of ECs in over 20 different mouse tissues was generated. These data show that uterine microvascular ECs express surface molecules that are not found on ECs in any other tissue. One of these markers, RXFP1, is also expressed on ECs in experimentally induced EM lesions in mice (FIG. 8A-8C).

[0226] Comparison of differential gene expression in human scRNA-Seq data from EM vs. normal endometrium8uncovered a second EC surface molecule, EVA1C, that is62MEl\57624905.v3Attorney Docket No. 117823-37820 expressed in EM lesions, but not in endometrium or any other EC population in the atlas (not shown). Using commercial antibodies to murine and human EVA1C, it was confirmed that EVA1C expression at the protein level on ECs in EM lesions, but not in uterus (FIG. 9 and FIG. 10) or any other tissue analyzed (e.g. heart, thymus, ovary and fallopian tubes; not shown). Notably, as shown in FIG. 9, robust staining for EVA1C was observed on ECs in EM lesions (indicated by yellow color in the right panel), whereas ECs in endometrium were only stainable for CD31 (green structures in the left panel). Further, as shown in FIG. 10, robust staining for EVA1C was observed in EM microvessels, but not in uterine endometrium.

[0227] High-resolution Spatial Transcriptomics and Proteomics platforms was established (FIGS. 11A-11F).

[0228] The lOx Genomics Xenium platform in SNUCM was used to obtain high- resolution, target-specific spatial transcriptome data at the single-cell level. By creating custom marker panels derived from differentially expressed genes identified through singlecell transcriptomics, it was found that the platform is capable of precisely localizing and characterizing pathogenic cell clusters within skin tissue of Hidradenitis suppurativa (HS) lesions. This approach revealed interactions between pathogenic cells and their microenvironment and a comprehensive view of RNA markers at subcellular resolution. The data obtained through Xenium is highly correlated with scRNA-seq data, enabling cross- validation and enhancing the reliability of the findings.

[0229] Complementing the lOx Genomics Xenium platform, the MAC Sima™ platform at SNUH offers single-cell proteomic analysis, allowing the identification of over 100 protein markers simultaneously within tissue samples. Transcriptomic findings are evaluated at the proteomic level. The use of the MACSima™ platform promises to provide a detailed map of protein expression in EM lesions, further validating the impact of EMECTs and the functional state of immune cells, including NK cells and Tregs, within these lesions.

[0230] First, the disclosure allows for the development of NAb panels targeting RXFP1 and 2 for non-invasive imaging of EM lesions (HMS).

[0231] Having identified two distinct proteins that are continuously exposed to the flowing blood on ECs in EM lesions and endometrium (RXFP1) or EM lesions only (EVA1C), NAbs are generated against RXFP1 and EVA1C as molecular imaging and drug63MEl\57624905.v3Attorney Docket No. 117823-37820 targeting reagents (FIG. 12). EVA1C is more selective for EM lesions, however, an imaging agent targeting RXFP1 could also have utility and may be pursued. Using a well characterized yeast display library encoding camelid NAbs9, a panel of NAbs against another EC surface marker was produced for a research program that targets solid tumors. As the present project makes use of essentially the same technology, it is not expected that major technical obstacles to produce and characterize EMECT targeting NAbs.

[0232] Briefly, candidate NAbs are assessed by ELISA and surface plasmon resonance (SPR) for their antigen binding properties. Specificity for intact EMECT ectodomains are assessed using flow cytometry of EMECT transfected cell lines and appropriate controls. High affinity clones are further evaluated by IHC of frozen or paraffin sections of human and murine EM as in FIGS. 8-10. In vivo targeting are initially assessed by injecting NAbs (or NAb-coated fluorescent microbeads) into EM lesion bearing mice and assessing vascular staining in lesions, endometrium and control tissues (liver, lung, heart, brain, spleen, skin, lymph nodes, bone marrow, intra-abdominal organs) by IHC and / or FACS. A high affinity clone with robust binding characteristics is modified to allow PET imaging as outlined in FIG. 12

[0233] Further, the disclosure allows for the definition of the role of NK cells in EM and evaluation of the potential for targeted NK cell therapy.

[0234] There are numerous clinical reports implying that dysfunctional or reduced numbers of NK cells in EM patients may contribute to disease pathogenesis10'13. For example, compared to healthy individuals, NK cells in EM patients display lower cytotoxicity14, 15, reduced chemotaxis16and higher levels of inhibitory receptors17. Thus, it has been proposed that infusions of NK cells may be beneficial for NK cell therapy10, 18. However, these correlative human studies fall short of establishing mechanistic proof of a causative role of NK cell defects in EM.

[0235] A mouse model of experimentally induced EM19is established that offers the opportunity for rigorous mechanistic exploration of the role of NK cells in this disease. Indeed, prior studies in similar mouse models provide important clues. For example, depletion of NK cells with anti-asialo-GMl or an anti-IL2Rp MAb increased the weight of EM lesions as compared to NK cell sufficient controls20, 21. However, anti-asialo-GMl also affects macrophages22, and IL2RP is expressed on many lymphoid cells other than NK cells,64MEl\57624905.v3Attorney Docket No. 117823-37820 so the observed effects of these antibodies on EM lesions could have involved alternative immunological mechanisms. It is believed that the effects on adoptive NK cell therapy has not been explored in EM models. Therefore, in aim 2 herein, it is proposed to address this issue in a mouse model of experimental EM19.

[0236] EM lesion establishment and growth are monitored in the presence and absence of NK cells using both selective antibody depletion, genetic strategies and adoptive transfers to ask whether and to what extent NK cells may protect against EM. Briefly, female donor mice can be once treated with P-estradiol (3 pg SC) to synchronizes uteri to the preovulatory stage. Uteri can be harvested, finely minced and injected IP through an 18G cannula into congenic female recipients. After challenge, peritoneal EM lesions usually develop within 4 weeks and can be harvested for quantitative analysis of number, size, weight, histological features (H4C and omics, aim 3), leukocyte and EC composition, phenotype and function (FACS, H4C and omics).

[0237] A reasonably selective and proven tool for MAb deletion of NK cells (in C57BL6 mice) are MAb against NK1.123, a marker that is highly expressed on all NK cells, but is also found on NKT cells, ILC1 and some effector T cells. NKp46 is arguably the most selectively expressed marker on NK cells (and at low level on a subset of ILC3; rstats.immgen.org). However, although anti-NKp46 MAbs are commercially available, it is believes that these MAbs have not been tested for in vivo murine NK cell depletion. Whether commercial anti- NKp46 can be used to delete NK cells can be determined. If this approach is ineffective, using anti-NKl. l can be resorted to. EM lesion development can be quantified in groups of mice that either receive NK-depleting or isotype control MAb immediately before or 2, 7, or 14 days after EM challenge. If lesions can be visualized non-invasively (aim 1), cohorts of mice can be treated after lesions have become detectable to ask whether acute NK depletion alters lesion growth. In parallel, EM formation can be tested in a genetic model of NK cell deficiency using Nfil3- / - mice which lack conventional NK (cNK) cells, but retain tissue resident NK (trNK) cells24, IL15R- / - mice which lack all NK cells25. EM formation can be compared in these two strains as differences in disease burden may provide hints on the relative importance of trNK vs. cNK cells.

[0238] Depletion of NK cells enhances EM lesions, therefore it can be determined whether NK cell adoptive transfer can reduce or prevent EM lesion formation. To this end,65MEl\57624905.v3Attorney Docket No. 117823-37820NK cells can be purified from spleen (contains only cNK) and liver (contains -50:50 cNK and trNK) and transferred to IL15R- / - recipients prior to EM challenge. Previously, it was shown that a subset of CXCR6+ NK cells in liver, but not spleen have the capacity to develop long-lived antigen-specific immune memory23, 26. Thus, the activity of sort-purified hepatic CXCR6+ NK cells to CXCR6neg NK cells from spleen and liver can be compared. Aside from monitoring EM lesions, the fate of the transferred NK cells can be tracked in regard to distribution (number in blood, spleen, liver, uterus and EM lesions) effector function (cytokine profile, CD 107a, Gzma and perforin expression) and phenotype (NKG2D, CD 137, PD-1, Tim-3). By using IL15R- / - recipients, the effect of transferred NK cells in the absence of potentially confounding endogenous NK cells can be assessed. However, adoptive transfers to congenic (CD45.1) wildtype (NK sufficient) recipients can also be performed which would be more similar to a clinical setting.

[0239] Since NK sufficient mice develop EM lesions (albeit smaller than after NK cell depletion20, 21), it would be surprising if transfers of unmodified exogenous NK cells could completely abrogate EM lesions. More likely, these strategies may show partial efficacy with reduced lesion size and growth rate. The expected inability to clear lesions completely could potentially reflect insufficient homing of migratory NK cells to EM lesions. Thus, it can be asked whether use of a synthetic homing receptor (SHORE) that enables blood-borne NK cells to bind to EVA1C on ECs within EM lesions could enhance the therapeutic effect (FIG. 13). Competitive homing experiments can be performed to ask whether EMECT-SHORE modified NK cells migrate to EM lesions at a higher frequency than control NK cells expressing a non-binding SHORE. If this approach enables enhanced NK trafficking to EM lesions, it can be asked whether this effect can boost the therapeutic impact of adoptive NK therapy in the model.

[0240] Finally, the disclosure allows for the characterization of the immunological landscape of EM lesions using high resolution spatial transcriptoraics and proteomics.

[0241] This synergistic effort allows for the assessment of the immunological role of specific immune cell subsets in EM with single-cell resolution using spatial transcriptomics and spatial proteomics in both mouse tissues and biopsies from a cohort of severe EM patients.66MEl\57624905.v3Attorney Docket No. 117823-37820

[0242] A Korean EM patient cohort can be established to obtain tissue samples with approval from the SNUH IRB. Murine EM lesion samples can be generated and murine samples can be subjected to a battery of 'omics' analytical technologies, including:

[0243] a) Single-cell spatial transcriptomics using the lOx Genomics Xenium platform. Given the importance of immune dysregulation in EM where Tregs are often elevated and NK cell activity is suppressed, contributing to lesion persistence and progression10, 27, 28, this analysis initially focuses on mapping the immune cell landscape in EM. To explore the role of NK cells in disease progression, spatial and single-cell transcriptomic data can be used to assess the impact of NK cell depletion. Additionally, it is hypothesized that among the various NK cell subclusters, there may be specific clusters crucial for suppression of EM development. Reductions in specific NK cell subpopulations can be screened for and key molecules among these subtypes can be defined.

[0244] b) Protein-level target validation. Findings from the transcriptomics analysis and in vivo functional studies at the protein level can be validated using the MACSima™ platform, which allows for high-dimensional, single-cell proteomic analysis, enabling the examination of the expression of hundreds of proteins simultaneously within EM lesions. Detailed protein mapping may validate the impact of EMECT targeting therapies and assess the functional states of immune cells within treated lesions. This approach can provide a comprehensive understanding of how the therapeutic interventions herein affect the molecular and cellular environment of EM.

[0245] c) By correlating human EM omics results with clinical symptoms (e.g. infertility, progression to endometrial clear cell carcinoma), validation of EMECT and NK cells as diagnostic and / or therapeutic targets is sought.References1. Zondervan, K.T., Becker, C.M. & Missmer, S.A. Endometriosis. N Engl J Med 382, 1244-1256 (2020).2. Arruda, M.S., Petta, C.A., Abrao, M.S. & Benetti-Pinto, C.L. Time elapsed from onset of symptoms to diagnosis of endometriosis in a cohort study of Brazilian women. Hum Reprod 18, 756-759 (2003).67MEl\57624905.v3Attorney Docket No. 117823-378203. Weir, E., Mustard, C., Cohen, M. & Kung, R. Endometriosis: what is the risk of hospital admission, readmission, and major surgical intervention? J Minim Invasive Gynecol 12, 486-493 (2005).4. Soliman, A.M., Surrey, E., Bonafede, M., Nelson, J.K. & Castelli-Haley, J. Real- World Evaluation of Direct and Indirect Economic Burden Among Endometriosis Patients in the United States. Adv Ther 35, 408-423 (2018).5. Nisenblat, V. et al. Blood biomarkers for the non-invasive diagnosis of endometriosis. Cochrane Database Syst Rev 2016, CD012179 (2016).6. Warren, L.A. et al. Analysis of menstrual effluent: diagnostic potential for endometriosis. Mol Med 24, 1 (2018).7. Thiriot, A. et al. Differential DARC / ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues. BMC Biol 15, 45 (2017).8. Tan, Y. et al. Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol 24, 1306-1318 (2022).9. McMahon, C. et al. Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol 25, 289-296 (2018).10. Hoogstad-van Evert, J., Paap, R., Nap, A. & van der Molen, R. The Promises of Natural Killer Cell Therapy in Endometriosis. Int J Mol Sci 23 (2022).11. Yang, S., Wang, H., Li, D. & Li, M. An Estrogen-NK Cells Regulatory Axis in Endometriosis, Related Infertility, and Miscarriage. Int J Mol Sci 25 (2024).12. Thiruchelvam, U., Wingfield, M. & O'Farrelly, C. Natural Killer Cells: Key Players in Endometriosis. Am J Reprod Immunol 74, 291-301 (2015).13. Sikora, J., Mielczarek-Palacz, A. & Kondera-Anasz, Z. Role of natural killer cell activity in the pathogenesis of endometriosis. Curr Med Chem 18, 200-208 (2011).14. Jeung, I., Cheon, K. & Kim, M.R. Decreased Cytotoxicity of Peripheral and Peritoneal Natural Killer Cell in Endometriosis. Biomed Res Int 2016, 2916070 (2016).15. Wang, L. et al. A History of Endometriosis Is Associated With Decreased Peripheral NK Cytotoxicity and Increased Infiltration of Uterine CD68(+) Macrophages. Front Immunol 12, 711231 (2021).68MEl\57624905.v3Attorney Docket No. 117823-3782016. Ushiwaka, T. et al. Peritoneal natural killer cell chemotaxis is decreased in women with pelvic endometriosis. Am J Reprod Immunol 88, el3556 (2022).17. Wu, M.Y. et al. Increase in the expression of killer cell inhibitory receptors on peritoneal natural killer cells in women with endometriosis. Fertil Steril 74, 1187-1191 (2000).18. Artemova, D. et al. The prospects of cell therapy for endometriosis. J Assist Reprod Genet 40, 955-967 (2023).19. Fattori, V. et al. Nonsurgical mouse model of endometriosis-associated pain that responds to clinically active drugs. Pain 161, 1321-1331 (2020).20. Du, Y., Liu, X. & Guo, S.W. Platelets impair natural killer cell reactivity and function in endometriosis through multiple mechanisms. Hum Reprod 32, 794-810 (2017).21. Itoh, H., Sashihara, T., Hosono, A., Kaminogawa, S. & Uchida, M. Interleukin- 12 inhibits development of ectopic endometriotic tissues in peritoneal cavity via activation of NK cells in a murine endometriosis model. Cytotechnology 63, 133-141 (2011).22. Wiltrout, R.H. et al. Reactivity of anti-asialo GM1 serum with tumoricidal and non-tumoricidal mouse macrophages. J Leukoc Biol 37, 597-614 (1985).23. O'Leary, J.G., Goodarzi, M., Drayton, D.L. & von Andrian, U.H. T cell- and B cell-independent adaptive immunity mediated by natural killer cells. Nat Immunol 7, 507-516 (2006).24. Sojka, D.K. et al. Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells. Elife 3, e01659 (2014).25. Lodolce, J.P. et al. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9, 669-676 (1998).26. Paust, S. et al. Critical role for the chemokine receptor CXCR6 in NK cell- mediated antigen-specific memory of haptens and viruses. Nat Immunol 11, 1127-1135 (2010).27. Fazleabas, A.T., Braundmeier, A. & Parkin, K. Endometriosis-induced changes in regulatory T cells - insights towards developing permanent contraception. Contraception 92, 116-119 (2015).69MEl\57624905.v3Attorney Docket No. 117823-3782028. Chang, L.Y., Shan, J., Hou, X.X., Li, D.J. & Wang, X.Q. Synergy between Thl and Th2 responses during endometriosis: A review of current understanding. J Reprod Immunol 158, 103975 (2023).EXAMPLE 3

[0246] This example provides data demonstrating successful generation of nanobodies (NAbs) targeting human and mouse enhancer of ventral-axon guidance defects homolog C (EVA1C) protein.

[0247] First, recombinant ectodomains for both mouse and human EVA1C were synthesized (data not shown). The recombinant EVA1C ectodomains were tagged with a FLAG peptide and then used as a bait by incubating them with NAb-expressing yeast cells (identified by staining for an HA tag) followed by immuno-magnetic bead selection. Bait binding was detected by subsequent staining with a fluorescent anti-FLAG 2ndstage monoclonal antibody. Six rounds of selection were performed to isolate clones with high affinity and specificity for EVA1C. FIG. 14A provides an illustration of the yeast display library screening strategy. The results of this screening study are provided in FIG. 14B. EVA1C-FLAG binders were incrementally enriched in the total yeast population with successive selection steps on human (FIG. 14B, selections 1 to 4) followed by mouse (FIG. 14B, selections 5 to 6) recombinant EVA1C proteins.

[0248] After the sixth round of selection, cells were subcloned by single cell FACS sorting for the highest binders to human and mouse EVA1C. The sorted subclones were then screened by FACS for binding of soluble human and mouse EVA1C-FLAG. The results of this experiment are provided in FIG. 15. As shown in the figure, binding of NAb-expressing yeast (determined by an HA tag) to EVA1C protein (determined by FLAG tag) was demonstrated for several individual subclones. Notably, high expression of FLAG (up to a 100%) was observed on HA-high cells.

[0249] The amino acid sequences of sorted subclones A3, A5, B4, C3, C7, and C12 were determined and are presented in Table 1.70MEl\57624905.v3Attorney Docket No. 117823-37820Table 1. NAbs amino acid sequences

[0250] Next, coding genes of the highest binders to human and mouse EVA1C-FLAG were transferred into expression vectors for high yield protein expression in E. coli. Proteins were purified from bacterial supernatant by Ni resin columns, and the presence of NAbs in eluates was confirmed by SDS-PAGE (data not shown).

[0251] The purified NAbs were then characterized for specificity for EVA1C ectodomain using flow cytometry of mouse and human EVA1C transfected 293T cell lines. 293T EVA1C knock-out cells were used as a control. The results of this experiment are provided in FIGS.16A-16C. Shown in the figures are FACS histograms demonstrating binding of various NAb sclones to EVA1C KO 293T cells (FIG. 16A), human EVA1C transfected 293T cells (FIG. 16B), and mouse EVA1C transfected 293T cells (FIG. 16C). Notably, high binding of NAb clone B4 to both human and mouse EVA1C transfected cells was observed.

[0252] Candidate NAbs were also assessed by ELISA for their antigen binding properties. Briefly, ELISA plates were coated with human EVA1C-FLAG protein or negative control protein (both at 1 ug / ml), incubated with various concentrations of NAbs (range from 5uM to InM), and absorbance at 450 nm wavelength was assessed by spectrophotometry.FIGS. 17A-17B provide the results from this experiment, showing binding of NAb subclones71MEl\57624905.v3Attorney Docket No. 117823-37820 to plate-bound human EVA1C-FLAG protein. FIG. 17A shows the raw data as absorbance at 450 nm wavelength. FIG. 17B shows the percent of maximum absorbance, which is determined to calculate apparent Kd. The Kd represents the affinity of a tested NAb. Thus, the apparent Kd for B4 sublone is ~8nM, for A3 ~60nM. Notably, subclone B4, which demonstrated the highest binding ability to EVA1C ectodomain (FIGS. 16A-16C), also exhibited the highest antigen binding properties in the ELISA assay.

[0253] Based on the results from the flow cytometry and ELISA data (FIGS. 16A-16C and FIGS. 17A-17B, respectively), the most prominent NAb, subclone B4, was tested using an immunofluorescence approach. Specifically, anti-EVAlC NAb clone B4 was used to identify EVA1C expression on ECs of murine endometriotic lesions. Briefly, frozen tissue sections were co-stained with anti-CD31 to identify ECs and either control anti-GFP NAb or anti-EVAlC NAb clone B4. NAb staining was followed by staining with anti-VHH polyclonal antibody. The results from this experiment are provided in FIGS. 18A-18B. As shown in FIG. 18A, there was a notable absence of staining with the control anti-GFP NAb. However, as shown in FIG. 18B, robust staining of EVA1C on ECs of murine endometriotic lesions was demonstrated with the anti-EVAlC B4 NAb.EXAMPLE 4

[0254] This example provides data demonstrating that EVA1C expression in normal human endometrium is dependent on the estrous cycle.

[0255] Briefly, tissue sections of normal human endometrium from the proliferative and secretory stages of the estrous cycle were prepared and co-stained with anti-CD31 (to demarcate the vessels) and anti-EVAlC antibody followed by a 2nd stage antibody. Results from this experiment are provided in FIGS. 19A-19C and 20A-20C.

[0256] FIGS. 19A-19C provide a micrograph of normal human endometrium in the proliferative stage shown in grey scale. FIG. 19A shows the absence of co-localization of anti-CD31 and anti-EVAlC staining. FIG. 19B shows no expression of EVA1C on stromal cells. FIG. 19C shows single color staining of blood vessels by anti-CD31. Thus, EVA1C is not expressed in normal human endometrium at the proliferative stage of the estrous cycle.

[0257] FIGS. 20A-20C provides a micrograph of normal human endometrium in the secretory stage of the estrous cycle shown in grey scale. FIG. 20A shows co-localization of anti-CD31 and anti-EVAlC expression depicted by the white arrow. FIG. 20B shows low to72MEl\57624905.v3Attorney Docket No. 117823-37820 moderate expression of EVA1C on stromal cells. In addition, weak expression of EVA1C on ECs in one microvessel (indicated by the white arrow) is shown. FIG. 20C shows single color staining of blood vessels by anti-CD31. Thus, only weak expression of EVA1C is observed on EECs in normal human endometrium at the secretory stage of the estrous cycle.73MEl\57624905.v3

Claims

Attorney Docket No. 117823-37820CLAIMSWhat is claimed is:

1. A targeting molecule, wherein the targeting molecule binds to a surface molecule on an endometrial endothelial cell (EEC) in which expression of the surface molecule is upregulated.

2. The targeting molecule of claim 1, wherein the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein-coupled receptor 37 (GPR37), Germ cellspecific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C).

3. The targeting molecule of claim 1 or 2, wherein the expression of the surface molecule is upregulated in normal endometrium.

4. The targeting molecule of claim 3, wherein the expression of the surface molecule is upregulated in normal endometrium as compared to expression in non- endometrial tissue.

5. The targeting molecule of claim 4, wherein the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in normal endometrium as compared to expression in non-endometrial tissue.

6. The targeting molecule of claim 1 or 2, wherein the expression of the surface molecule is upregulated in endometriotic (EM) lesions.74MEl\57624905.v3Attorney Docket No. 117823-378207. The targeting molecule of claim 6, wherein the expression of the surface molecule is upregulated in EM lesions as compared to expression in non-endometrial tissue.

8. The targeting molecule of claim 7, wherein the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in EM lesions as compared to expression in non-endometrial tissue.

9. The targeting molecule of claim 1 or 2, wherein the expression of the surface molecule is upregulated in EM lesions as compared to expression in normal endometrium.

10. The targeting molecule of claim 9, wherein the expression of the surface molecule is upregulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold more in EM lesions as compared to expression in normal endometrium.

11. The targeting molecule of any one of claims 1, 2, 9, and 10, wherein the surface molecule is not expressed in normal endometrium.

12. The targeting molecule of claim 11, wherein the surface molecule is not expressed in normal endometrium in the proliferative stage of the estrous cycle.

13. The targeting molecule of any one of claims 1, 2, 9, and 10, wherein the surface molecule is weakly expressed in normal endometrium.75MEl\57624905.v3Attorney Docket No. 117823-3782014. The targeting molecule of claim 13, wherein the surface molecule is weakly expressed in normal endometrium in the secretory stage of the estrous cycle.

15. The targeting molecule of any one of claims 1, 2, and 6-14, wherein the surface molecule is EVA1C.

16. The targeting molecule of any one of claims 1-15, wherein the targeting molecule is an antibody or antigen-binding fragment thereof.

17. The targeting molecule of claim 16, wherein the antibody or antigen-binding fragment is a monoclonal antibody, human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a multispecific antibody, or an antigen-binding fragment thereof.

18. The targeting molecule of claim 16 or 17, wherein the antigen-binding fragment is 1) an Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2; 2) a diabody, ScFv, SMIP, single chain antibody, affibody, avimer, or nanobody; 3) a fusion of one or more heavy chains and one or more nanobodies; or 4) a single domain antibody.

19. The targeting molecule of claim 18, wherein the antigen-binding fragment is a nanobody.

20. The targeting molecule of claim 19, wherein the nanobody specifically binds to EVA1C.

21. The targeting molecule of claim 20, wherein the nanobody comprises an amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of:76MEl\57624905.v3Attorney Docket No. 117823-37820GTISTWRDELVAAITQGGTTYYAVYYCAAVGTRKTTGEPYDYAY (SEQ ID NO: 1); GSISSEYDELVAAISGGGTTYYAVYYCAAPGYHRSSYYY (SEQ ID NO: 2); GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3); GTISARRTEFVAGINIGGITYYAVYYCAAPGGPDREHNY (SEQ ID NO: 4); GTISAVQDELVAAINIGTTTYYAVYYCAAVYESKGYYALSHYY (SEQ ID NO: 5); and GTIFWYGDELVAAISNGGTTYYAVYYCAVVVIASIRGHPPHYY (SEQ ID NO: 6).

22. The targeting molecule of claim 20, wherein the nanobody comprises an amino acid sequence selected from the group consisting of: GTISTWRDELVAAITQGGTTYYAVYYCAAVGTRKTTGEPYDYAY (SEQ ID NO: 1); GSISSEYDELVAAISGGGTTYYAVYYCAAPGYHRSSYYY (SEQ ID NO: 2); GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3); GTISARRTEFVAGINIGGITYYAVYYCAAPGGPDREHNY (SEQ ID NO: 4); GTISAVQDELVAAINIGTTTYYAVYYCAAVYESKGYYALSHYY (SEQ ID NO: 5); and GTIFWYGDELVAAISNGGTTYYAVYYCAVVVIASIRGHPPHYY (SEQ ID NO: 6).

23. The targeting molecule of claim 20, wherein the nanobody comprises the amino acid sequence of GNIFWDYYEFVAAIDVGTNTNYAVYYCAAPDFFLWVNAQLYTY (SEQ ID NO: 3).

24. A composition comprising1) the targeting molecule of any one of claims 1-23, and2) an agent.

25. The composition of claim 24, wherein the agent is selected from the group consisting of radioisotopes, small molecules, biologies, nanoparticles, or a second targeting molecule.

26. The composition of claim 25, wherein the second targeting molecule specifically binds to an antigen on the surface of an NK cell or a nanoparticle.77MEl\57624905.v3Attorney Docket No. 117823-3782027. An engineered immune cell comprising:1) a first polynucleotide encoding a first targeting molecule that specifically binds to a surface molecule on an endometrial endothelial cell (EEC); and2) a second polynucleotide encoding a second targeting molecule that specifically binds to an immune cell.

28. The engineered immune cell of claim 27, wherein the first targeting molecule is the targeting molecule of any one of claims 1-23.

29. The engineered immune cell of claim 27 or 28, wherein the immune cell is a T cell, macrophage, monocyte, granulocyte, natural killer (NK) cell, or natural killer T (NKT) cell.

30. A pharmaceutical composition comprising 1) the targeting molecule of any one of claims 1-23, the composition of any one of claims 24-26, or the engineered immune cell of any one of claims 27-29; and 2) a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition comprises a lipid formulation.

32. A method of treating a disease or disorder of the uterus in a subject in need thereof, comprising administering to the subject the composition of any one of claims 24-26, the engineered immune cell of claim 27-29, or the pharmaceutical composition of claim 30 or 31.78MEl\57624905.v3Attorney Docket No. 117823-3782033. The method of claim 32, wherein the disease or disorder of the uterus is endometriosis.

34. A method of treating endometriosis in a subject in need thereof, wherein the endometriosis is characterized by endometriotic (EM) lesions, comprising administering to the subject a composition comprising a targeting molecule which binds to the surface molecule on an endometrial endothelial cell (EEC) and an agent to treat the endometriotic (EM) lesions.

35. A medical imaging method comprising (i) administering to a subject having endometriosis characterized by endometriotic (EM) lesions a composition comprising a targeting molecule which binds to a surface molecule on an endometrial endothelial cell (EEC) and an agent to treat the EM lesions, wherein the targeting molecule is associated with a detectable marker, and (ii) detecting the detectable marker in the body of the subject.

36. A medical imaging method comprising (i) administering to a subject a composition comprising a targeting molecule which binds to a surface molecule on an endometrial endothelial cell (EEC), wherein the targeting molecule is associated with a detectable marker, and (ii) detecting the detectable marker in the body of the subject.

37. The method of claim 35 or 36, wherein the detectable marker is selected from the group consisting of fluorescent labels, phosphorescent labels, chemiluminescent labels or bioluminescent labels, radio-isotopes, metals, metals chelates or metallic cations, chromophores and enzymes.

38. A method of diagnosing or prognosing a disease or disorder of the uterus in a subject, comprising determining the expression in vivo of a surface molecule on an endometrial endothelial cell (EEC) located within an endometriotic (EM) lesion, wherein79MEl\57624905.v3Attorney Docket No. 117823-37820 upregulation of expression of the surface molecule on the EEC, as compared to a control level, is indicative of the presence or progression of the disease or disorder of the uterus.

39. The method of any one of claims 34-38, wherein the targeting molecule is the targeting molecule of any one of claims 1-23.

40. The method of any one of claims 34-39, wherein the composition is the composition of any one of claims 24-26 or the pharmaceutical composition of claim 30 or 31.

41. The method of any one of claims 32-40, wherein the surface molecule is selected from G-protein coupled receptor (GPCR), Alpha-2B adrenergic receptor (ADRA2B), Purinergic Receptor P2X2 (P2RX2), G protein-coupled receptor 37 (GPR37), Germ cell-specific gene 1 (GSG1) protein, Relaxin / insulin-like family peptide receptor 1 (RXFP1), and Enhancer of ventral-axon guidance defects homolog C (EVA1C).80MEl\57624905.v3

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