Antibodies and conjugates against prostaglandin F2 receptor inhibitors and uses thereof

Abstract

The present disclosure provides antibodies and methods of making and using the antibodies, wherein the antibodies bind to PTGFRN on a cell.

Description

[0001] Related applications This application claims priority to U.S. Patent Application Serial No. 63 / 495,835, filed April 13, 2023, which is incorporated herein by reference in its entirety.

[0002] open field This disclosure relates to fully human antibodies and related molecules that bind to prostaglandin F2 receptor inhibitors (PTGFRN). This disclosure also relates to molecules comprising full-length antibodies, antibody fragments, or variants thereof, or alternatively molecules composed of full-length antibodies, antibody fragments, or variants thereof. This disclosure further relates to amino acid and nucleic acid sequences encoding such antibodies. This disclosure also relates to antibodies against PTGFRN (anti-PTGFRN antibodies), and in some particularly preferred embodiments, to antibody conjugates (e.g., antibody-drug conjugates or immunoconjugates) comprising anti-PTGFRN antibodies, compositions comprising anti-PTGFRN antibodies, and methods for using anti-PTGFRN antibodies and their conjugates to treat conditions (e.g., cancer) associated with PTGFRN expression. This disclosure further includes the use of said antibodies, their antigen-binding fragments, or antibody-drug conjugates, and corresponding methods for detecting and diagnosing pathological diseases associated with PTGFRN expression. This disclosure ultimately includes products and / or compositions or kits comprising at least such antibodies or antibody-drug conjugates for prognostic or diagnostic or therapeutic monitoring of such diseases.

[0003] Public background Prostaglandin F2 receptor negative regulator (“PTGFRN”) is a member of a subfamily of proteins known as tetraspan proteins. Tetraspan proteins are proteins that bind to each other and interact with multiple partners, forming what is called a “tetraspan protein network.” This “network” and its members participate as signaling molecules in multiple processes, such as fertilization (Glazar et al.). Immunoglobulin superfamily members IgSF8 (EWI-2) and CD9 in fertilization: Evidence that CD9 and CD9-related proteins have different functions in mammalian sperm-egg interactions. Reprod Fertil Dev. 2009;21(2):293–303; Swegen et al., From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule. Proteomics. 2017;17(17–18):1–13), migration (Chambrion et al., The tetraspanins CD9 and CD81 regulate CD9P1- induced effects on cell migration. PLoS One. 2010;5(6)), and lipid accumulation in preadipocytes (Orlicky et al., Synthesis and accumulation of areceptor regulatory protein associated with lipid droplet accumulation in 3T3-L1 cells. J Lipid Res. 1998;39(6):1152–61).In addition, PTGFRN has been found to affect extracellular vesicle bioactivity (Xu et al., Human perivascular stem cell-derived extracellular vesicles mediate bone repair. Elife. 2019;8:1–23), non-alcoholic fatty liver disease (Hotta et al., Identification of the genomic region under epigenetic regulation during non‐alcoholic fatty liver disease progression. Hepatol Res. 2018;(48):320–34), and Alzheimer's disease (Gerber et al., The APMAP interactome reveals new modulators of APP processing and beta-amyloid production that are altered in Alzheimer's disease. Acta Neuropathol Commun. 2019;7(1):13). Although the molecular mechanisms of these interactions are not fully elucidated, it is hypothesized that the binding of tetraspanin members to each other may promote and enhance their binding to other partners (Charrin et al., Multiple levels of interactions within the tetraspanin web. Biochem Biophys Res Commun. 2003;304(1):107–12, 2003; Mazurov et al., Tetraspanin protein CD9 interacts with metalloprotease CD10 and enhances its release via exosomes. FEBS J. 2013;280(5):1200–13).

[0004] Tetraspan proteins are the basis of complexes known as tetraspan protein enrichment microdomains (TEMs). TEMs have been found to promote signaling in a variety of different cellular pathways by acting as scaffolds for protein interactions and / or stabilization (Mazzocca et al., Tetraspanin-enriched microdomains and hepatocellular carcinomaprogression. Cancer Lett [Internet]. 2014;351(1):23–9). Available at: http: / / dx.doi.org / 10.1016 / j.canlet.2014.05.016; Yauch et al. The highly stoichiometric, stable and specific binding of integrin α3β1 to CD151 provides a major link for phosphatidylinositol 4-kinase and can regulate cell migration. Mol Biol Cell. 1998;9(10):2751–65). PTGFRN on the cell surface has been shown to be internalized after antibody binding, suggesting that PTGFRN may serve as a therapeutic target for certain cancer cell types that express PTGFRN (Marquez et al., Identification of Prostaglandin F2 Receptor Negative Regulator). (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development. PLoS One 16(1): e0246197). At the mRNA level, PTGFRN expression is increased in metastatic cancer cells (Karhemo et al., An optimized isolation of biotinylated cell surface proteins reveals novel players in cancer metastasis. J Proteomics [Internet]. 2012;77:87–100. Available at: http: / / dx.doi.org / 10.1016 / j.jprot.2012.07.009). In addition, PTGFRN expression is associated with biological functions that play an important role in tumor development.Aguila et al. demonstrated that PTGFRN is overexpressed in glioblastoma, and that higher PTGFRN expression in tumors is associated with poorer survival (Aguila et al., The Igsuperfamily protein PTGFRN coordinates survival signaling in Glioblastomamultiforme. Cancer Lett. 2019;462(April):33–42). Our laboratory, through immunohistochemical analysis of paraffin-embedded mesothelioma lesions using an anti-PTGFRN antibody developed in our laboratory, discovered, and discloses in this paper, that PTGFRN expression is negative in normal pleura, but increases in tumors progressing from benign to malignant (see, for example, Figure 1).

[0005] In a study published by Colin et al., PTGFRN was found to be crucial for angiogenesis, a necessary process for tumor growth (Colin et al., Br J Cancer. 2011;105(7):1002–11). It has also been shown that PTGFRN can be internalized, and that upregulation of PTGFRN is linked to the ability to target cancer cells via antibody-drug conjugates (ADCs) (Marquez et al., PLoS One 16(1): e0246197). Given the upregulation of PTGFRN in metastatic cancers, it would be beneficial to develop an antibody that can be used as is or as an antibody-drug conjugate (ADC) as a potential targeted therapy. Several antibody-drug conjugates (ADCs) have already been approved by the U.S. Food and Drug Administration for use in hematology and solid tumors. Antibody-drug conjugates (ADCs) are combinations of biological and small molecule drugs that have gained significant attention in recent years as a cancer treatment option. ADCs consist of a monoclonal antibody (mAb) that specifically binds to a cell surface target, an adapter, and a cytotoxic payload. Upon binding to its cell surface antigens, the mAb induces endocytosis of the antigens, shuttling the toxic payload within the cell. Depending on the linker type, the toxic payload is released from the mAb via a cleavable linker and leaves the lysosome before or during proteolysis (Tsuchikama et al., Antibody-drug conjugates: recent advances inconjugation and linker chemistries. Protein Cell. 2018;9(1):33–46). Alternatively, using an uncleavable linker, the payload cannot enter the cytosol until it has been completely degraded by the lysosome, at which point it can freely exert its anticancer effect (Jain et al., Current ADC Linker Chemistry. Pharm Res. 2015;32(11):3526–40). Furthermore, there is increasing interest in those that do not require internalization into the cell to achieve therapeutic efficacy. There are also ADCs with non-radioactive conjugates that still rely on extracellular cleavage of their linkers, allowing the cleaved drug to diffuse across the cell. In fact, the recently approved ADC Troveldy (Sacituzumab govitecan) operates in this manner (Cardillo et al., Clin Cancer Res. 2011;17(10):3157–69). The approved ADC for solid tumor targeting is Kadcyla, or T-DM1 (Diamantis et al., Br J Cancer 2016;114(4):362–7), for the treatment of HER-2 overexpressing breast cancer.It originates from the anti-HER2 monoclonal antibody trastuzumab, which was initially approved by the FDA as a non-conjugated therapeutic antibody for the treatment of HER2-overexpressing breast cancer. In Kadcyla... ®In this case, trastuzumab conjugates with emtansine via an irreducible thioether linker. Once T-DM1 enters the cell, emtansine binds to tubulin, leading to cell death through mitotic arrest (Teicher et al., The Promise of Antibody–Drug Conjugates. N Engl J Med. 2012;367(19):1847–8). T-DM1 has been reported to be more effective than trastuzumab. Compared to 77% of patients treated with trastuzumab, patients with HER2-positive cancer treated with T-DM1 had a 3-year disease-free survival rate of 88.3% (Von Minckwitz et al., Trastuzumab Emtansine for Residual Invasive HER2-Positive Breast Cancer. N Engl J Med. 2018;380(7):617). Trastuzumab is the latest generation of HER2-targeting ADCs, showing significant efficacy even in patients with HER2 expression immunohistochemical scores below 3+ and considered "HER2-negative" (Modi et al., (2022). "Trastuzumab Deruxtecan in Previously Treated HER2-Low Advanced Breast Cancer." New England Journal of Medicine 387(1): 9-20). Other FDA-approved ADCs include rituximab vedotin targeting CD30 / TNFRSF8; Gemtuzumab Ozogamici targeting CD33 / SIGLEC-3; Inotuzumab ozogamicin targeting CD22; pluutuzumab vedotin-piiq targeting CD79b; Enfortumab vedotin targeting Nectin-4; and Belantamab mafodotin targeting CD269.See, for example, Kitson et al., Antibody-Drug Conjugates (ADCs)–Biotherapeutic bullets. ChemToday. 2013;31(August):30–6; Kantarjian et al., N Engl J Med. 2016;375(8):740–53; Tilly et al., J Clin Oncol. 2019;37(15_suppl):TPS7571–TPS7571; Halford et al., Ann Pharmacother. 2020; and Tzogani et al., Oncologist. 2020;31(0):1–7). Recently, the FDA approved Sacituzumab Govitecan, a drug targeting Trop2 for the treatment of metastatic breast cancer. These data underscore the need to identify more cancer-related targets in addition to novel ADC therapies, particularly for solid tumors where targeted therapy needs are unmet (Zhao et al., Acta Pharm Sin B. 2020;10(9):1589–600). These results are encouraging for developing ADCs targeting PTGFRN in a variety of cancer types, such as squamous cell carcinoma, head and neck cancer, glioblastoma, pediatric medulloblastoma, or mesothelioma expressing PTGFRN, for which targeted therapy options are limited.

[0006] Despite the numerous examples described above, there remains a need in the art for monoclonal antibody-based therapeutics. In particular, there remains a need for fully human antibody therapeutics capable of binding to, detecting, imaging, treating, and / or eliminating cancer cells, thereby providing opportunities for the development of new therapeutics. This disclosure addresses this need, as well as other needs. This disclosure thus provides solutions to these and other known and unknown problems in the art. Brief description of the attached diagram Figure 1. Treatment of paraffin-embedded lungs (normal, normal) with anti-PTGFRN mouse monoclonal antibody [(1B4??)]. Figure 1A ) and mesothelioma (benign, Figure 1B Malignant Figure 1C Immunohistochemical staining (IHC) was performed on tissues to detect PTGFRN expression. IHC antibody 1B4 was used to stain tissue microarrays to assess PTGFRN expression levels. Healthy non-cancerous tissues, such as the colon ( Figure 1D ), breast ( Figure 1E ),lung( Figure 1F ), kidneys (Figure 1G), prostate ( Figure 1H ) and spleen ( Figure 1I), showing no detectable PTGFRN levels, while squamous cell carcinoma ( Figure 1J ), melanoma ( Figure 1K Tissue samples from pancreatic ductal adenocarcinoma (Fig. 1L), papillary renal cell carcinoma (Fig. 1M), and clear cell carcinoma (Fig. 1N) all showed elevated PTGFRN expression levels.

[0008] Figure 2. PTGFRN expression levels after shRNA or cDNA transfection. Western blot analysis showed that PTGFRN expression was significantly decreased after shRNA transfection in A431 and DAOY cells, while it was significantly increased after cDNA transfection in MSTO-211H cells. Relative increases or decreases in expression were assessed using commercial quantitative software and normalized to GAPDH expression. Data are presented as mean ± SD (n = 3).

[0009] Figure 3. Flow cytometry analysis of PTGFRN expression levels after transfection with shRNA or PTGFRN cDNA. Flow cytometry analysis using anti-PTGFRN antibody 8C7 confirmed that PTGFRN expression was significantly reduced after transfection with shRNA in (A) control shRNA A431, (B and C) A431 shRNA1 and shRNA2, (D) control shRNA DAOY, and (E and F) DAOY shRNA1 and shRNA2. Transfection of human PTGFRN cDNA into MSTO-211H cells resulted in a significant increase in PTGFRN expression, (G) empty vector MSTO-211H, (H) MSTO-PTG. Data are expressed as mean ± SD (n = 3).

[0010] Figure 4. Cell proliferation after PTGFRN silencing / overexpression. Cells were seeded in DME / F12 medium supplemented with 0.2% FBS as described in the Methods section. Silencing PTGFRN with shRNA at low serum levels (0.2% FBS) significantly reduced proliferation in (A) A431 and (B) DAOY cells, while overexpression of PTGFRN in (C) MSTO-211H cells resulted in significantly enhanced proliferation. Data are expressed as mean ± SD (n = 3). Significance: ****p < 0.0001, relative to control cells.

[0011] Figure 5. Effect of PTGFRN expression on cell migration. (A) Silencing PTGFRN via shRNA in A431 and DAOY cells significantly reduced cell migration, while (B) overexpression of PTGFRN in MSTO-211H cells significantly increased cell migration. Data are expressed as mean ± SD (n = 3). Significance: **p < 0.005, ****p < 0.0001, relative to control cells.

[0012] Figure 6. PTGFRN expression affects the ability of cells to form clones. (A) Reduced PTGFRN expression in A431 and DAOY cells resulted in fewer clones formed at low cell density seeding, while (B) PTGFRN overexpression in MSTO-211H cells showed a significant increase in clone number under the same conditions. Data are expressed as mean ± SD (n = 3). Significance: ***p < 0.001, ****p < 0.0001, relative to control cells.

[0013] Figure 7. PTGFRN expression knockdown inhibits globular formation. Figure 7A and Figure 7B Knocking down PTGFRN via shRNA prevents A431 and DAOY cells from forming spheroids under 3D culture conditions. Any aggregates formed in the DAOY shRNA clone disintegrate upon perturbation. Figure 7C A431 cells transfected with PTGFRN shRNA grown under globular culture conditions did not show an increase in integrin β1 and E. cadherin levels in control shRNA globular cells. This decrease in E. cadherin and integrin β1 following PTGFRN knockdown was not observed in cells grown under 2D culture conditions. Relative increases or decreases in expression were estimated using commercial quantitative software and normalized to GAPDH expression. Data are presented as mean ± SD (n = 3).

[0014] Figure 8. Co-localization of PTGFRN, E. cadherin, and integrin β1. Immunofluorescence using specific antibodies shows that E. cadherin (green) (… Figure 8A ), PTGFRN (red) Figure 8B ) and integrin β1 (purple) Figure 8C Colocalization was observed in the cell junction region (pink). The nuclei were counterstained with Hoechst 33342 (blue).

[0015] Figure 9. Effects of siRNA knockdown on the expression levels of other proteins. siRNA knockdown of E. cadherin and integrin β1 had no effect on PTGFRN expression. Figure 9AsiRNA knockdown of PTGFRN and integrin β1 had no effect on E. cadherin expression. Figure 9B siRNA knockdown of PTGFRN had no effect on integrin β1 expression, while siRNA knockdown of integrin β1 appeared to slightly increase E. cadherin expression. Figure 9C The relative increase or decrease in expression was assessed using commercial quantitative software and normalized to GAPDH expression. Data are expressed as mean ± SD (n = 3).

[0016] Figure 10. PTGFRN knockdown affects autophagy. PTGFRN knockdown in A431 cells led to increased LC3B transformation. This can be observed via fluorescence microscopy (…). Figure 10A Observations were made using an LC3B-specific fluorescent antibody (red) under microscopy; they were also made using Western blotting with an LC3B-specific antibody, including a positive control of A431 cells treated with metformin. Relative increases or decreases in expression were estimated using commercial quantitative software and normalized to GAPDH expression. Data are presented as mean ± SD (n = 3). Significance: ***p < 0.001, ****p < 0.0001, relative to control cells.

[0017] Figure 11. Mass spectrometry proteomic analysis after PTGFRN knockdown. The heatmap shows the top 20 cellular processes upregulated after PTGFRN knockdown based on upregulated protein expression. Figure 11A ) and the top 20 cellular processes downregulated after PTGFRN knockdown based on downregulated protein expression ( Figure 11B Statistical significance was determined by Student's T-test. All results met the threshold of p < 0.05.

[0018] Figure 12 Biological processes / pathways enriched after PTGFRN knockdown.

[0019] Figure 13 A431 cells were pre-incubated for 6 hours with 10 ug / ml hIgG, non-internalized anti-PTGFRN 3G3 or internalized 8C7, and then isolated and their migration was assessed by transwell assay.

[0020] Figure 14 Flow cytometry analysis using 8C7 antibody.

[0021] Figure 15. PTGFRN internalization via 8C7. 8C7 antibody binding to PTGFRN induces receptor endocytosis. Incubation with 8C7 (green) at 37°C for 0 hours (… Figure 15A ), three (3) hours ( Figure 15B) and five (5) hours ( Figure 15C Hoechst 33342 was used to counterstain cell nuclei (blue); 60X magnification.

[0022] Figure 16 The effect of the selected fully human anti-PTGFRN antibody on the proliferation and survival of A431 cells.

[0023] Figure 17 Effects of 8C7-Duocarmycin and 4F8-Duocarmycin conjugates on the proliferation and survival of various cell lines.

[0024] Figure 18 Effects of increased concentration of 6B2-duocarmycin conjugate on A431 proliferation and survival.

[0025] Figure 19. In vitro activity of 8C7-Duocarmycin ADC. Incubation of 8C7-Duocarmycin with PTGFRN-positive cancer cell lines produced significant anticancer efficacy. No efficacy was observed in the PTGFRN-negative cancer cell line (MDA-MB-231). Error bars represent standard deviation (** = P < 0.005; **** = P < 0.00005).

[0026] Figure 20. Administration of the fully human anti-PTGFRN 8C7-Duocarmycin antibody-drug conjugate inhibited three human cancer cell lines A431 in a dose-dependent manner. Figure 20A -B), MSTO-211H ( Figure 20C -D) and DAOY ( Figure 20E -F) tumor growth.

[0027] Figure 21. Western blot analysis of PTGFRN expression in seven types of head and neck cancer tumors from patients. 30 µg of seven different PDX tumor lysates were subjected to SDS-PAGE electrophoresis, then transferred to PVDF membranes for Western blot analysis using an anti-PTGFRN antibody. 30 µg of A431 cell lysates were analyzed as a positive control. GAPDH expression was used as an internal control to ensure consistent loading amounts.

[0028] Figure 22. Effects of 8C7-ADC and isotype control ADC on the growth of patient-derived head and neck tumors in NRG mice. Head and neck tumors JZ0628 were implanted into female NRG mice. When the tumor volume reached 100 mm³, the mice were randomly divided into two groups: the first group received intraperitoneal (IP) isotype control duocarmycin ADC once a week for 44 days, and the second group received 8C7-duocarmycin ADC.

[0029] Figure 23. Long-term effects of 8C7-ADC on H / N tumor growth after treatment cessation. On day 44, 8C7 treatment in the 8C7-ADC group was discontinued, and mice were observed for another 15 days to determine tumor growth.

[0030] Figure 24. Treatment of PTGFRN-negative tumors in nude mice. Treatment of nude mice carrying PTGFRN-negative MDA-MB-231 tumors with our 8C7-Duocarmycin antibody showed no difference in tumor growth or significant signs of toxicity compared to the control ADC, indicating that our 8C7 antibody has high specificity.

[0031] Figure 25. Exemplary 4F8-IgG (VH) antibody amino acid and nucleotide sequence.

[0032] Figure 26. Exemplary 4F8-IgK(VL) antibody amino acid and nucleotide sequences.

[0033] Figure 27. Exemplary 6B2-IgG (VH) antibody amino acid and nucleotide sequence.

[0034] Figure 28. Exemplary 6B2-IgK (VL) antibody amino acid and nucleotide sequences.

[0035] Figure 29. Exemplary 8C7-IgG (VH) antibody amino acid and nucleotide sequence.

[0036] Figure 30. Exemplary 8C7-IgK (VL) antibody amino acid and nucleotide sequences.

[0037] Figure 31. Exemplary 12D8-1-IgG (VH) antibody amino acid and nucleotide sequence.

[0038] Figure 32. Exemplary 12D8-1-IgK(VL) antibody amino acid and nucleotide sequences.

[0039] Public content This disclosure relates to and provides isolated antibodies, antigen-binding fragments, and / or derivatives thereof, said antibodies being antibodies 4F8, 6B2, 8C7, or 12D8, said antibodies comprising: a) a heavy chain variable region comprising a complementarity-determining region (CDR) sequence shown in any of Tables 1-13 (optionally including its framework (FR) amino acid sequence in some preferred embodiments); or a derivative of any of the above antibodies, optionally wherein said derivative comprises one to four amino acid substitutions in at least one of its CDRs; wherein said antibody or derivative specifically binds to a human prostaglandin F2 receptor inhibitor (PTGFRN). This disclosure also provides methods for preparing and using said antibodies or derivatives thereof. In some preferred embodiments, this disclosure provides antibody-drug conjugates (ADCs) of said antibodies. Polynucleotides and host cells comprising such polynucleotides are also provided. This disclosure also provides other embodiments, which will be apparent to those skilled in the art. Invention Details This disclosure provides isolated antibodies, antigen-binding fragments, and derivatives thereof that bind to prostaglandin F2 receptor inhibitors (PTGFRN), namely, anti-PTGFRN antibodies. Those skilled in the art also refer to PTGFRN as CD91P1 (CD9 chaperone protein 1), protein F containing the Glu-Trp-Ile EWI motif (EWIF), FPRP, KIAA1436, prostaglandin F2 receptor negative regulator, prostaglandin F2-α receptor regulatory protein, and prostaglandin F2-α receptor-associated protein, etc. Such antibodies, antigen-binding fragments, and derivatives thereof can be attached to one or more functional (or effector) parts (e.g., a detectable part, a cytotoxic part, etc.). This disclosure also includes the amino acid sequences of the variable heavy and light chains of the antibodies and their corresponding nucleic acid sequences. In one embodiment, the antibody of this disclosure may be a monoclonal antibody. In some embodiments, this disclosure provides isolated antibodies or antigen-binding fragments thereof that specifically bind to and are internalized by prostaglandin F2 receptor inhibitors (PTGFRN).

[0041] In some embodiments, this disclosure provides experiments using cell lines that overexpress PTGFRN using non-restrictive techniques, such as transfection with PTGFRN cDNA or transfection with siRNA or shRNA to inhibit PTGFRN expression. These experiments demonstrate that PTGFRN expression is associated with the ability to proliferate under low serum conditions, form clones at low cell densities, migrate via transwells, and form spheroids under three-dimensional culture conditions. Since these are biomarkers and characteristics of cancer stem cells, this suggests that PTGFRN is a biomarker and / or associated with the cancer stem cell phenotype. Therapeutic development targeting proteins associated with the cancer stem cell phenotype is known to provide a robust therapeutic approach to addressing drug resistance caused by maintaining cancer stem cells that cannot be eliminated by standard therapeutic agents. This makes targeting PTGFRN important because its expression is associated with or directly involved in cancer stem cells.

[0042] In some implementations, this disclosure provides information on the direct development of fully human monoclonal antibodies against PTGFRN. The advantage of this strategy is that it bypasses the need for maturation of humanized mouse monoclonal antibodies or phage-displayed antibody affinity. These processes are time-consuming and cumbersome, and can lead to alterations in antibody affinity, structure, or sequence, thereby reducing efficacy and increasing the risk of immunogenicity during patient administration.

[0043] Furthermore, in some embodiments, this disclosure provides human antibody-producing mice (TC-mAb) TM Uses of mice.

[0044] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the same prostaglandin F2 receptor inhibitor (PTGFRN) epitope as antibodies selected from the group consisting of any combination of complementarity-determining regions (CDRs) of each antibody referred to herein as 4F8, 6B2, 8C7, and 12D8 as shown in Table 1, and Figures 6-13 (alternative to (“ALT” CDRs, meaning not necessarily identified by the Kabat or Chothia methods), and their nucleotide-coding sequences. Framework and CDR sequences, along with their nucleotide-coding sequences, are also listed in Tables 2-13.

[0045] Table 1

[0046] Table 2 4F8 V H Chain framework (FR) (preferred) and CDR amino acid sequence (K) district Sequence fragments Residue length HFR1 EVQLVESGEGLVQPGRSLRLSCAASGFTFD (SEQ ID NO: 141) 1 - 30 30 CDR-H1 DYAMH (SEQ ID NO: 1) 31 - 35 5 HFR2 WVRQAPGKGLEWVS (SEQ ID NO: 142) 36 - 49 14 CDR-H2 GISWDSGRIGYADSVKG (SEQ ID NO: 2) 50 - 66 17 HFR3 RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK (SEQ ID NO: 143) 67 - 98 32 CDR-H3 DRGGGNWNYYYYGMDV (SEQ ID NO: 3) 99 - 114 16 HFR4 WGQGTTVTVSS (SEQ ID NO: 144) 115 - 125 11

[0047] Table 3 4F8 V H Chain framework (FR) (preferred) and CDR amino acid sequence (Ch) district Sequence fragments Residue length HFR1 EVQLVESGEGLVQPGRSLRLSCAAS (SEQ ID NO: 145) 1 - 25 25 CDR-H1(Ch) GFTFDDY (SEQ ID NO: 4) 26 - 32 7 HFR2 AMHWVRQAPGKGLEWVSGI (SEQ ID NO: 146) 33 - 51 19 CDR-H2(Ch) SWDSGR (SEQ ID NO: 5) 52 - 57 6 HFR3 IGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK (SEQ ID NO: 147) 58 - 98 41 CDR-H3(Ch) DRGGGNWNYYYYGMDV (SEQ ID NO: 6) 99 - 114 16 HFR4 WGQGTTVTVSS (SEQ ID NO: 148) 115 - 125 11

[0048] Table 4 4F8 V H Chain framework (FR) (preferred) and CDR amino acid sequence (K / Ch) district Sequence fragments Residue length LFR1 DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO: 149) 1 - 23 23 CDR-L1 RASQGISNYLA (SEQ ID NO: 7) 24 - 34 11 LFR2 WFQQKPGKAPKSLIY (SEQ ID NO: 150) 35 - 49 15 CDR-L2 AASSLQS (SEQ ID NO: 8) 50 - 56 7 LFR3 GVPSKFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 151) 57 - 88 32 CDR-L3 QQYNSFPFT (SEQ ID NO: 9) 89 - 97 9 LFR4 FGPGTKVDIK (SEQ ID NO: 152) 98 - 107 10

[0049] Table 5 6B2 V H Framework (FR) (preferred) and CDR amino acid sequence (K) district Sequence fragments Residue length HFR1 QLQLQESGPGLVKPSETLSLTCTVSGGSIS (SEQ ID NO: 153) 1 - 30 30 CDR-H1(K) SSSYYWD (SEQ ID NO: 18) 31 - 37 7 HFR2 WIRQPPGKGLEWIG (SEQ ID NO: 154) 38 - 51 14 CDR-H2(K) TIYYGGSTYYNPSLKS (SEQ ID NO: 19) 52 - 67 16 HFR3 RVTISVDTSKNQFSLKLNSVTAADTAVYYCAR (SEQ ID NO: 155) 68 - 99 32 CDR-H3(K) GNSYGLDY (SEQ ID NO: 20) 100 - 107 8 HFR4 WGQGTLVTVSS (SEQ ID NO: 156) 108 - 118 11

[0050] Table 6 6B2 V H Framework (FR) (preferred) and CDR amino acid sequence (Ch) district Sequence fragments Residue length HFR1 QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 157) 1 - 25 25 CDR-H1 (Ch) GGSISSSSY (SEQ ID NO: 21) 26 - 34 9 HFR2 YWDWIRQPPGKGLEWIGTI (SEQ ID NO: 158) 35 - 53 19 CDR-H2 (Ch) YYGGS (SEQ ID NO: 22) 54 - 58 5 HFR3 TYYNPSLKSRVTISSVDTSKNQFSLKLNSVTAADTAVYYCAR (SEQ ID NO: 159) 59 - 99 41 CDR-H3 (Ch) GNSYGLDY (SEQ ID NO: 23) 100 - 107 8 HFR4 WGQGTLVTVSS (SEQ ID NO: 160) 108 - 118 11

[0051] Table 7 6B2 V L Chain framework (FR) (preferred) and CDR amino acid sequence district Sequence fragments Residue length LFR1 DVVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 161) 1 - 23 23 CDR-L1 RSSQSLVHSDGNMYLN (SEQ ID NO: 24) 24 - 39 16 LFR2 WFQQRPGQSPRRLIY (SEQ ID NO: 162) 40 - 54 15 CDR-L2 KVSNRDS (SEQ ID NO: 25) 55 - 61 7 LFR3 GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC (SEQ ID NO: 163) 62 - 93 32 CDR-L3 MQSTHWPPVT (SEQ ID NO: 26) 94 - 103 10 LFR4 FGQGTKLEIK (SEQ ID NO: 164) 104 - 113 10

[0052] Table 8 8C7 V H Chain framework (FR) (preferred) and CDR amino acid sequence (K) district Sequence fragments Residue length HFR1 QLQLQESGPGLVKPSETLSLTCTVSGGSIS (SEQ ID NO: 165) 1 - 30 30 CDR-H1(K) SSSYYWG (SEQ ID NO: 36) 31 - 37 7 HFR2 WIRQPPGKGLEWIG (SEQ ID NO: 166) 38 - 51 14 CDR-H2(K) SIYYGGSTYYNPSLKS (SEQ ID NO: 37) 52 - 67 16 HFR3 RVTISVDTSTNQFSLKLNSVTAADTAVYYCAR (SEQ ID NO: 167) 68 - 99 32 CDR-H3(K) QGLGSFDC (SEQ ID NO: 38) 100 - 107 8 HFR4 WGQGTLVTVSS (SEQ ID NO: 168) 108 - 118 11

[0053] Table 9 8C7 V H Chain framework (FR) (preferred) and CDR amino acid sequence (Ch) district Sequence fragments Residue length HFR1 QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 169) 1 - 25 25 CDR-H1(Ch) GGSISSSSY (SEQ ID NO: 39) 26 - 34 9 HFR2 YWGWIRQPPGKGLEWIGSI (SEQ ID NO: 170) 35 - 53 19 CDR-H2(Ch) YYGGS (SEQ ID NO: 40) 54 - 58 5 HFR3 TYYNPSLKSRVTISVDTSTNQFSLKLNSVTAADTAVYYCAR (SEQ ID NO: 171) 59 - 99 41 CDR-H3(Ch) QGLGSFDC (SEQ ID NO: 41) 100 - 107 8 HFR4 WGQGTLVTVSS (SEQ ID NO: 172) 108 - 118 11

[0054] Table 10 8C7 V L Chain framework (FR) (preferred) and CDR amino acid sequence district Sequence fragments Residue length LFR1 DVVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 173) 1 - 23 23 CDR-L1 RSSQSLVHSDGNTYLN (SEQ ID NO: 42) 24 - 39 16 LFR2 WFQQRPGQSPRRLIY (SEQ ID NO: 174) 40 - 54 15 CDR-L2 KVSNRDS (SEQ ID NO: 43) 55 - 61 7 LFR3 GVPDRFSGSGSGTDFTLKISGVEAEDVGIYYC (SEQ ID NO: 175) 62 - 93 32 CDR-L3 MQGTHWPPLT (SEQ ID NO: 44) 94 - 103 10 LFR4 FGGGTKVEIK (SEQ ID NO: 176) 104 - 113 10

[0055] Table 11 12D8 V H Chain framework (FR) (preferred) and CDR amino acid sequence (K) district Sequence fragments Residue length HFR1 EVQLVESGGGLVQPGRSLRLSCAASGFTFD (SEQ ID NO: 177) 1-30 30 CDR-H1(K) DYAMH (SEQ ID NO: 54) 31-35 5 HFR2 WVRQAPGKGLEWVS (SEQ ID NO: 178) 36-50 14 CDR-H2(K) GISWSSGSLGYEDSVKG (SEQ ID NO: 55) 51-68 17 HFR3 RFTISRDNAKKTLYLQMNSLRAEDTALYYCAK (SEQ ID NO: 179) 69-80 32 CDR-H3(K) DMGFGDFLYYYGMDV (SEQ ID NO: 56) 81-96 15 HFR4 WGQGTTVTVSS (SEQ ID NO: 180) 97-108 11

[0056] Table 12 12D8 V H Chain framework (FR) (preferred) and CDR amino acid sequence (Ch) district Sequence fragments Residue length HFR1 EVQLVESGGGLVQPGRSLRLSCAAS (SEQ ID NO: 181) 1 - 25 25 CDR-H1(Ch) GFTFDDY (SEQ ID NO: 57) 26 - 32 7 HFR2 AMHWVRQAPGKGLEWVSGI (SEQ ID NO: 182) 33 - 51 19 CDR-H2(Ch) SWSSGS (SEQ ID NO: 58) 52 - 57 6 HFR3 LGYEDSVKGRFTISRDNAKKTLYLQMNSLRAEDTALYYCAK (SEQ ID NO: 183) 58 - 98 41 CDR-H3(Ch) DMGFGDFLYYYGMDV (SEQ ID NO: 59) 99 - 113 15 HFR4 WGQGTTVTVSS (SEQ ID NO: 184) 114 - 124 11

[0057] Table 13 12D8 V L Chain framework (FR) (preferred) and CDR amino acid sequence (K / Ch) district Sequence fragments Residue length LFR1 EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 185) 1-23 23 CDR-L1 RASQSVSSSYLT (SEQ ID NO: 60) 24-35 12 LFR2 WYQQKPGQAPRLLIY (SEQ ID NO: 186) 36-50 15 CDR-L2 GASSRAT (SEQ ID NO: 61) 52-59 7 LFR3 GIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYC (SEQ ID NO: 187) 60-89 32 CDR-L3 QQYGDSPPWT (SEQ ID NO: 62) 90-99 10 LFR4 FGQGTKVEIK (SEQ ID NO: 188) 100-109 10

[0058] The following shows the light and heavy chain sequence comparison results for antibodies 4F8, 6B2, 8C7, and 12D8, with CDRs highlighted. The amino acid sequences of CDR1, CDR2, and CDR3 are labeled and underlined. The variable weight (V) of antibodies 4F8, 6B2, 8C7, and 12D8 is compared below. H The CDR amino acid sequence determined by the Kabat method is underlined. The following compares the variable lightness (V) of the 4F8, 6B2, 8C7, and 12D8 antibodies. L The CDR amino acid sequence determined by the Kabat method is underlined. The following shows the preferred nucleotide sequences encoding each variable region: 4F8 V H Preferred DNA sequence (Kabat (K) CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGAAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTGAT GATTATGCCATGCAC TGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCA GGTATTAGTTGGGATAGTGGTCGCATAGGCTAT GCGGACTCTGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GATCGG GGCGGTGGTAACTGGAACTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACG GTCACCGTCTCCTCAG (SEQ ID NO: 197) 4F8 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGAAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCT GGATTCACCTTTGATGATTAT GCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATT AGTTGGGATAGTGGTCGC ATAGGCTAT GCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GATCGG GGCGGTGGTAACTGGAACTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACG GTCACCGTCTCCTCAG (SEQ ID NO: 198) 4F8 V L DNA sequence (Kabat (K) / Chothia (Ch) CDR coding sequence is underlined) gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcacc atcacttgt cgggcgagtcagggcattagcaattatttagcc tggtttcagcagaaacca gggaaagcccctaagtccctgatctat gctgcatccagtttgcaaagt ggggtcccatca aagttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct gaagattttgcaacttattactgc caacagtataatagtttccccttcact ttcggccct gggaccaaagtggatatcaaac (SEQ ID NO: 199) 6B2 V H Preferred DNA sequence (Kabat (K) CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCATCAGC AGTAGTAGTTACTACTGGGAC TGGATCCGC CAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGG ACTATCTATTATGGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGT CGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTC TCTCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA GGG AACAGCTATGGCCTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:200) 6B2 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCT GGTGGCTCCATCAGCAGTAGTAGTTAC TACTGGGACTGGATCCGC CAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGACTATC TATTATGGTGGGAGC ACCTAC TACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTC TCTCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA GGG AACAGCTATGGCCTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:201) 6B2 V L Preferred DNA sequence (Kabat (K) / Chothia (Ch) CDR coding sequence is underlined) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGC AGGTCAAGTCAAAGCCTCGTACACAGTGATGGAAACATGTACTTGAAT TGG TTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGTCTAATTTAT AAGGTTTCTAACCGGGAC TCT GGGGTCCCAGACAGATTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGGATTTATTACTGC ATGCAAAGTACACACTGGCCT CCCGTCACT TTTGGCCAGGGGACCAAGCTGGAGATCAAAC (SEQ ID NO: 202) 8C7 V H Preferred DNA sequence (Kabat (K) CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCATCAGC AGTAGTAGTTACTACTGGGGC TGGATCCGC CAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG AGTATCTATTATGGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGT CGAGTCACCATATCCGTAGACACGTCCACGAACCAGTTC TCCCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA CAA GGGCTGGGGTCCTTTGACTGC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:203) 8C7 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCT GGTGGCTCCATCAGCAGTAGTAGTTAC TACTGGGGCTGGATCCGC CAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGAGTATC TATTATGGTGGGAGC ACCTAC TACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCACGAACCAGTTC TCCCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA CAA GGGCTGGGGTCCTTTGACTGC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:204) 8C7 V L Preferred DNA sequence (Kabat(K) / Chothia(Ch) CDR coding sequence is underlined) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGC AGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAACACCTACTTGAAT TGG TTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTAT AAGGTTTCTAACCGGGAC TCT GGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCGGGGTCGAGGCTGAGGATGTTGGAATTTATTACTGC ATGCAAGGTACACACTGGCCT CCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAAC (SEQ ID NO: 205) 1 2D8 V H Preferred DNA sequence (Kabat (K) CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGAT GATTATGCCATGCAC TGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCA GGTATTAGCTGGAGTAGTGGTAGCTTAGGCTATGAGGACTCTGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAAAACCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GA TATGGGGTTCGGGGACTTCCTCTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 206) 12D8 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTGATGATTAT GCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATT AGCTGGAGTAGTGGTAGC TTAGGCTATGAGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAAACCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GA TATGGGGTTCGGGGACTTCCTCTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 207) 12D8 V L DNA sequences (Kabat and Chothia CDR sequences are underlined) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGC AGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAACC TGGTACCAGCAGAAA CCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCCAGCAGGGCCACT GGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGGAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGT CAGCAGTATGGTGACTCACCTCCGTGGACG TTC GGCCAAGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 208) In a preferred embodiment, the invention covers any CDR described herein, or any CDR identified by any other method known in the art from the VH and VL amino acid peptides of the corresponding antibody and the preferred nucleotide sequence. Variations and derivatives thereof may also be considered as described herein.

[0059] In some embodiments, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to a prostaglandin F2 receptor inhibitor (PTGFRN), wherein the antibody or fragment thereof competitively inhibits the antibody as determined using any standard competitive binding assay. In some embodiments, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to a prostaglandin F2 receptor inhibitor (PTGFRN); the antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of a 4F8, 6B2, 8C7, and 12D8 antibody; the VH and VL comprise complementarity-determining regions CDR1, CDR2, and CDR3 of the 4F8, 6B2, 8C7, and 12D8 antibody; VH-CDR1, VH-CDR2, and VH-CDR3 of the 4F8, 6B2, 8C7, and 12D8 antibody, as well as the VL-CDR1, VL-CDR2, and VL-CDR3, and / or include 1, 2, 3, or 4 conserved amino acid substitutions to provide substantially maintained binding to PTGFRN; and / or the antibody or antigen-binding fragment thereof of this disclosure comprises a polypeptide sequence having at least 90%, 95%, 99%, or 100% identity with the polypeptide sequence PTGFRN.

[0060] Those skilled in the art will understand that the use of standard three-letter abbreviations or other amino acid abbreviations for conserved and non-conserved amino acid substitutions is shown in Table 14 below: Table 14 Original amino acid residues Exemplary Conserved Substitution of Original Amino Acid Residues Preferred conservative substitution of original amino acid residues Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Leucine Leu Leu Leucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4-diaminobutyric acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Leucine Leu

[0061] In some embodiments, the nucleic acid molecule encoding one or more antibodies described herein can be inserted into one or more expression vectors, as will be discussed in more detail below. In such embodiments, the antibody can be encoded by nucleotides corresponding to an amino acid sequence. Specific combinations of nucleotides (codons) encoding various amino acids (AAs) are well known in the art, as described in various references used by those skilled in the art (e.g., Lewin, B., Genes V (Oxford University Press, 1994). For example, the nucleotide sequence encoding the amino acids of the antibody can be determined with reference to Table 15. Nucleic acid variants can use any combination of nucleotides encoding the antibody.

[0062] Table 15 The codon (AA) that encodes an amino acid. *TERM: Termination codon.

[0063] In some embodiments, the antibody or its antigen-binding fragment (collectively referred to herein as "antibody" or "antibodies") is internalized. In some embodiments, the antibody or its antigen-binding fragment is murine, human, humanized, or chimeric. In some embodiments, the antibody or its antigen-binding fragment is CDR-grafted, recombinant, or surface-repaired. In some embodiments, the antibody or its antigen-binding fragment further comprises a human or human-derived heavy and light chain variable region framework. In some embodiments, the antibody or its antigen-binding fragment comprises an IgG1 or IgG2 constant region. In some embodiments, the antibody or its antigen-binding fragment is capable of inhibiting cell proliferation and inducing cell death. In some embodiments, the antibody or its antigen-binding fragment binds to human PTGFRN. In some preferred embodiments, the antibody and / or its derivatives inhibit one or more cellular functions of PTGFRN, including but not limited to migration and cell proliferation. In some embodiments, the antibody or its antigen-binding fragment binds to murine PTGFRN. In some embodiments, the antibody is a full-length antibody. In some embodiments, it is an antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0064] This disclosure further provides antibody conjugates of the following formula: Ab-LM, wherein: (a) Ab is an antibody that specifically binds to PTGFRN or an antigen-binding fragment thereof; (b) L is a linker; and (c) M is a functional portion. In some embodiments, Ab in the antibody conjugate is an antibody or an antigen-binding fragment thereof described herein. In some embodiments, M in the antibody conjugate is selected from cytotoxic agents, immunomodulators, imaging agents, therapeutic proteins, biopolymers, ionizing agents, radioisotopes, and oligonucleotides. In some embodiments, M is a cytotoxic agent. Many different types of cytotoxic agents can be used as M, either bound to the same antibody or as a combination of antibodies (ADCs), each antibody containing one or more different cytotoxic agents. In some embodiments, the cytotoxic agent is selected from anthracyclines, auristatin, camptothecin, combrestatin, dolastine, pyroxine, enediyne, geldmycin, indole-benzodiazepine dimer, maytansine, puromycin, pyrrole-benzodiazepine dimer, taxane, vinca alkaloids, tubulysin, hemiasterlin, spliceostatin, pladienolide, and calicimycin. In some embodiments, the cytotoxic agent is selected from monomethylauristatin E, monomethylauristatin F, maytansine DM1, maytansine DM4, calicimycin, oxazomicin, α-amanitrine, yttrium-90 and iodine-131, topoisomerase inhibitors (e.g., esaxatecan, delutec), DNA replication inhibitors, and / or DNA repair inhibitors. In some embodiments, the linker in the antibody conjugate is selected from cleavable linkers, non-cleavable linkers, hydrophilic linkers, and dicarboxylic acid-based linkers. In some embodiments, the antibody conjugates provided in this disclosure bind to PTGFRN and are internalized.

[0065] This disclosure further provides pharmaceutical compositions comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof covered by this disclosure, or an antibody conjugate, and a pharmaceutically acceptable carrier.

[0066] This disclosure further provides a method for treating a disease in a subject associated with PTGFRN function or expression, comprising administering to the subject in need an effective amount of an antibody covered by this disclosure or an antigen-binding fragment thereof, antibody conjugate, or pharmaceutical composition. In some embodiments, the disease is cancer.

[0067] This disclosure also provides a method for reducing or inhibiting tumor growth or progression in a subject with a tumor expressing PTGFRN, comprising administering to the subject in need an effective amount of an antibody covered by this disclosure or an antigen-binding fragment thereof, antibody conjugate, or pharmaceutical composition.

[0068] This disclosure further provides a method for reducing or inhibiting the metastasis of cancer cells expressing PTGFRN in a subject, comprising administering to the subject in need an effective amount of an antibody covered by this disclosure or an antigen-binding fragment thereof, antibody conjugate, or pharmaceutical composition.

[0069] This disclosure provides a method for inducing tumor regression in a subject with a tumor expressing PTGFRN, comprising administering to the subject in need an effective amount of an antibody covered by this disclosure or an antigen-binding fragment thereof, antibody conjugate, or pharmaceutical composition.

[0070] This disclosure also provides a method for imaging cells, comprising: (1) contacting the cells with an antibody or antigen-binding fragment or antibody conjugate covered by this disclosure; and (2) detecting the antibody or antigen-binding fragment or conjugate.

[0071] This disclosure also provides a method for identifying PTGFRN expression in tumors, comprising: (1) obtaining a sample of the tumor; (2) contacting the sample with an antibody or antigen-binding fragment thereof or antibody conjugate covered by this disclosure; and (3) detecting the antibody or antigen-binding fragment thereof or conjugate. In some embodiments, the detection is performed by immunochemistry.

[0072] This disclosure also provides a method for preventing tumor regrowth in subjects who have or already have tumors expressing PTGFRN, comprising administering to the subject in need an effective amount of an antibody covered by this disclosure or an antigen-binding fragment thereof, antibody conjugate or pharmaceutical composition thereof.

[0073] This disclosure also provides a method for improving symptoms in subjects who have or already have tumors expressing PTGFRN, comprising administering to the subject in need an effective amount of an antibody covered by this disclosure or an antigen-binding fragment thereof, antibody conjugate or pharmaceutical composition thereof.

[0074] This disclosure further provides diagnostic reagents comprising the antibodies or antigen-binding fragments thereof disclosed herein. In some embodiments, the antibodies or antigen-binding fragments thereof are labeled. In some embodiments, the label is selected from radiolabelers, fluorophores, chromophores, imaging agents, and metal ions.

[0075] This disclosure also provides kits containing antibodies or antigen-binding fragments thereof, antibody conjugates or pharmaceutical compositions covered by this disclosure.

[0076] This disclosure also provides isolated polynucleotides. In some embodiments, the polynucleotide comprises a sequence encoding a polypeptide having at least 90%, 95%, 99%, or 100% identity with sequences selected from those shown herein. In some embodiments, the polynucleotide comprises a sequence having at least 90%, 95%, 99%, or 100% identity with those shown herein. This disclosure also provides vectors containing the polynucleotides and / or host cells containing such vectors.

[0077] In some embodiments, this disclosure includes the use of the complementarity-determining region (CDR) sequence of the antibody of this disclosure for obtaining a binding molecule that binds to PTGFRN. Such binding molecules typically contain one or more CDR regions or CDR-derived regions of the antibody of this disclosure.

[0078] In some embodiments, this disclosure provides antibody-drug conjugates (ADCs) comprising the anti-PTGFRN antibody disclosed herein. In another aspect, this disclosure includes the use of anti-PTGFRN antibodies, their antigen-binding fragments, antibody-drug conjugates, and corresponding methods for detecting and diagnosing diseases related to the expression or function of PTGFRN.

[0079] In another aspect, this disclosure includes products and / or compositions or kits comprising at least one such antibody, antigen-binding fragment, or antibody-drug conjugate for prognostic or diagnostic or therapeutic monitoring of certain cancers.

[0080] In some embodiments, this disclosure provides pharmaceutical compositions comprising the anti-PTGFRN antibody disclosed herein, its antigen-binding fragment or antibody-drug conjugate, and a pharmaceutically acceptable carrier.

[0081] In some embodiments, this disclosure provides methods for treating cancers expressing PTGFRN in a subject. Such methods may include administering to the subject a composition comprising an anti-PTGFRN antibody or an antigen-binding fragment thereof. Typically, such anti-PTGFRN antibodies and / or fragments are conjugated via a linker to a functional portion comprising a cytotoxic payload. The conjugate may be administered in an amount sufficient to prevent, reduce, or inhibit the growth of the subject's cancer (and / or tumor). In some embodiments, the anti-PTGFRN antibody or antigen-binding fragment comprises: (a) three heavy chain complementarity-determining regions (VH CDR1, VHCDR2, and VH CDR3) comprising the amino acid sequences of 4F8, 6B2, 8C7, and 12D8 antibodies; and (b) three light chain complementarity-determining regions (VL CDR1, VL CDR2, and VL CDR3) comprising the amino acid sequences of 4F8, 6B2, 8C7, and 12D8 antibodies; and / or derivatives thereof (see, for example, Tables 1-15). Cancer cells and / or tissues and / or body fluids expressing PTGFRN can be measured by measuring PTGFRN protein expression in cancer tissues or by measuring PTGFRN DNA or RNA expression using an anti-PTGFRN antibody.

[0082] Any suitable functional moiety (“M”) may be conjugated to the antibodies of this disclosure. In some preferred embodiments, the antibody conjugates of this disclosure may comprise the functional moiety M, which may be a cytotoxic agent, immunomodulator, imaging agent, therapeutic protein, biopolymer, or oligonucleotide. In some embodiments, M may be a drug, particularly a drug for treating cancer. In some preferred embodiments, M may be any cytotoxic agent that can be used in ADCs. Any cytotoxic agents known to those skilled in the art (e.g., enzymes, toxins, peptides, and anthracyclines) may be used in the practice of this disclosure. In some preferred embodiments, M may be one or more agents that cause reduced cell growth and / or cell death, block DNA replication, DNA repair, or protein synthesis (any of which is considered cytotoxic). Furthermore, in some preferred embodiments, M can be any nucleoside antagonist (e.g., 5-fluorouracil, 6-mercaptopurine, arabinosylcytosine, capecitabine, clofarabine, cytarabine, dacarbazine, fludarabine, gemcitabine, and nelarabine), any intercalating agent (e.g., oxaliplatin, cisplatin, and carboplatin), or any microtubule assembly inhibitor (e.g., ozogluconate). Statins (auristatins, monomethylauristatin E, monomethylauristatin F, taxanes, docetaxel, paclitaxel, ixabepilone, vinblastine alkaloids, vinblastine, vinorelbine, vinblastine and maytansine), any folic acid inhibitors (e.g., methotrexate and pemetrexed), any ribosome-inactivating protein (e.g., saponins), and / or any toxins (e.g., ricin, cholera toxin).In the most preferred embodiment, M is selected from therapeutic agents, cytotoxic agents, abrinogen A chain, anthracyclines, amanita (α-amanita), oliquistatin (e.g., monomethyloliquistatin E, monomethyloliquistatin F), calicimycin, camptothecin, comprbetastain, crotonin, candidin, curcin, dolalastatin, duocarmycin, DNA alkylating agents, DNA repair inhibitors, duocarmycin, enediyne, essanotecan or its derivatives (e.g., DX-8951), exotoxin A chain, delutecan, diphtheria A chain, enoxacin, gerdemycin, hemizoxin (a type of sclerosing agent). asterlin), ataxia-telangiectasia and Rad3-related kinase inhibitors (ATR inhibitors, e.g., bezoceletine), indole-benzodiazepine dimers, maytansine, maytansine DM1, maytansine DM4, ozomicin, phenolmycin, pladienolide, phytotoxins, puromycin, pyrrolobenzodiazepine dimers, ricin A chain, splicing statin, taxanes, toxins, tubulolysin, tumor-activating prodrugs, topoisomerase inhibitors (e.g., topoisomerase I inhibitors used for delutecan), vinca alkaloids, radiochemical substances (or radioisotopes) (e.g., iodine-131, yttrium-90). Other cytotoxic agents may also be suitable as disclosed herein or available to those skilled in the art.

[0083] Antibodies or antigen-binding fragments thereof suitable for use in the methods of this disclosure include, but are not limited to, human antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, CDR-transplanted antibodies or antigen-binding fragments thereof, and chimeric antibodies or antigen-binding fragments thereof. In some embodiments, the anti-PTGFRN antibody may comprise a human or human-derived heavy and light chain variable region framework (which may be part of a bispecific antibody or other type of target antibody). In some embodiments, the antibodies of this disclosure may comprise a heavy chain variable region comprising one or more amino acid sequences of 4F8, 6B2, 8C7, and 12D8 antibodies and / or their derivatives.

[0084] The antibody or antigen-binding fragment suitable for forming the antibody conjugate of this disclosure can be a humanized antibody, a chimeric antibody, a CDR-grafted antibody, or a recombinant human antibody. Preferably, the antibody is a fully human antibody.

[0085] In some embodiments, this disclosure provides pharmaceutical compositions comprising a therapeutically effective amount of an antibody of this disclosure or an antigen-binding fragment thereof or an antibody conjugate of this disclosure, and a pharmaceutically acceptable carrier.

[0086] In some embodiments, this disclosure provides a method for treating a disease in a subject associated with PTGFRN function or expression, comprising administering an effective amount of the pharmaceutical composition of this disclosure to a subject in need. Treatable diseases include cancer.

[0087] In some embodiments, this disclosure provides a method for reducing or inhibiting tumor growth or progression in a subject with a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need.

[0088] In some embodiments, this disclosure provides a method for reducing or inhibiting the metastasis of cancer cells expressing PTGFRN in a subject, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need.

[0089] In some embodiments, this disclosure provides a method for inducing tumor regression in a subject with a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need of doing so.

[0090] In some embodiments, this disclosure provides a method for preventing tumor regrowth in a subject who already has a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need.

[0091] In some embodiments, this disclosure provides a method for improving symptoms in subjects who have or already have tumors expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need.

[0092] In some embodiments, this disclosure provides a method for imaging cells, comprising: contacting the cells with an antibody of the present disclosure or an antigen-binding fragment thereof or an antibody conjugate thereof; and detecting the antibody or the antigen-binding fragment thereof or the antibody conjugate thereof. In some embodiments, this disclosure provides a method for determining PTGFRN expression in a tissue, comprising contacting the tissue with an antibody of the present disclosure or an antigen-binding fragment thereof or an antibody conjugate thereof; and detecting the antibody or the antigen-binding fragment thereof or the antibody conjugate thereof.

[0093] This disclosure provides antibodies and antibody conjugates (e.g., antibody-drug conjugates) that bind to PTGFRN (e.g., human PTGFRN, mouse PTGFRN, goose PTGFRN). An example of such antibodies is 33B7. This disclosure also provides polynucleotides encoding antibodies of 4F8, 6B2, 8C7, or 12D8, compositions comprising the antibodies of this disclosure, and methods for preparing and using these antibodies. In some embodiments, the antibodies of this disclosure comprise all or part of the variable regions of the specific heavy and light chain sequences disclosed herein. In some embodiments, the antibodies of this disclosure comprise one or more amino acid sequences of the CDR regions disclosed herein.

[0094] This disclosure further includes methods for using antibodies of this disclosure, such as to detect PTGFRN, modulate PTGFRN activity, and / or target PTGFRN-expressing cells for killing (e.g., ADCs) (e.g., for treating and / or preventing cancer). In some preferred embodiments, the methods may be and / or include methods for measuring PTGFRN expression in cells and / or tissues to determine the cell, tissue, and / or cancer type susceptible to treatment with anti-PTGFRN antibody-ADC conjugates. The measurement step can be implemented by detecting and measuring the expression of RNA or protein encoding PTGFRN in cells and / or tissues.

[0095] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0096] Prostaglandin F2 receptor inhibitors (PTGFRN) are known in the art by several names, such as FPRP; CD315; EWI-F; CD9P-1; SMAP-6; KIAA1436; prostaglandin F2-α receptor regulatory protein; prostaglandin F2-α receptor-associated protein; prostaglandin F2 receptor negative regulator; and protein F containing the Glu-Trp-Ile EWI motif. PTGFRN has the following accession numbers: UniProt Q9P2B2, Entrez Gene ID: 5738, Ensemble: ENSG00000134247, OMIM: 601204, and HGNC: 9601.

[0097] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of recognizing and binding to a specific target or antigen. The antibodies of this disclosure typically contain at least one antigen recognition site located in a variable region of an immunoglobulin molecule. The antibodies of this disclosure can be monoclonal antibodies, polyclonal antibodies, and antigen-binding fragments thereof, retaining the ability to specifically bind PTGFRN. In some embodiments, the antibodies of this disclosure can be single-chain (ScFv) and single-domain antibodies (e.g., shark and camel antibodies). The antibodies of this disclosure can be humanized antibodies, chimeric antibodies, or fully human antibodies. Fully human antibodies do not contain mouse sequences (see, for example, adalimumab (Humira), panitumab (Vectib), golimumab (Simponi)). Fully human antibodies can be prepared using transgenic mice (as illustrated in the examples herein), phage display, B cell cloning, phage display, and / or other techniques available to those skilled in the art. The antibodies disclosed herein may be derived from any source known to those skilled in the art; for example, the antibodies disclosed herein may be of mouse, rat, camel, human, or any other origin, or may be synthetic.

[0098] As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments, all of which are antigen-binding fragments), single-chain Fv (scFv) mutants, fully human antibodies, multispecific antibodies (such as bispecific antibodies generated from at least two complete antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits desired biological activity. Antibodies can belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on their heavy chain constant domains, respectively designated α, δ, ε, γ, and μ. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules, such as toxins, radioisotopes, etc. For example, in some embodiments, the antibody disclosed herein may be a humanized antibody conjugated to a drug molecule.

[0099] In some preferred embodiments, the antibody may be a "naked" antibody, i.e., an antibody not conjugated to a cytotoxic portion. In some preferred embodiments, the antibody may be an antibody-drug conjugate (ADC), i.e., an antibody conjugated to a cytotoxic portion. In some preferred embodiments, the "naked" antibody or ADC may be used to treat and / or prevent cancer in a preferred embodiment. In some preferred embodiments, the "naked" antibody and ADC may be used in combination (in the same or different compositions, substantially simultaneously or not simultaneously) to treat and / or prevent cancer in a preferred embodiment.

[0100] In some embodiments, the antibodies of the present invention can be humanized. The term "humanized antibody" refers to a non-human (e.g., mouse) antibody as a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing a minimum non-human (e.g., mouse) sequence. Typically, a humanized antibody is a human immunoglobulin in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) with desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding residues from an antibody of a non-human species with desired specificity, affinity, and capability. Humanized antibodies can be further modified by substituting additional residues within the Fv framework region and / or within the substituted non-human residues to refine and optimize the antibody's specificity, affinity, and / or potency. Typically, humanized antibodies will contain at least one, usually two or three, variable domains, which contain all or substantially all of the CDR regions corresponding to non-human immunoglobulins, while all or substantially all of the FR regions are regions of the human immunoglobulin common sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region or domain (Fc) (typically the immunoglobulin constant region or domain (Fc) of human immunoglobulins). Humanization can be performed by any method known in the art, for example, using the methods disclosed in: Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988). As used herein, antibodies are humanized by replacing all or part of one or more of the CDRs of human antibodies with all or part of one or more of the CDRs of nonhuman antibodies of this disclosure. U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; and 5,859,205, the contents of which relate to the humanization of antibodies, are incorporated herein by reference.

[0101] In some embodiments of this disclosure, the antibodies of this disclosure can be prepared in cells of animals other than mice, for example, antibodies of this disclosure can be prepared in cells from chickens, pigs, guinea pigs, hamsters, horses, rats, camels, alpacas, goats, rabbits, donkeys, sheep and / or other suitable animals. The antibodies of this disclosure can be synthetic antibodies.

[0102] The term "anti-PTGFRN antibody" or "PTGFRN-binding antibody" refers to an antibody that binds to PTGFRN with sufficient affinity, making the antibody usable as a diagnostic and / or therapeutic agent targeting PTGFRN. The degree of binding of the anti-PTGFRN antibody to unrelated non-PTGFRN proteins may be less than about 10%, as measured, for example, by an immunoassay. In some embodiments, the PTGFRN-binding antibody has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. The anti-PTGFRN antibody of this disclosure can be any type of antibody, and in a preferred embodiment is a fully human antibody.

[0103] The term "antibody fragment" refers to a portion of a complete antibody and specifically to the antigenic determination variable region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single-chain antibodies, single-chain scFv, and multispecific antibodies formed from antibody fragments.

[0104] "Monoclonal antibody" refers to a homogeneous group of antibodies that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically contain different antibodies targeting different antigenic determinants. The term "monoclonal antibody" includes full-length and complete monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to such antibodies prepared in any number of ways, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

[0105] The “variable region” of an antibody refers to the variable region of either the antibody light chain or the antibody heavy chain, either alone or in combination. The variable regions of both the heavy and light chains are each composed of four frame regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together closely by the FRs and, together with CDRs from the other chain, contribute to the formation of the antibody’s antigen-binding site. At least two techniques exist for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., (1997) J. Molec. Biol. 273:927-948). Additionally, a combination of these two methods is sometimes used in the art to determine CDRs.

[0106] The term "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence corresponding to that of an antibody produced by a human (preferably without any amino acid sequence from any source, such as mice or rats) using any technique known in the art. This definition of human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide, such as, for example, antibodies containing mouse light chain and human heavy chain polypeptides.

[0107] The term "humanized antibody" refers to a non-human (e.g., mouse) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human (e.g., mouse) sequence. Typically, a humanized antibody is a human immunoglobulin in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) with desired specificity, affinity, and capability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding residues from an antibody of a non-human species with desired specificity, affinity, and capability. Humanized antibodies can be further modified by substituting additional residues in the Fv frame region and / or within the substituted non-human residues to refine and optimize the antibody's specificity, affinity, and / or capability. Typically, a humanized antibody will contain at least one, typically two or three, variable domains containing all or substantially all of the CDR regions corresponding to the non-human immunoglobulin, while all or substantially all of the FR regions are regions of the human immunoglobulin's common sequence. Humanized antibodies may also contain at least a portion of an immunoglobulin constant region or structural domain (Fc) (typically the immunoglobulin constant region or structural domain (Fc) of human immunoglobulins). Examples of methods for generating humanized antibodies are described in U.S. Patent No. 5,225,539.

[0108] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with desired specificity, affinity, and capability, while the constant regions are sequence-homogeneous with those in antibodies derived from another mammalian species (typically human) to avoid triggering an immune response in that species.

[0109] The terms “epitope” or “antigenic determinant” are used interchangeably herein and refer to the portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, an epitope can be formed from consecutive amino acids and discontinuous amino acids juxtaposed by the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained after protein denaturation, while epitopes formed by ternary folding are generally lost after protein denaturation. Epitopes typically contain at least three, and more usually at least five or eight to ten amino acids in a unique spatial conformation.

[0110] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies typically bind antigens slowly and tend to dissociate readily, while high-affinity antibodies typically bind antigens more quickly and tend to remain bound for longer periods. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific illustrative embodiments are described in the examples herein.

[0111] When used herein to refer to binding affinity, "or better" means a stronger binding between a molecule and its binding partner. When used herein, "or better" means a stronger binding represented by a smaller numerical value (Kd). For example, an antibody with an affinity of "0.3 nM or better" for an antigen has an affinity <0.3 nM, i.e., 0.29 nM, 0.28 nM, 0.27 nM, etc., or any value less than 0.3 nM.

[0112] "Specific binding" generally refers to an antibody binding to an epitope via its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to it more easily than to a random, unrelated epitope. The term "specificity" is used in this paper to quantify the relative affinity of an antibody for a particular epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to have higher specificity for epitope "C" than for related epitope "D."

[0113] "Preferential binding" means that an antibody is more likely to bind to an epitope than to bind to related, similar, homologous, or analogous epitopes. Therefore, an antibody that "prefers binding" to a given epitope is more likely to bind to that epitope than to a related epitope, even if the antibody may cross-react with the related epitope.

[0114] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope to a degree that blocks the binding of a reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art (e.g., competitive ELISA assay). An antibody may be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0115] As used herein, the phrase "substantially similar" or "substantially identical" indicates a sufficiently high degree of similarity between two values ​​(typically one relating to the antibody of this disclosure and the other to a reference / comparative antibody) such that a person skilled in the art would consider that the difference between the two values ​​is small or not biologically and / or statistically significant in the context of the biological characteristic measured by said values ​​(e.g., Kd values). Depending on the value of the reference / comparative antibody, the difference between said two values ​​may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.

[0116] "Isolated" peptides, antibodies, polynucleotides, vectors, cells, or compositions are peptides, antibodies, polynucleotides, vectors, cells, or compositions that are not found in nature. Isolated peptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the point that they are no longer found in nature. In some embodiments, the isolated antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0117] As used in this article, “substantially pure” means material that is at least 50% pure (i.e., pure means free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0118] In some embodiments, the antibodies of this disclosure can be conjugated to a functional moiety to form an antibody conjugate. As used herein, the terms “immunoconjugate,” “conjugate,” or “antibody-drug conjugate” interchangeably refer to a compound or derivative thereof linked to a cell-binding agent (i.e., an anti-PTGFRN antibody or a fragment thereof). The antibody conjugates of this disclosure may have the formula Ab-LM, wherein: (a) Ab is the PTGFRN-binding antibody of this disclosure or an antigen-binding fragment thereof; (b) L is a linker; and (c) M is a functional moiety. The conjugation can be direct, in which case L is simply a chemical bond between Ab and M. Conjugation may involve the use of a linker. Examples of functional moieties include, but are not limited to, drugs, fluorescent molecules, radioactive molecules, chemiluminescent molecules, molecules for imaging, epitopes, ligands, etc. The functional moiety can be conjugated to the antibodies of this disclosure using any technique known to those skilled in the art. When a linker is used, the linker can be cleavable or incleavable and can have any length and any composition. When a linker is used in practice with this disclosure, the linker can be conjugated to any portion of an immunoglobulin molecule or any amino acid of an immunoglobulin molecule. The adapter can be attached to the antibodies of this disclosure using any techniques known to those skilled in the art, such as via surface lysine, reductive conjugation to oxidized carbohydrates, release of cysteine ​​residues via reductive interchain disulfide bonds, engineered reactive cysteine ​​residues at specific sites, and reactive tags containing acyl donor glutamine or endogenous glutamine via peptide engineering in the presence of transglutaminase and amines. Various ADC bond systems are known in the art, including hydrazone-, disulfide-, and peptide-based bonds. The adapter and toxin can be expressed as part of the antibody via DNA recombination.

[0119] A "connector" is any chemical or protein moiety (such as a dipeptide or tripeptide connector) that can stably and covalently link a compound (typically a drug, such as maytansine or a toxin) to a cell binder, such as an anti-PTGFRN antibody, or a fragment thereof. While the compound or antibody remains active, the connector may be sensitive to or substantially resistant to acid-induced cleavage, light-induced cleavage, enzyme-induced cleavage such as peptidase-induced cleavage, esterase-induced cleavage, transglutaminase-induced cleavage, cathepsin-induced cleavage, and disulfide bond cleavage. Suitable connectors are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. Connectors also include charged connectors and their hydrophilic forms, as described herein and known in the art. The connector can be, for example, a cleavable connector, an incleavable connector, a hydrophilic connector, or a dicarboxylic acid-based connector.

[0120] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. “Tumor” and “vesicle” refer to one or more cells resulting from excessive cell growth or proliferation, including benign (non-cancerous) or malignant (cancerous), including precancerous lesions and metastatic sites. Any cancer expressing PTGFRN can be detected and / or treated using the methods disclosed herein. In a preferred embodiment, the types of cancer that can be treated and / or prevented by antibodies, ADCs, or combinations thereof (between each other, and / or with at least one other drug and / or regimen) include, but are not limited to: solid tumors, lung adenocarcinoma, bladder cancer, blastoma, breast cancer (including triple-negative breast cancer), carcinoma, choriocarcinoma, colon cancer, rectal cancer, cervical cancer, endometrial cancer, epidermoid carcinoma, gastrointestinal cancer, glioblastoma, head and neck cancer, gastric cancer, primary liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, kidney (renal) cancer, leiomyosarcoma, liver cancer, lung cancer, medulloblastoma, mesothelioma, neuroblastoma, non-small cell lung cancer and small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer (including castration-resistant prostate cancer). Testicular cancer, bone cancer, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, salivary gland cancer, sarcoma, small cell lung cancer, spindle cell sarcomatoid carcinoma, squamous cell carcinoma (including lung cancer), squamous cell carcinoma, lung squamous cell carcinoma, thyroid cancer, uterine cancer, vulvar cancer, hematopoietic system cancers (e.g., lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous B-cell or T-cell lymphoma, primary macroglobulinemia), leukemias (e.g., chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL)) and / or any of their local, adjacent, or distant metastases. Other types of cancer may also be treated, as can be determined by a person skilled in the art.

[0121] The terms “cancer cell,” “tumor cell,” and their grammatical equivalents refer to the total population of cells originating from a tumor, precancerous lesion, or metastatic site, including both non-tumorigenic cells that constitute the majority of the tumor cell population and tumorigenic stem cells (cancer stem cells) matrix and surrounding cells. As used herein, the term “tumor cell” will be modified by the term “non-tumorigenic” when referring only to those tumor cells that lack the capacity for renewal and differentiation to distinguish them from cancer stem cells.

[0122] The term “subject” refers to any animal (e.g., a mammal) to be a recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, canines (dogs), felines (cats), etc. Generally, the terms “subject” and “patient” are used interchangeably in this document for the purpose of referring to human subjects.

[0123] Administering in combination with one or more other therapeutic agents and / or modes of treatment and / or regimens includes simultaneous (concurrent) and / or sequential administration, in any order of administration and / or at any physical site of administration. The antibodies and ADCs of this disclosure may be combined with any suitable additives, such as one or more chemotherapeutic agents, one or more anti-payload antibodies (i.e., binding regions of the ADC to the cytotoxic portion (or one or more cytotoxic portions (M)) and / or antibodies) to reduce the toxicity of the free payload, other types of anti-id antibodies, and immunotherapy regimens (e.g., CAR-T therapy).

[0124] The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the bioactivity of the active ingredient to be effective and does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation is administered. The formulation may be sterile.

[0125] The “effective amount” of an antibody, as disclosed herein, is an amount sufficient to perform the clearly stated purpose. The “effective amount” can be determined empirically and in a conventional manner for the stated purpose.

[0126] The term "therapeuticly effective amount" refers to the amount of an antibody or other drug that effectively "treats" a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow or stop) the invasion of cancer cells into surrounding organs; inhibit (i.e., to some extent slow or stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. See the definition of "therapeutic" in this document. The extent to which the drug can prevent the growth and / or kill existing cancer cells can be inhibitory of cell growth and / or cytotoxic. "Prophylacticly effective amount" refers to the amount that effectively achieves a desired preventive outcome within the necessary dosage and time period. It is usually, but not necessary, because a preventive dose is used in the subject before or at an earlier stage of the disease, so the preventively effective amount will be less than the therapeutically effective amount.

[0127] When used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to generate a "labeled" antibody. The label itself is detectable (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, can catalyze a chemical change in a detectable substrate compound or composition.

[0128] "Chemotherapy agents" are chemical compounds that can be used to treat cancer, regardless of their mechanism of action. Chemotherapy agents include, but are not limited to, for example, CD20 antagonists such as rituximab and cyclophosphamide, doxorubicin, vincristine, prednisone, fludarabine, etoposide, methotrexate, lenalidomide, chlorambucil, bendamustine, platinum-based drugs, taxanes, tubulin, enzyme inhibitors, inhibitors of cellular processes essential for cell growth and survival, and / or modified forms of such chemotherapy agents.

[0129] Terms such as “treating,” “treatment,” “to treat,” “alleviating,” or “to alleviate” refer to 1) therapeutic measures that cure, slow, or alleviate the symptoms of a diagnosed pathological condition or symptom and / or stop the progression of the diagnosed pathological condition or symptom; and 2) preventive or preventative measures that prevent and / or slow the development of a targeted pathological condition or symptom; and / or 3) typically, for example, prolonging disease-free survival or overall survival. Therefore, those requiring treatment include those who already have the condition; those who are susceptible to the condition; and those among them who need to prevent the condition. In some implementations, a subject's cancer is considered successfully "treated" according to the method of this disclosure if the patient exhibits one or more of the following: a reduction or complete absence of cancer cell numbers; no disease progression; a reduction in tumor size; inhibition or absence of cancer cell infiltration into surrounding organs (including, for example, spread of cancer to soft tissues and bones); inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with a specific cancer; reduced morbidity and mortality; improved quality of life; a reduction in tumorigenicity, frequency, or capacity for tumorigenesis; a reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells into a non-tumorigenic state; or some combination of these effects.

[0130] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a nucleotide polymer of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure can be conferred before or after polymer assembly, if present. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components. Other types of modifications include, for example, “caps”; substitution of one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, triphosphates, phosphoamidates, carbamates, etc.) and those with charged bonds (e.g., thiophosphates, dithiophosphates, etc.); those containing overhanging portions (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.)); those with intercalating agents (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); those containing alkylating agents; those with modified bonds (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of one or more polynucleotides. Furthermore, any hydroxyl group normally present in sugars can be, for example, replaced by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages with other nucleotides, or can be conjugated to a solid support. The 5' and 3' terminal OH groups may be phosphorylated or partially substituted with an amine or an organic end-capping group of 1 to 20 carbon atoms. Other hydroxyl groups may also be derived as standard protecting groups. The polynucleotide may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methylribose, 2'-O-allylribose, 2'-fluororibose, or 2'-azidoribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose, or lythose), pyranoses, furans, sedoheptulose, acyclic analogs, and non-basic nucleoside analogs (such as methylriboside). One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), P(O)NR2 (“amide”), P(O)R, P(O)OR, CO or CH2 (“formacetal”), wherein each R or R is independently H or a substituted or unsubstituted alkyl group (1-20 Cs) (optionally containing an ether (-O-) bond), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl.Not all bonds in a polynucleotide must be identical. The foregoing description applies to all polynucleotides mentioned in this article, including RNA and DNA.

[0131] The term "vector" refers to a construct capable of delivering and optionally expressing one or more target genes or sequences within a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, granule, or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (such as production cells).

[0132] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to amino acid polymers of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also covers amino acid polymers, whether naturally occurring or modified through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that because the polypeptides of this disclosure are antibody-based, in some embodiments, the polypeptide may exist as a single chain or a related chain.

[0133] In the context of two or more nucleic acids or peptides, the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned (introducing vacancies, if necessary) to obtain maximum correspondence, without considering any conserved amino acid substitutions as part of sequence identity. Percentage identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. A non-limiting example of such sequence alignment algorithms is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, modified as in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST procedures (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In some implementations, Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, can be used. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR) are other publicly available software programs that can be used for sequence alignment. In some implementations, the percentage of identity between two nucleotide sequences is determined using the GAP procedure in GCG software (e.g., using the NWSgapdna.CMP matrix with vacancy weights of 40, 50, 60, 70, or 90 and length weights of 1, 2, 3, 4, 5, or 6). In some alternative implementations, the GAP procedure in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)), can be used to determine the percentage identity between two amino acid sequences (e.g., using a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, or 5). Alternatively, in some implementations, the percentage identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).For example, the percentage identity can be determined using the ALIGN program (version 2.0) and with PAM120 using a residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Those skilled in the art can determine appropriate parameters for maximizing alignment using specific alignment software. In some embodiments, the default parameters of the alignment software are used. In some embodiments, the percentage identity “X” between the first and second amino acid sequences is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (e.g., by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percentage identity between the first and second sequences will be longer than the percentage identity between the second and first sequences.

[0134] As a non-limiting example, the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis 5371 1) can be used in some embodiments to determine whether any particular polynucleotide has a specific percentage sequence identity with a reference sequence (e.g., at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99%). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981) to find the optimal homology region between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to this disclosure, parameters are set such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence and allows for homology vacancies of up to 5% of the total number of nucleotides in the reference sequence.

[0135] In some embodiments, the two nucleic acids or polypeptides of this disclosure are substantially identical, meaning that when compared and aligned to obtain maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, as measured by using sequence comparison algorithms or by visual inspection. Identity can be present in regions of sequence lengths of at least about 10, about 20, about 40-60, about 60-80, about 90-100 residues, or any value in between, and in some embodiments, the sequences are substantially identical across the entire length of the compared sequences (such as, for example, coding regions of nucleotide sequences).

[0136] "Conservative amino acid substitution" is the substitution of an amino acid residue by another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, replacing tyrosine with phenylalanine is a conservative substitution. In some embodiments, the conservative substitution in the sequence of the peptides and antibodies of this disclosure does not eliminate the binding of the peptide or antibody containing that amino acid sequence to one or more antigens (e.g., PTGFRN to which the peptide or antibody binds). Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32: 1 180-1 187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0137] As used in this disclosure and claims, the singular forms “a”, “an”, and “the” include the plural forms unless the context clearly indicates otherwise.

[0138] It should be understood that whenever the implementation is described in this document using the language "comprising", other similar implementations described in terms of "consisting of" and / or "substantially consisting of" are also provided.

[0139] As used in phrases such as “A and / or B” herein, the term “and / or” is intended to include both “A and B”, “A or B”, and “A and B”. Similarly, as used in phrases such as “A, B and / or C”, the term “and / or” is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0140] This disclosure provides reagents that specifically bind to PTGFRN. These reagents are referred to herein as "PTGFRN binders". In some embodiments, the PTGFRN binder is an antibody, an immunoconjugate, or a peptide. In some embodiments, the PTGFRN binder is a human or humanized antibody. In some embodiments, the anti-PTGFRN binding molecule may be an antibody or antigen-binding fragment having a CDR sequence of antibody 4F8, 6B2, 8C7, or 12D8; or a derivative thereof; in some embodiments, each CDR includes up to four (i.e., 0, 1, 2, 3, or 4) conserved amino acid substitutions.

[0141] In some embodiments, the antibody or its antigen-binding fragment is internalized. In some embodiments, the antibody or its antigen-binding fragment is murine, human, humanized, or chimeric. In some embodiments, the antibody or its antigen-binding fragment is CDR-grafted, recombinant, or surface-repaired. In some embodiments, the antibody or its antigen-binding fragment further comprises a human or human-derived heavy and light chain variable region framework. In some embodiments, the antibody or its antigen-binding fragment comprises an IgG1 or IgG2 constant region. In some embodiments, the antibody or its antigen-binding fragment is capable of inducing cell death. In some embodiments, the antibody or its antigen-binding fragment binds to human PTGFRN. In some embodiments, the antibody or its antigen-binding fragment binds to murine PTGFRN. In some embodiments, the antibody is a full-length antibody. In some embodiments, it is an antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, camelid, nanobody, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0142] In some embodiments, one or more functional moieties may be conjugated to the antibodies of this disclosure. Those skilled in the art can use known techniques to vary the number of functional moieties in each antibody. Typically, each antibody will be conjugated with at least one functional moiety. In some embodiments, the ratio of functional moieties in each antibody may be about 1 to about 8, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, or about 7 to about 8. In some embodiments, the antibody conjugates of this disclosure may comprise 1, 2, 3, 4, 5, 6, 7, or 8 functional moieties. Typically, the number of functional moieties in each antibody may be expressed as a drug-antibody ratio (DAR). The number of functional moieties in each antibody can be determined using any technique known in the art (e.g., UV spectroscopy, mass spectrometry, immunoassay, radiometric assay, hydrophobic interaction chromatography (HIC), electrophoresis, or HPLC).

[0143] According to some embodiments described herein, the immunoconjugate can be internalized into cells. Therefore, when the immunoconjugate is taken up or internalized by cells expressing PTGFRN, it can exert a therapeutic effect. In some specific embodiments, the immunoconjugate comprises an antibody, antibody fragment, or peptide linked to a cytotoxic agent via a cleavable linker, and the cytotoxic agent is cleaved from the antibody, antibody fragment, or peptide, wherein it is internalized by cells expressing PTGFRN.

[0144] This disclosure further provides antibody conjugates of the following formula: Ab-LM, wherein: (a) Ab is an antibody that specifically binds to PTGFRN or an antigen-binding fragment thereof; (b) L is a linker; and (c) M is a functional portion. In some embodiments, Ab in the antibody conjugate is an antibody or an antigen-binding fragment thereof from any of the embodiments and / or claims of this disclosure. In some embodiments, M in the antibody conjugate is selected from cytotoxic agents, immunomodulators, imaging agents, therapeutic proteins, biopolymers, and oligonucleotides. In some embodiments, M is a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from anthracyclines, auristatin, camptothecin, combrestatin, dolastine, pyroxine, enediyne, galdecycin, indole-benzodiazepine dimer, maytansine, puromycin, pyrrolobenzodiazepine dimer, taxane, vinca alkaloids, tubulysin, hemiasterlin, spliceostatin, pladienolide, and galicarmycin, topoisomerase inhibitors, DNA synthesis inhibitors, and DNA repair inhibitors. In some embodiments, the cytotoxic agent is selected from monomethylauristatin E, monomethylauristatin F, maytansine DM1, maytansine DM4, galicarmycin, oxazomicin, α-amanitin, yttrium-90, iodine-131, and / or topoisomerase inhibitors. In some embodiments, the linker in the antibody conjugate is selected from cleavable linkers, non-cleavable linkers, hydrophilic linkers, and dicarboxylic acid-based linkers. In some embodiments, the antibody conjugates provided in this disclosure bind to PTGFRN and are internalized.

[0145] In some embodiments, the PTGFRN binder (e.g., an antibody, its antigen-binding fragment, and / or antibody-drug conjugate) has one or more of the following effects: inhibiting tumor cell proliferation, reducing tumorigenicity by decreasing the frequency of cancer stem cells in the tumor, inhibiting tumor growth, inhibiting migration to distant locations, reducing survival, triggering cell death of tumor cells, differentiating tumorigenic cells into a non-tumorigenic state, or preventing tumor cell metastasis.

[0146] In some embodiments, the PTGFRN binder binds to cell surface PTGFRN antigens. In some embodiments, the PTGFRN binder binds to the extracellular domain (ECD) of PTGFRN. In some embodiments, cancer cells have multiple cell surface PTGFRN antigens. In some embodiments, the PTGFRN and the PTGFRN binder are internalized into the cells.

[0147] In some embodiments, the PTGFRN binding agent binds to the same epitopes on PTGFRN on the cell surface with 4F8, 6B2, 8C7, or 12D8 antibodies; or derivatives thereof (see, for example, preferred embodiments of antibodies with CDRs and / or derivatives provided in Tables 1-15). In some embodiments, the PTGFRN binding agent binds to different epitopes on PTGFRN on the cell surface with 4F8, 6B2, 8C7, or 12D8 antibodies; or derivatives thereof (see, for example, preferred embodiments of antibodies with CDRs and / or derivatives provided in Tables 1-15).

[0148] In some embodiments, the PTGFRN binder is capable of killing cells. In some embodiments, cell killing is achieved by an anti-PTGFRN antibody. In some embodiments, cell killing is achieved by a cytotoxic agent conjugated to an anti-PTGFRN antibody. In some embodiments, cell killing is achieved by an anti-PTGFRN antibody and a cytotoxic agent conjugated to the anti-PTGFRN antibody.

[0149] In some embodiments, this disclosure covers polynucleotides comprising a polynucleotide encoding a polypeptide or a fragment of such a polypeptide that specifically binds to PTGFRN. For example, this disclosure provides polynucleotides comprising a nucleic acid sequence encoding an antibody against human PTGFRN or a fragment encoding such an antibody. The polynucleotides of this disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. This disclosure also provides isolated polynucleotides. In some embodiments, the polynucleotide comprises a sequence encoding a polypeptide (and / or its CDR or variable region) of 4F8, 6B2, 8C7, or 12D8. In some embodiments, the polynucleotide comprises a sequence encoding a polypeptide having at least 90%, 95%, 99%, or 100% sequence identity with a preferred polynucleotide disclosed herein encoding an antibody (or its CDR and / or variable region) of at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity. In some embodiments, the polynucleotide comprises a sequence encoding a mature polypeptide fused to the polynucleotide in the same reading frame, the polynucleotide facilitating, for example, expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence for controlling the transport of the polypeptide from the cell). The polypeptide having the leader sequence is a preprotein and may have a leader sequence that can be cleaved by the host cell to form a mature form of the polypeptide. The polynucleotide may also encode a preprotein, which is a mature protein with an additional 5' amino acid residue. The mature protein having the presequence is a preprotein and is the inactive form of the protein. Once the pre-sequence is cleaved, the active mature protein is retained. In some embodiments, the polynucleotide comprises the coding sequence of a mature polypeptide fused with a marker sequence in the same reading frame, the marker sequence allowing, for example, purification of the encoded polypeptide. This disclosure further relates to variants of the polynucleotides described above that encode, for example, fragments, analogs, and derivatives. The polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for various reasons, such as to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by bacterial hosts, such as E. coli). Vectors and cells containing the polynucleotides described herein are also covered by this disclosure.

[0150] In some embodiments, the antibodies and / or antibody conjugates of this disclosure may be formulated as pharmaceutical compositions. Pharmaceutical compositions of this disclosure will typically comprise about 0.1 wt% to about 75 wt% of the antibodies or antibody-pharmaceutical conjugates of this disclosure. In some embodiments, pharmaceutical compositions of this disclosure may comprise about 0.2 wt% to about 75 wt%, about 0.5 wt% to about 75 wt%, about 1 wt% to about 75 wt%, about 2 wt% to about 75 wt%, about 5 wt% to about 75 wt%, about 10 wt% to about 75 wt%, about 20 wt% to about 75 wt%, or about 50 wt% to about 75 wt% of the antibodies or antibody-pharmaceutical conjugates of this disclosure. Pharmaceutical compositions of this disclosure may be formulated in any manner known to those skilled in the art. Typically, pharmaceutical compositions of this disclosure are formulated as injectable compositions, which may be liquid solutions or suspensions or solid forms to be dissolved or suspended in a liquid prior to injection. Pharmaceutical compositions of this disclosure may be formulated for delayed release, for example, for reservoir injection. Pharmaceutical compositions of this disclosure will typically comprise one or more pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral media such as sodium chloride, Ringer's dextran, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluids, and nutritional supplements, as well as similar materials and combinations thereof, as known to those skilled in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0151] This disclosure also provides methods for treatment using the antibodies of this disclosure. The methods of this disclosure generally involve administering the pharmaceutical composition of this disclosure to a patient in need. The amount to be administered, depending on the number of treatments and dosage, can be determined by those skilled in the art. The determination of the amount and timing of administration of the pharmaceutical composition of this disclosure can be made by considering factors such as: the subject's weight, age, health, and sex; the type of disease being treated; the extent of disease penetration; prior or concurrent therapeutic interventions; the patient's idiopathic condition; the route of administration; and the efficacy, stability, and toxicity of the specific therapeutic substance.

[0152] In some embodiments, the antibodies of this disclosure can be used for imaging applications. Typically, for imaging applications, the antibodies of this disclosure can be conjugated to functional portions suitable for imaging. Functional portions suitable for imaging include, but are not limited to, portions detectable by microscopy, such as fluorescence microscopy, confocal microscopy, or electron microscopy. Other functional portions can be detected by other analytical techniques, such as magnetic resonance imaging, tomography, gamma (SPECT / CT, planar) and positron emission tomography (PET / CT), radiography, or ultrasound. Functional portions suitable for use in imaging applications of this disclosure can include luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorescence quenchers, colored molecules, radioisotopes, scintillators, mass markers (for detection via mass change), biotin, avidin, streptoavidin, protein A, protein G, antibodies or fragments thereof, Grb2, polyhistidines, Ni2. + Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donor / acceptor, acridine esters, and colorimetric substrates.

[0153] The PTGFRN binders disclosed herein (including antibodies, immunoconjugates, and peptides) can be used in a variety of applications, including but not limited to therapeutic treatments such as cancer treatment. In some embodiments, the reagents can be used to inhibit tumor growth, induce differentiation, reduce tumor volume and / or reduce tumorigenicity, and inhibit metastasis. Methods of use may include in vitro, ex vivo, or in vivo methods and for patient use.

[0154] In some embodiments, the anti-PTGFRN antibody and immunoconjugate of this disclosure can be used to detect the presence of PTGFRN in a biological sample. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some embodiments, the biological sample comprises cells or tissues or biological fluids. In some embodiments, such tissues include normal and / or cancerous tissues that express PTGFRN at higher levels relative to other tissues.

[0155] In some embodiments, this disclosure provides a method for detecting the presence of PTGFRN in a biological sample. In some embodiments, the method includes contacting the biological sample with an anti-PTGFRN antibody under conditions that allow the anti-PTGFRN antibody to bind to PTGFRN, and detecting whether a complex is formed between the anti-PTGFRN antibody and PTGFRN.

[0156] In some embodiments, this disclosure provides methods for diagnosing conditions such as cancer. In some embodiments, the method includes contacting test cells or tissue with an anti-PTGFRN antibody; determining the expression level of PTGFRN in the test cells (quantitative or qualitative) by detecting the binding of the anti-PTGFRN antibody to PTGFRN; and comparing the expression level of PTGFRN in the test cells with the expression level of PTGFRN in control cells (e.g., normal cells of the same tissue origin as the test cells or cells expressing PTGFRN at a level comparable to that of such normal cells), wherein a higher PTGFRN expression level in the test cells compared to the control cells demonstrates the presence of a condition associated with increased PTGFRN expression. In some embodiments, the test cells are obtained from an individual suspected of having a condition associated with increased PTGFRN expression. In some embodiments, the condition is a proliferative condition, such as cancer or a tumor.

[0157] In some embodiments, diagnostic or detection methods (such as those described above) include detecting the binding of an anti-PTGFRN antibody to PTGFRN expressed on the surface of a cell or in a membrane preparation obtained from cells expressing PTGFRN on their surface. In some embodiments, the method includes contacting a cell with an anti-PTGFRN antibody under conditions allowing binding of the anti-PTGFRN antibody to PTGFRN, and detecting whether a complex is formed between the anti-PTGFRN antibody and PTGFRN on the cell surface. An exemplary assay for detecting the binding of an anti-PTGFRN antibody to PTGFRN expressed on the cell surface is a flow binding assay using flow cytometry. Certain other methods can be used to detect the binding of an anti-PTGFRN antibody to PTGFRN. Such methods include, but are not limited to, antigen binding assays well known in the art, such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation assay, fluorescence immunoassay, protein A immunoassay, lateral flow assays, and immunohistochemistry (IHC).

[0158] In some embodiments, the anti-PTGFRN antibody is labeled. The label includes, but is not limited to, labels or portions that are directly detectable (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and portions that are indirectly detected (e.g., by enzymatic reactions or molecular interactions), such as enzymes or ligands.

[0159] In some embodiments, the disease treated with a PTGFRN binder (e.g., an anti-PTGFRN antibody or conjugate) is one or more types of cancer (preferably human cancer). In some embodiments, the cancer is characterized by PTGFRN-expressing cells to which the PTGFRN binder (e.g., an antibody or conjugate) binds. In some preferred embodiments, the cancer type is head and / or neck cancer, squamous cell carcinoma, epidermoid carcinoma, medulloblastoma, mesothelioma, and hematopoietic system cancer, and / or as disclosed herein, or otherwise as can be determined by those skilled in the art. This disclosure provides such methods of treating cancer, comprising administering a therapeutically effective amount of a PTGFRN binder to a subject (e.g., a subject requiring treatment). As disclosed herein, in some preferred embodiments, the method comprises determining the overexpression of the PTGFRN protein in cancer cells to be treated and comparing it to a control / non-cancerous sample (e.g., cells or tissue). In a preferred embodiment, overexpression is determined by measuring the expression of RNA encoding PTGFRN and / or the PTGFRN protein.

[0160] This disclosure further provides methods for inhibiting tumor growth using the antibodies or conjugates described herein. In some embodiments, the method of inhibiting tumor growth includes contacting cells in vitro with a PTGFRN conjugate (e.g., an antibody or conjugate). For example, immortalized cell lines or cancer cell lines expressing PTGFRN are cultured in a medium in which an antibody or other agent for inhibiting tumor growth is added. In some embodiments, tumor cells are isolated from patient samples, such as patient-derived cell or tissue biopsy samples for, for example, patient-derived xenografts, pleural effusion, or blood samples, and cultured in a medium in which a PTGFRN conjugate is added to inhibit tumor growth.

[0161] In some embodiments, methods for inhibiting tumor growth include contacting a tumor or tumor cells with a PTGFRN binder (e.g., an antibody or conjugate) in vivo. In some embodiments, contacting a tumor or tumor cells with a PTGFRN binder is performed in an animal model. For example, a PTGFRN binder may be administered to xenografts of PTGFRN-expressing human cell lines or patient-derived cells that have already been grown in immunocompromised mice to inhibit tumor growth.

[0162] In some embodiments, the method of inhibiting tumor growth includes administering a therapeutically effective amount of a PTGFRN binder to a subject. In some embodiments, the subject is a human being. In some embodiments, the subject has a tumor or has had a tumor removed.

[0163] In some embodiments, the tumor expresses PTGFRN bound by the PTGFRN binder or antibody. In some embodiments, the tumor overexpresses human PTGFRN. In some embodiments, the method may include isolating cancerous tissue and / or cells and determining whether the cancerous tissue overexpresses PTGFRN compared to non-cancer cells, optionally wherein PTGFRN expression is determined by measuring the expression of PTGFRN protein and / or RNA encoding PTGFRN.

[0164] In some embodiments, this disclosure provides a method for treating a condition in a subject related to PTGFRN function or expression, comprising administering an effective amount of the pharmaceutical composition of this disclosure to a subject in need. Treatable conditions include cancer.

[0165] In some embodiments, this disclosure provides a method for reducing tumor growth or progression in a subject with a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need. In some embodiments, this disclosure provides a method for reducing metastasis of cancer cells expressing PTGFRN in a subject, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need. In some embodiments, this disclosure provides a method for inducing tumor regression in a subject with a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need. In some embodiments, this disclosure provides a method for inhibiting the growth of cancer stem cells in a subject who previously had a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need. In some embodiments, this disclosure provides a method for preventing tumor recurrence in a subject who previously had a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need. In some embodiments, this disclosure provides a method for relieving symptoms in a subject who has or previously had a tumor expressing PTGFRN, comprising administering an effective amount of the pharmaceutical composition of this disclosure to the subject in need.

[0166] In some embodiments, this disclosure provides methods for imaging cells, comprising: contacting the cells with an antibody of the present disclosure or an antigen-binding fragment thereof or an antibody conjugate thereof; and detecting the antibody or the antigen-binding fragment thereof or the antibody conjugate thereof. In some embodiments, this disclosure provides methods for identifying PTGFRN expression in tumors, comprising: (1) obtaining a sample of the tumor; (2) contacting the sample with an antibody covered by the present disclosure or an antigen-binding fragment thereof or an antibody conjugate thereof; and (3) detecting the antibody or the antigen-binding fragment thereof or the conjugate thereof. In some embodiments, the detection is performed by any immunochemical method. In some embodiments, the method for identifying PTGFRN expression in tumors can be performed by immunochemistry (IHC). In other embodiments, such methods can be performed by, but are not limited to, antigen-binding assays well known in the art, such as Western blot, lateral flow chromatography, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation assay, fluorescence immunoassay, and protein A immunoassay. In some embodiments, the method includes in vivo imaging, for example, for identifying the presence and / or location of tumor cells and / or tumors. In some such in vivo embodiments, the method may include targeted therapy, delivering a toxic compound bound to an antibody to the tumor, or using an antibody to perform targeted radiotherapy on the tumor.

[0167] This disclosure provides monoclonal antibodies (mAbs) specifically binding to prostaglandin F2 receptor inhibitors (PTGFRN), named 4FB, 6B2, 8C7, and 12D8, PTGFRN being preferentially expressed in certain cancer cells, particularly medulloblastoma and mesothelioma. The monoclonal antibodies of this disclosure bind to cells naturally expressing PTGFRN and cells transfected to express PTGFRN. The monoclonal antibodies of this disclosure can also be internalized by cancer cells expressing PTGFRN, and are therefore potential payloads for killing targeted cells. An example of this use of the monoclonal antibodies of this disclosure is provided below in the form of a general type of antibody-drug conjugate (ADC), which consists of the monoclonal antibody of this disclosure linked to duocarmycin. Duocarmycin is widely used in proof-of-concept ADC studies. Using this ADC, selective action on target cells has been demonstrated both in vitro and in vivo, while cells not expressing the target PTGFRN remain unaffected. These data suggest that PTGFRN bound by these monoclonal antibodies is a valuable target for developing novel anticancer agents for certain cancers.

[0168] Therefore, this disclosure provides the following preferred embodiments. In some preferred embodiments, this disclosure provides isolated antibodies or antigen-binding fragments thereof comprising: heavy chain variable regions comprising complementarity-determining region (CDR) sequences SEQ ID NOs: 1, 2, and 3, and light chain variable regions comprising CDR sequences SEQ ID NOs: 7, 8, and 9, respectively (4F8 K); heavy chain variable regions comprising complementarity-determining region (CDR) sequences SEQ ID NOs: 4, 5, and 6, and light chain variable regions comprising CDR sequences SEQ ID NOs: 7, 8, and 9, respectively (4F8 Ch); heavy chain variable regions comprising complementarity-determining region (CDR) sequences SEQ ID NOs: 4, 5, and 6, and light chain variable regions comprising CDR sequences SEQ ID NOs: 7, 8, and 9, respectively (4F8 Alt); heavy chain variable regions of SEQ ID NO: 189 and light chain variable regions of SEQ ID NO: 190 (4F8); and complementarity-determining region (CDR) sequences SEQ ID NOs: 18, 19, and 20. The heavy chain variable region and the light chain variable region containing CDR sequences SEQ ID NOs: 24, 25 and 26, respectively (6B2 K); the heavy chain variable region containing complementarity determinant (CDR) sequences SEQ ID NOs: 21, 22 and 23, and the light chain variable region containing CDR sequences SEQ ID NOs: 24, 25 and 26, respectively (6B2 Ch); the heavy chain variable region containing complementarity determinant (CDR) sequences SEQ ID NOs: 27, 28 and 29 or 30, and the light chain variable region containing CDR sequences SEQ ID NOs: 31 or 32, and 33 and 34 or 35, respectively (6B2 Alt); the heavy chain variable region of SEQ ID NO: 191 and the light chain variable region of SEQ ID NO: 192 (6B2); the heavy chain variable region containing complementarity determinant (CDR) sequences SEQ ID NOs: 36, 37 and 38, and the light chain variable region containing CDR sequences SEQ ID NOs: 36, 37 and 38, and the light chain variable region containing CDR sequences SEQ ID NOs: 36, 37 and 38, respectively (6B2 Ch); Light chain variable regions of NOs: 42, 43 and 44, respectively (8C7K); Heavy chain variable regions containing complementarity-determining region (CDR) sequences SEQ ID NOs: 39, 40 and 41, and light chain variable regions containing CDR sequences SEQ ID NOs: 42, 43 and 44, respectively (8C7Ch); Heavy chain variable regions containing complementarity-determining region (CDR) sequences SEQ ID NOs: 45, and 46 or 47, and 48 or 49, and light chain variable regions containing CDR sequences SEQ ID NOs: 50, and 51, and 52 or 53, respectively (8C7Al); Heavy chain variable regions of SEQ ID NO: and light chain variable regions of SEQ ID NO: 193 and 194 (8C7);The heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NOs: 54, 55, and 56, and the light chain variable regions containing the CDR sequences SEQ ID NOs: 60, 61, and 62, are respectively (12D8 K); the heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NOs: 57, 58, and 59, and the light chain variable regions containing the CDR sequences SEQ ID NOs: 60, 61, and 62, are respectively (12D8 Ch); the heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NOs: 63, and 64 or 65, and 66, and the light chain variable regions containing the CDR sequences SEQ ID NOs: 67, 68, and 69 or 70, are respectively (12D8 Alt); the heavy chain variable region of SEQ ID NO: 195 and SEQ ID NO: 196. The antibody or its derivative comprises the light chain variable region (12D8); or a derivative thereof, optionally wherein the derivative comprises one to four amino acid substitutions in at least one CDR, preferably wherein the substitutions are conserved amino acid sequences; wherein the antibody or its derivative specifically binds to a human prostaglandin F2 receptor inhibitor (PTGFRN). In some preferred embodiments, the antibody binds to cells expressing PTGFRN in vitro and / or in vivo. In some preferred embodiments, this disclosure provides an antibody that competes with any of the above antibodies for binding to PTGFRN on cells. In some preferred embodiments, this disclosure provides combinations of the above antibodies, as well as other reagents and methods. In some preferred embodiments, the antibody is an isolated monoclonal antibody, preferably a human monoclonal antibody. In some preferred embodiments, the antibody is derived from human antibodies, human IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, human IgE, canine antibodies, canine IgGA, canine IgGB, canine IgGC, canine IgGD, chicken antibodies, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, cat antibodies, goat antibodies, goat IgG, mouse antibodies, mouse IgG, porcine antibodies, rat antibodies, alpaca antibodies, llama antibodies, shark antibodies, and camel antibodies. In some preferred embodiments, this disclosure provides derivatives of such antibodies, optionally selected from F; ab F ab2 Fab' single-chain antibody, F vSingle-chain, monospecific, bispecific, trimeric, multispecific, multivalent, chimeric, canine-human, canine-mouse, antibodies containing canine Fc, humanized antibodies, human antibodies, canine-derived antibodies, CDR transplantation antibodies, shark antibodies, and nanobodies. In some preferred embodiments, any antibody derivative disclosed herein may comprise a detectable label that can be immobilized and attached thereto, optionally said detectable label being selected from fluorescein, DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein, 5-FAM, serotonin, 5-hydroxytryptamine (5-HAT), 6-carboxyfluorescein (6-FAM), FITC, 6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET), 6-carboxy-1,4-dichloro-2',4',5',7'-tetrachlorofluorescein (HEX), and 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (6-JOE), Alexa Fluor, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa... Fluor488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor700, Alexa Fluor 750, BODIPY fluorescent dye, BODIPY 492 / 515, BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 505 / 515, BODIPY 530 / 550, BODIPY 542 / 563, BODIPY 558 / 568、BODIPY 564 / 570、BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650-X, BODIPY 650 / 665-X, BODIPY665 / 676, FL, FL ATP, FI-ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-XSE, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine B, Rhodamine B200, Rhodamine BB, Rhodamine BG, Rhodamine Bextra, 5-CarboxytetramethylRhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Rhodamine, Rhodamine B, Phallicidine, Phalloidine, Rhodamine Red, Rhodamine-2, 6-Carboxy-X-Rhodamine (ROX), Carboxy-X-Rhodamine (5-ROX), Sulforhodamine B and C, Sulforhodamine G Extra, 6-CarboxytetramethylRhodamine (TAMRA), TetramethylRhodamine (TRITC), Rhodamine WT, Texas Red, and Texas Red-X. In some preferred embodiments, the antibody may include an effector moiety attached thereto, optionally wherein the effector moiety is selected from cytotoxic drugs, toxins, diphtheria A chain, exotoxin A chain, ricin A chain, absinthecin A chain, curcin, crotonin, phenylmycin, enzyme, and radiochemical substances. In some preferred embodiments, the cytotoxic drug is duocarmycin. In some preferred embodiments, an uncleavable or cleavable linker is provided between the antibody and the effector moiety, wherein the cleavable linker releases the effector moiety into or within cells. In some preferred embodiments, this disclosure provides an isolated polynucleotide encoding an antibody disclosed herein, or a polynucleotide comprising at least one polynucleotide having at least about 90% sequence identity with any of the polynucleotides herein. In some preferred embodiments, the polynucleotide may be contained in an expression vector. In some preferred embodiments, this disclosure provides a host cell comprising an isolated polynucleotide and / or an expression vector. In some preferred embodiments, this disclosure provides a composition comprising at least one antibody or derivative disclosed herein; at least one isolated polynucleotide and / or one or more expression vectors containing the polynucleotide; and / or at least one host cell containing the polynucleotide; or a combination thereof; and a pharmaceutically acceptable vector.

[0169] In some preferred embodiments, this disclosure provides a method for detecting PTGFRN on cells and / or tissues, the method comprising contacting a test biological sample with an antibody or derivative of this disclosure and detecting the antibody binding to the biological sample or a component thereof. In some preferred embodiments, the method comprises comparing the amount bound to the test biological sample or a component thereof with the amount bound to a control biological sample or a component thereof, wherein an increase in the amount bound to the test biological sample or a component thereof compared to the control biological sample or a component thereof indicates the presence of cells expressing PTGFRN in the test biological sample (preferably, for example, mammalian cells, tissues, or biological fluids, such as blood). In some preferred embodiments, the method is an in vivo method or an in vitro method. In some preferred embodiments, this disclosure provides an in vivo method for detecting tumor cells and / or diagnosing cancer, the method comprising administering at least one antibody, combination, or derivative and detecting at least one antibody binding to tumor cells. In some preferred embodiments, the at least one antibody or derivative comprises at least one detectable marker. In some preferred embodiments, the method comprises using at least two antibodies and / or derivatives, each antibody and / or derivative comprising at least one detectable marker, the detectable markers of each antibody and / or derivative may be the same or different. In some preferred embodiments, the method includes imaging a tumor for targeted cancer therapy. In some preferred embodiments, the method further includes treating cancer. In some preferred embodiments, this disclosure provides a method for treating cancer, the method comprising administering at least one antibody, combination, or derivative of this disclosure to a mammal. In some preferred embodiments, the at least one antibody or derivative comprises an effector portion. In some preferred embodiments, the method comprises using at least two antibodies or derivatives, each antibody or derivative comprising at least one effector portion, the effector portion of each antibody or derivative may be the same or different. In some preferred embodiments, the method comprises administering at least two antibodies to a mammal, wherein at least one antibody is a naked antibody without a cytotoxic effector portion attached thereto, and at least one antibody comprises a cytotoxic effector portion attached thereto. In some preferred embodiments, this disclosure provides a method for detecting, diagnosing, and treating cancer, the method comprising imaging a tumor with an antibody, composition, or derivative of this disclosure attached thereto, and targeting the cancer treatment to the tumor or its cells. In some preferred embodiments, the diagnostic methods of this disclosure may include measuring PTGFRN protein in biological fluids (e.g., blood, plasma, exosomes) and / or tissues using imaging and / or other techniques (e.g., immunohistochemistry).

[0170] In some preferred embodiments, the antibody or a derivative thereof comprises a detectable marker and / or an effector moiety. In some preferred embodiments, this disclosure provides methods for treating, preventing, and / or improving mammalian cancer, comprising administering to the mammal at least one effective dose of a pharmaceutical composition comprising at least one antibody and / or derivative of this disclosure. In some preferred embodiments, the antibody comprises a cytotoxic effector moiety attached thereto (preferably an "M" group in the general formula provided herein), optionally wherein the effector moiety is as disclosed herein, or in other forms available to those skilled in the art (e.g., cytotoxic drugs, toxins, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotonin, phenylmycin, entromine, and radiochemical substances). In some preferred embodiments, the cytotoxic drug is duocarmycin. Other cytotoxic drugs are also applicable, as described herein, and in other forms available to those skilled in the art. In some preferred embodiments, an uncleavable or cleavable linker is located between the antibody and the effector moiety, wherein the cleavable linker releases the effector moiety into or within cells. In some preferred embodiments, the antibody is administered as an antibody-drug conjugate. In some preferred embodiments, the antibody is administered to the animal multiple times; and / or, the antibody is administered at a dose of about 1 to 50 mg / kg.

[0171] In some preferred embodiments, this disclosure provides a kit for detecting PTGFRN expression in cells, tissues, or biological fluids, the kit comprising one or more antibodies (antibodies disclosed herein or derivatives thereof) and instructions for use. In some preferred embodiments, the antibodies or derivatives thereof are in lyophilized form.

[0172] Other implementation methods are also disclosed herein, which will be understood by those skilled in the art.

[0173] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or variations thereof will be apparent to those skilled in the art and should be included within the spirit and scope of this disclosure. When preceded by a list of values ​​or ranges, the terms “about,” “approximately,” etc., independently refer to each individual value in the list or range as if each individual value in the list or range were immediately followed by the term. These terms mean that the value they refer to is exactly, close to, or similar to it. Optionally, it implies that an event or condition subsequently described may or may not occur, and the description includes instances where the event or condition occurs and instances where it does not occur. A range herein may be expressed as from about one particular value and / or to about another particular value. When such a range is expressed, another aspect includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, it can be understood by using the antecedent of about or approximation that the particular value forms another aspect. It will be further understood that each endpoint of a range is significant relative to the other endpoint and independent of the other endpoint. A range (e.g., 90-100%) means including the range itself as well as each individual value within the range, as if each value were listed separately. All references cited in this disclosure are incorporated herein by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided by way of example only and are not intended to limit the scope of the claims in any way. Example

[0174] As these examples illustrate, a unique fully human anti-PTGFRN monoclonal antibody has been developed in Tc mice (TC-mAb mice) that produce human Abs by inoculating them with recombinant human PTFRN ECD protein. TC-mAb mice stably maintain mouse-derived engineered chromosomes containing complete human Ig heavy chain and κ chain loci in humanized mice with a mouse Ig gene knockout background (Moriwaki et al., Exp. Cell. Res. 390(2): 111914). These TC-mAb mice carrying miniature chromosomes containing human immunoglobulin (Ig) loci can facilitate the development of fully human therapeutic monoclonal antibodies (Abs) by fusing mouse B cells with myeloma cells to form hybridomas after immunization with target antigens and then plate these cells in semi-solid culture media to obtain selectable and evaluable single hybridoma clones. Of the 1248 clones examined, we narrowed down to four (4) hybridomas producing fully human anti-PTGFRN antibodies through a series of progressive iterations encompassing multiple biochemical and biological assays, including flow cytometry binding and internalization analyses, and sequenced their heavy and light chains. Two antibody families were identified. The first group, represented by 8C7, binds to a variety of cell types expressing different levels of PTGFRN on their cell surface. The second group, represented by 4F8, 12D8, and 6B2, appears to preferentially bind to cells expressing very high levels of PTGFRN. These distinct antibodies have been shown to inhibit the proliferation of human cancer cells expressing PTGFRN when present in ADC form, thus opening up therapeutic applications for human diseases with PTGFRN overexpression, including but not limited to cancer and metastasis.

[0175] Example 1. PTGFRN expression is associated with metastatic phenotypes This embodiment demonstrates the use of stable shRNA and cDNA transfection to knock down and overexpress PTGFRN in three different cancer cell lines, two of which represent rare and aggressive cancers (mesothelioma and pediatric medulloblastoma), to generate new cell lines (clones) with altered PTGFRN expression. The resulting clones were characterized by decreased proliferation, migration, colony formation, and globular growth in cells with suppressed PTGFRN expression, while the opposite was observed in cells overexpressing PTGFRN. This embodiment also shows that PTGFRN can directly bind to two protein chaperones (integrin β1 and E. cadherin), with E. cadherin being a novel direct binding chaperone for PTGFRN. Furthermore, this embodiment shows that silencing PTGFRN expression affects autophagy, providing another potential pathway for PTGFRN to promote cancer cell phenotypes. These findings suggest a potential role for PTGFRN in cancer metastasis and hint at PTGFRN as a future target for drug development to treat metastatic cancers. This embodiment also shows that PTGFRN can provide a pre-screening target for identifying individuals who may be candidates for PTGFRN-related therapies, some of which have been disclosed herein.

[0176] A. Materials and Methods 1. Cell lines Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). A431 (CRL-1555), DAOY (HTB-186), and MSTO-211H (CRL-2081) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) / Ham's F12 medium (DMEM / F12 1:1 mixture) supplemented with 50 μg / ml gentamicin and 5% FBS, and maintained in an incubator at 37°C and 5% CO2.

[0177] 2. Silencing / Overexpression of PTGFRN and Clonal Screening To silence PTGFRN, A431 and DAOY cells were transfected with two different human PTGFRN shRNAs (FenicsBio, HSH321177-100) to silence PTGFRN expression (shRNA #1 sequence: TAGCCTTAAGAATGAATATGAA; shRNA #2 sequence: GTGGTATGTTTTGCTTTCCTAA), and a scrambled shRNA as a control.

[0178] For PTGFRN overexpression, MSTO-211H cells were transfected with a pcDNA3.1 plasmid vector (Thermo Fisher, V79020) containing a human PTGFRN gene insert to overexpress PTGFRN. An empty vector served as a negative control. All transfections were performed using Lipofectamine 3000 reagent (Thermo Fisher, L3000015) according to the manufacturer's instructions. After transfection, the mixed cells were sorted using a Hana single-cell sorter (Namocell, #NI004) and aliquoted into 96-well plates. These single-cell clones were then amplified, and the lowest and highest PTGFRN expression clones in each cell line were screened by flow cytometry and Western blotting.

[0179] For flow cytometry, we used a human anti-PTGFRN monoclonal antibody, 8C7, which was generated in our laboratory by inoculating humanized TC mice (Takauchi et al., 2005), targeting the extracellular domain of PTGFRN (PTGFRN-ECD). All cells were isolated and collected using PBS-5mM EDTA. Cells were incubated at 4°C at 5 x 10⁻⁶ cells / mL. 5 Cells were incubated for 1 hour in DMEM + 1% BSA medium with increased concentrations of human IgG or anti-PTGFRN monoclonal antibody 8C7. Afterwards, the cells were washed three times with cold PBS and incubated for 1 hour at 4°C with 20 μg / mL goat anti-human IgG-Alexa Fluor 647 (Jackson Immuno Research, 109-605-088) in DMEM + 1% BSA medium. Subsequently, the cells were washed three times with cold PBS, resuspended in PBS, and binding was measured using an Intellicyt flow cytometer (Intellicyt HTFC screening system).

[0180] For Western blot analysis, cells were lysed in RIPA buffer containing a mixture of protease inhibitors (Roche). 20 µg of total cell lysate protein was prepared and diluted with reducing SDS+DTT sample buffer. After heating to 100 °C for 5 min, the mixture was cooled on ice, and all samples were run on 4–12% Bis Tris gels (Genscript, M00653). After separation, proteins were transferred to PVDF membranes and blocked with 5% milk-PBST for 1 h at room temperature. Following blocking, the membranes were incubated overnight at 4 °C with 0.2 µg / mL anti-PTGFRN goat antibody (Bio-techne, AF4495). The next day, the membranes were washed with PBST and then incubated for 1 h at room temperature with rabbit-anti-goat-HRP conjugate antibody (Jackson ImmunoResearch, 313-035-045) in 5% milk-PBST (1:2000). Subsequently, the cells were developed using ECL chemiluminescence solution in an Azure Biosystems 280 chemiluminescence unit.

[0181] 3. siRNA knockdown of PTGFRN, E. cadherin, and integrin β1 A431 cells were transfected with siRNAs (Thermo Fisher) encoding PTGFRN, E. cadherin, and integrin β1. Transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher, L3000015) according to the manufacturer's instructions. After transfection and incubation for 48 hours, cells were washed three times with PBS and lysed in a 1% Brij-O10 + protease inhibitor mixture. Specific knockdown was confirmed using Western blot analysis.

[0182] 4. Proliferation assay with decreased serum concentration All clones selected for functional assays were isolated and collected with PBS-5mM EDTA. Cells were then plated at 250 cells / well (triple) in multiple 96-well DMEM / F12 plates containing 0.2% fetal bovine serum (FBS). These plates were then incubated at 37°C in a 5% CO2 incubator. Proliferation was assessed at each time point by measuring ATP levels using a Molecular Devices LMaxII chemiluminescence analyzer and CellTiter-Glo assay kit (Promega, G9683).

[0183] 5. Migration Measurement Transwell chambers and 96-well plates were coated overnight at 4°C with type II rat collagen (corning) (40 µg / mL) in sterile deionized water. The coating was aspirated the following morning and allowed to air dry completely. For MSTO-211H cells, a 24-hour serum starvation step was performed before cell collection. Cells were separated with 5 mM EDTA and treated with DMEM / F... 12 Wash and resuspend with 0.1% BSA. (7.5 x 10) 4 1 cell (for A431 cells) or 5 x 10 4 One cell (for DAOY and MSTO-211H cells) is seeded in the inner chamber of the chamber. The outer chamber is filled with DMEM / F. 12 +5% FBS medium. A431 cells were allowed to migrate for 18 hours, DAOY cells for 6 hours, and MSTO-211H cells for 48 hours. For each cell line, a pre-defined number of cells were seeded in 96-well plates, a standard curve was established, and cells were allowed to adhere for 5 hours before fixation and crystal violet staining, as described below. All cultures were performed in a humidified, 5% CO2, 37°C incubator.

[0184] After their respective incubation periods, the inserts were removed from the chambers, and unmigrated cells at the top of the Transwell chambers were wiped away with cotton swabs. The standard curve and inserts were fixed in 4% paraformaldehyde for 10 minutes, then placed in 0.05% crystal violet solution (Fisher Scientific, C581-25) and stained overnight at room temperature. Images of the inserts were captured, and the dye was eluted with 0.1M citric acid solution dissolved in 50% ethanol to quantify the number of migrating cells and compare them with the cell standard curve in a 96-well plate. The eluted crystal violet solution was measured at OD590 nm.

[0185] 6. Clonogenesis assay A431 and DAOY shRNA transfected clones and MSTO-211H overexpression clones were encoded in DMEM / F. 12 Cells were seeded repeatedly in 6-well plates containing +5% FBS at densities of 200, 100, and 50 cells / well. These plates were incubated at 37°C for 10 days. Cell culture medium was then aspirated, the plates were washed with PBS, and cells were fixed with 4% paraformaldehyde for 10 minutes. After fixation, the paraformaldehyde was removed, and the cells were stained in 0.005% crystal violet solution for 20 minutes. The plates were washed three times with H2O, images of the stained clones were captured, and the clone count was performed.

[0186] 7. 3D Spheroid Culture To generate an A431 sphere, use an 8x10 4 Cells / well in DMEM / F 12 Coated in 6-well ultra-low adhesion plates (Costar, 3471) with +0.4% BSA and +4µg / mL bovine insulin. Spheroids were allowed to form and persist in culture for 10 days.

[0187] For DAOY spheroids, 500 cells / well were plated in 96-well ultra-low adhesion plates (Costar, 7007) in complete 3D tumor spheroid medium XF (Promo Cell, C-28070). Imaging and lysis buffer collection were performed 10 days after spheroid formation.

[0188] 8. Immunofluorescence The anti-PTGFRN human monoclonal antibody 8C7 was generated in our laboratory (the manuscript has been submitted and is awaiting publication). Following the manufacturer's instructions, 8C7 was conjugated with Alexa Fluor 555 using the Zip Alexa Fluor™ Rapid Antibody Labeling Kit (Thermo Fisher, Z11234).

[0189] Cover slips with chambers (Thermo Fisher, #155380) were coated overnight at 4°C in sterile deionized water containing 50 µg / mL poly-D-lysine (Millipore Sigma, P6407). The next day, the collagen solution on the cover slips was aspirated and air-dried at room temperature for 2 hours. 8 x 10-1 cells were inoculated into each chamber. 4 Cells were collected and incubated overnight at 37°C with 5% CO2 to allow them to adhere. The next day, the cells were washed once with PBS and fixed with 100% methanol at -20°C for 10 minutes. After washing three times with PBS, the cells were incubated overnight at 4°C with 1 µg / mL 8C7-Alexa Fluor 555, 1:200 anti-E. cadherin-Alexa Fluor 488 (Cell Signaling, 3199S), 1:100 diluted anti-integrin β1-Alexa Fluor 647 (Abcam, ab214706), and 1 µg / mL Hoechst 33342 (Thermo Fisher, H1399) diluted with 0.2% PBST + 1% BSA. The next day, the coverslips were washed three times with 0.2% PBST and then mounted with ProLong™ anti-fading glass mounting medium (Thermo Fisher Scientific, P36982). The slides were stored overnight at 4°C and then observed using a Nikon A1 point scanning laser confocal microscope (NIS-Elements, 100X).

[0190] For autophagy immunofluorescence, coverslips and cells were prepared using the same method, except for the fixation steps. Cells were fixed with 4% paraformaldehyde for 10 minutes, then permeabilized with 0.2% Triton X-100 and washed three times with PBS. Cells were incubated overnight at 4°C with 1 µg / mL anti-LC3B antibody (Abcam, ab48394) and Hoechst 33342. After washing three times with PBST, cells were incubated with 5 µg / mL goat anti-rabbit Alexa Fluor 555 antibody (Abcam, ab150078) at 4°C for 2 hours. Washing, mounting, and development were performed as described above.

[0191] 9. Co-immunoprecipitation Cells were lysed in 1% Brij-O10 lysis buffer containing a mixture of protease inhibitors, and the lysis buffer was pre-cleaned overnight at 4°C with 100 µL of compacted Protein G agarose beads (Protein Mods). The next day, aliquots of the lysis buffer (250 µg) were incubated overnight at 4°C with 10 µg of control human IgG, 8C7, anti-E. cadherin antibody (Novus Biologicals, NBP3-14687), or anti-integrin β1 antibody (Bio-techne, MAB1778). The following day, Protein G agarose beads were added to the lysis buffer and mixed continuously at 4°C for 2 hours. After washing five times with 1% Brij lysis buffer, 2X SDS+DTT sample buffer was added, the beads were heated to 100°C for 5 minutes, and then cooled on ice. Electrophoresis was then performed on 4–12% Bis-Tris polyacrylamide gels using eluent, and Western blot analysis was performed using their respective Western blot antibodies. Anti-E. cadherin Western blot (WB) antibody was purchased from Cell Signaling (3195S). Anti-integrin β1 WB antibody was purchased from Fortis Life Sciences (A303-735A). Anti-GAPDH WB antibody was purchased from Cell Signaling (2118S). Goat-anti-rabbit-HRP antibody (111-035-144) was purchased from Jackson Immuno Research Laboratories.

[0192] 10. Statistical Analysis Statistical analysis was performed using GraphPad Prism version 8.3. Flow cytometry and functional analysis were performed, repeated three times. Proliferation assays were repeated three times each, with three parallel samples per assay. Welsh's T-test was used to analyze the statistical significance of the results.

[0193] B. PTGFRN expression and proliferation potential Previous studies have shown that A431 and DAOY cells are two cell lines with high PTGFRN expression levels (Marquez et al., 2021). For these two cell lines, we generated clonal derivatives in which PTGFRN expression was significantly and stably suppressed by transfection with PTGFRN shRNA plasmids, as described in the Methods section. These clones were designated A431 shRNA1 and A431 shRNA2, DAOY shRNA1 and DAOY shRNA2, respectively. Control cells were transfected with scrambled shRNA plasmids. Furthermore, transfection of human PTGFRN cDNA into the MSTO-211H cell line, which exhibits low PTGFRN expression, yielded a stable clone with enhanced PTGFRN expression (designated MSTO-PTG). Figure 2 The results of Western blot analysis of PTGFRN expression in the obtained clone are shown.

[0194] We also performed flow cytometry analysis to validate our Western blot results. By measuring the flow cytometry binding of the anti-PTGFRN antibody (8C7), we observed a decrease in PTGFRN expression of approximately 90% in the A431 shRNA and DAOY shRNA clones, while transfection of PTGFRN cDNA into MSTO-211H cells resulted in a 10-fold increase in PTGFRN expression. Figure 3 ).

[0195] C. Effect of PTGFRN expression on cell proliferation potential under low serum conditions When their proliferation capacity was tested at low serum concentrations, compared with the control clone, the proliferation capacity of A431 shRNA1 and shRNA2 clones was reduced by 75% under low serum conditions. Figure 4A In DAOY shRNA1 and shRNA2 cells, PTGFRN knockdown resulted in a 90% reduction in proliferation. Figure 4B Compared with empty vector-transfected cells that did not proliferate or even died under the low serum conditions detected, MSTO-PTG cells showed a significantly increased proliferation level (Figure 4C).

[0196] D. Effects of PTGFRN expression on cell migration The migration ability of cells with suppressed or overexpressed PTGFRN expression was also evaluated. Figure 5A As shown, the migration ability of A431 shRNA1 and shRNA2 cells decreased by 90%, while that of DAOY shRNA1 and shRNA2 cells ( Figure 5B The migration ability of PTGFRN was reduced by approximately 65% ​​compared to the control group. In contrast to PTGFRN knockdown, overexpression of PTGFRN in MSTO-PTG cells (MSTO-211H cells expressing PTGFRN via transfection with PTGFRN cDNA) increased cell migration by 4-fold compared to the empty vector control clone. Figure 5C ).

[0197] E. PTGFRN expression and colony formation ability PTGFRN expression also affected the colony-forming ability of the tested cells. As shown in Figure 6A, the number of clones formed by A431 and DAOY shRNA1 and shRNA2 clones was significantly reduced (by 80% and 66%, respectively). Alternatively, overexpression of PTGFRN in MSTO-PTG cells significantly increased the number of clones formed, showing 50+ clones, while the MSTO-211H control cell line showed almost no clone formation even at the highest cell count tested (Figure 6B).

[0198] G. PTGFRN knockdown inhibits globular formation. The ability of cells to form globules in 3D culture is a hallmark of cancer cells, and especially cancer stem cells. The effect of PTGFRN expression on globule formation was assessed. Figure 7A As shown, after PTGFRN knockdown, the A431 shRNA clone failed to form spheroids, while cells transfected with the control shRNA were able to form spheroids. The DAOY shRNA clone also failed to form spheroids of the same size or shape as the control shRNA clone. Figure 7B For the DAOY shRNA clone, although cell aggregates formed and appeared spherical, they immediately broke down into single-cell suspensions once the culture plate was disturbed or we attempted to collect these aggregates. In contrast, the spheroids of the DAOY control shRNA clone were maneuverable and did not break down into individual cells. This suggests that positive expression of PTGFRN is associated with the ability of cells to form spheroids in 3D culture.

[0199] H. PTGFRN knockdown inhibited the accumulation of integrin β1 and E. cadherin under 3D culture conditions, but had no effect under 2D culture conditions. Based on the observation that PTGFRN expression affects globule formation, we investigated the mechanisms and potential protein interactions associated with this process. As detailed by Lin et al., the two main drivers of globule formation are E. cadherin and integrin β1. The binding of integrin β1 to extracellular matrix (ECM) proteins triggers the upregulation and accumulation of E. cadherin, which is responsible for tightening cell-cell junctions, leading to cell aggregation and the formation of compact, dense globules (Lin et al., Cell Tissue Res. 324(3): 411-22 (2006)). Based on this observation, we determined whether PTGFRN knockdown had any effect on the expression of these two proteins under 2D and 3D culture conditions.

[0200] exist Figure 7C In this study, the expression levels of integrin β1 and E. cadherin in 3D culture of control shRNA A431 cells that formed spheroids were compared with those in 3D culture of A431 shRNA cells that did not form spheroids. The results showed that both integrin β1 and E. cadherin maintained high expression levels in control A431 spheroids, while the expression levels of these two proteins were significantly reduced in PTGFRN shRNA A431 cells that could not form spheroids. Furthermore, this reduction in expression was not observed in shRNA1 and shRNA2 cells grown under 2D culture conditions compared to control shRNA A431 cells cultured for 2D.

[0201] I. PTGFRN, integrin β1, and E. cadherin are co-localized at cell-cell junctions and can bind directly to each other. To assess the relationship (or association) between PTGFRN, integrin β1, and E. cadherin, fluorescence microscopy was used to determine the presence of these three proteins in the same subcellular region. Figure 8 shows the results of using confocal fluorescence microscopy to determine the presence of E. cadherin (…). Figure 8A ), PTGFRN ( Figure 8B ) and integrin β1 ( Figure 8C The fluorescent antibodies produced co-localize at the junctions between cells grown in 2D culture. Figure 8D ).

[0202] To maintain direct protein-protein interactions, cell lysis was performed using a weak Brij O10 detergent for co-immunoprecipitation assays. Immunoprecipitation with anti-PTGFRN antibody 8C7 revealed the presence of integrin β1 and E. cadherin in the IP fraction. IP with anti-integrin β1 antibody showed the presence of PTGFRN and E. cadherin, while IP with anti-E. cadherin antibody showed the presence of PTGFRN and integrin β1. These results indicate an association between these three proteins and support the findings of co-localization.

[0203] The next step was to investigate whether the formation of this potential complex required all three proteins. To this end, the expression of each protein was suppressed via siRNA transfection, and the co-immunoprecipitation of the other two proteins was determined. Analysis showed that E. cadherin binding was dependent on the binding of integrin β1 and PTGFRN. Data showed that when PTGFRN expression was knocked down via siRNA transfection, E. cadherin could no longer undergo co-immunoprecipitation with integrin β1. Similarly, siRNA knockdown of integrin β1 prevented co-immunoprecipitation of PTGFRN and E. cadherin. However, siRNA transfection knockdown of E. cadherin had no effect on the co-immunoprecipitation ability of PTGFRN and integrin β1.

[0204] To confirm that the changes in the co-immunoprecipitation profile were due to the loss of binding partners rather than alterations in protein expression following transient siRNA transfection, the expression of these three proteins—PTGFRN, E. cadherin, and integrin β1—was assessed by knocking down PTGFRN, E. cadherin, and integrin β1 with siRNA. Figure 9 shows that PTGFRN knockdown had no effect on the expression levels of E. cadherin and integrin β1; E. cadherin knockdown had no effect on either PTGFRN or integrin β1 expression; and integrin β1 knockdown had no effect on PTGFRN expression. However, E. cadherin expression appeared to increase slightly after integrin β1 knockdown.

[0205] J. Loss of PTGFRN expression leads to increased LC3B accumulation, indicating enhanced autophagy. Compared to the control shRNA clone, knockdown of PTGFRN in A431 cells resulted in altered morphological features, specifically an increase in large vacuolar structures. These structures are generally considered a hallmark of autophagy. To verify this hypothesis, immunofluorescence was used (…). Figure 10A ) and Western blot analysis ( Figure 10B The expression level of the autophagy marker LC3B was measured. In both cases, LC3B expression was increased in the PTGFRN shRNA knockdown clones compared to the control shRNA clones. This provides another potential pathway for PTGFRN to influence cell phenotype.

[0206] K. Discussion Identifying proteins associated with or involved in the metastasis process and understanding their functions is crucial. Therefore, the properties of prostaglandin F2 receptor negative regulator (PTGFRN) and its potential role in promoting metastasis-like phenotypes were further evaluated.

[0207] PTGFRN is the primary binding partner for several members of the tetraspannocyte protein family. This family comprises 33 unique proteins that can bind not only to each other but also to proteins outside the tetraspannocyte protein family (Jiang, Zhang, & Huang, 2015). The primary binding partner of PTGFRN, the tetraspannocyte protein CD9, has also been found to promote proliferation in certain cell types but is generally still classified as a metastasis inhibitor; while the secondary binding partner of PTGFRN, CD151, is classified as a metastasis promoter (Lazareth et al., 2019; Zöller, 2009). However, cell type may play a role in determining the metastatic effects of tetraspannocyte proteins. For example, CD9 has been shown to both promote and inhibit cell migration, depending on the cell type studied. CD9 has been reported to inhibit migration and metastasis in small cell lung cancer (Funakoshi et al., 2003; Zheng et al., 2005). However, CD9 plays a completely opposite role in melanoma and breast cancer cell lines: it promotes the migration and metastasis of cutaneous melanoma and collagen-induced migration in triple-negative breast cancer, but inhibits the migration and metastasis of these cancers upon fibronectin stimulation (Castro-Sanchez, Soto-Guzman, Navarro-Tito, Martinez-Orozco and Salazar, 2010; Longo et al., 2001; Powner, Kopp, Monkley, Critchley and Berditchevski, 2011; Yin et al., 2014).

[0208] Recently, Mala et al. investigated the effects of PTGFRN expression on glioblastoma cells and reported similar results to ours, namely, PTGFRN silencing led to decreased GBM cell proliferation, migration, and colony formation. They found that PTGFRN shRNA silencing reduced the activity of the ERK, AKT, and mTOR signaling pathways (Mala, Baral, & Somasundaram, 2022). However, in all three cell lines, no changes in ERK and AKT phosphorylation levels were observed due to PTGFRN inhibition or overexpression (data not shown).

[0209] Interestingly, similar to glioblastoma (GBM) reported by Mala et al., the two cell lines tested in this study originated from rare cancers (medulloblastoma and mesothelioma). This work evaluated the effects of increasing PTGFRN expression in naturally low-expression cell lines, rather than focusing solely on PTGFRN silencing. The results showed that increasing PTGFRN expression enhanced cell migration, proliferation, and colony formation.

[0210] Previous studies have shown that the transmembrane and cytoplasmic domains of PTGFRN regulate cell motility, an essential feature of metastasis (Chambrion & le Naour, 2010). In leukocytes, PTGFRN has been found to interact with ERM (Ezrin-Radixin-Moesin) proteins, which act as intermediates between cell membrane and cytoskeleton components (Sala-Valdés et al., 2006). Rearrangement of cytoskeleton proteins is a prerequisite for cell motility and has been found to be driven not only in part by ERM but also in part by the inhibition of E. cadherin, which leads to reduced cell binding tightness and altered membrane protrusion activity (Alt-Holland et al., 2008).

[0211] The ability of cancer cells to proliferate and form secondary tumors is a property that can be measured in vitro using a clonogenic assay. Clonogenic capacity is considered a hallmark characteristic of malignant cancer cells or cells that transform from benign to malignant (Brix, Samaga, Belka, Lauber, & Zitzelsberger, 2021; Fiebig, Maier, & Burger, 2004). Studies have found that cells with enhanced clonogenic capacity also express higher levels of stem cell markers such as ALDH, CD31, and OCT4 (Toledo-Guzmán, Hernández, Gómez-Gallegos, & Ortiz-Sánchez, 2018; Yang et al., 2022; Zhang et al., 2023). Data in this study show that cells with suppressed PTGFRN expression have lower clonogenic capacity.

[0212] Similar to colony-forming ability, globule-forming ability has previously been shown to be associated with increased expression of stemness markers (Rozenberg et al., 2021). Research has primarily focused on the key proteomic drivers of globule formation, particularly E. cadherin and integrin β1. Studies by Lin et al. have shown that the binding of integrin β1 to extracellular matrix (ECM) proteins triggers what they call a “lag phase,” during which cells begin to upregulate E. cadherin expression, which is crucial for globule compaction and growth (R. Lin et al., 2006). This paper demonstrates that PTGFRN and E. cadherin can co-IP. This suggests that E. cadherin and PTGFRN may be interconnected, either directly or through a larger complex. Silencing PTGFRN inhibited the accumulation of integrin β1 and E. cadherin in 3D culture, further demonstrating that PTGFRN can regulate the activity of multiple proteins and protein complexes involved in cell migration and globule growth.

[0213] These experiments determined the levels of interaction among the three proteins. Knockdown of E. cadherin had no effect on co-immunoprecipitation of PTGFRN and integrin β1. Conversely, knockdown of PTGFRN inhibited co-immunoprecipitation of E. cadherin and integrin β1, while knockdown of integrin β1 prevented co-immunoprecipitation of PTGFRN and E. cadherin. Data (not shown) confirm this is due to the lack of a binding partner, as knockdown of each protein separately via siRNA transfection did not affect the expression of the other two proteins. The only observed change was a slight increase in E. cadherin expression after integrin β1 knockdown. Following integrin β1 knockdown, PTGFRN did not co-immunoprecipitate with E. cadherin, indicating that PTGFRN and integrin β1 must be present and interact with each other for E. cadherin to form a complex with these proteins.

[0214] In A431 cells, stable knockdown of PTGFRN via shRNA significantly increased the conversion of LC3I to LC3B, a widely studied biomarker of autophagy (Lazova et al., 2012). Autophagy is a form of cell death in which cells selectively isolate protein aggregates, lipids, and organelles under stress or starvation, delivering them to lysosomes for degradation. The degraded substances are then recycled by the cell (Levine, Kroemer, & Roussy, 2008; P. Lin, Chu, & Liu, 2020; Mizushima, 2010). Studies have found that autophagy plays a crucial role in suppressing cancer, as demonstrated in the development of liver cancer (Qu et al., 2003; Yue, Jin, Yang, Levine, & Heintz, 2003). However, the survival role of autophagy during starvation also suggests that cancer cells could utilize it as a protective mechanism (Degenhardt et al., 2006). In breast cancer models, autophagy has been found to support the growth of primary tumors but inhibit their metastasis to secondary sites (Marsh et al., 2020).

[0215] The integrin family plays a crucial role in mediating cell attachment to the extracellular matrix (ECM), and its disruption can trigger anoikis (a form of cell death) (Frisch & Screaton, 2001). In this context, autophagy is typically activated as a survival mechanism to counteract anoikis-induced detachment (Vlahakis & Debnath, 2017). Furthermore, integrin β1 has been found to specifically participate in the regulation of autophagy (Zhou, Liu, Zhou, & Li, 2022). E. cadherin is also associated with autophagy, as it is isolated into autophagosomes in breast and lung cancer (45, 46). This autophagy-driven degradation of E. cadherin is thought to regulate tumor progression, as loss of E. cadherin is commonly observed in migrating cells, poorly prognostic breast cancer, and dormant cancer cells (Lazova et al., 2012; Vera-Ramirez, 2020). Determining whether the autophagy observed after PTGFRN knockdown is protective or induces cell death will provide valuable insights. If this autophagy is determined to be pro-death, it supports a positive correlation between PTGFRN expression and metastatic-like phenotype. It is hypothesized that PTGFRN knockdown leads to disruption of the integrin β1 complex, thereby preventing the accumulation of E. cadherin (essential for globular formation), and simultaneously inducing cellular stress in cancer cells, which in turn activates pro-survival autophagy signaling pathways in response to anoikis.

[0216] Using three different cancer cell lines, the study showed that PTGFRN expression levels were positively correlated with cell proliferation under low serum conditions, migration across the Transwell membrane, clonal formation at low cell numbers, and the ability to form multicellular 3D spheroids—all characteristics associated with metastasis. These studies also found that the adhesion molecule E. cadherin may be a direct binding partner of PTGFRN, making it a novel binding partner for this tetraspan membrane protein. Furthermore, the simultaneous occurrence of PTGFRN loss and increased conversion of the key autophagy marker LC3B may be another potential factor influencing PTGFRN's role in cancer metastasis.

[0217] Mass spectrometry proteomics analysis can be used to further study the effects of PTGFRN on cancer cells and provide a global, non-targeted approach to better evaluate not only proteins affected by PTGFRN expression but also PTGFRN complex chaperones and identify the overall pathways affected by PTGFRN expression.

[0218] In summary, the expression of the cell surface transmembrane protein PTGFRN is associated with multiple key cellular functions in epidermoid carcinoma (A431), pediatric medulloblastoma (DAOY), and mesothelioma (MSTO-211H), such as proliferation, migration, colony formation, and 3D globule formation. These effects involve the binding of PTGFRN to integrin β1 (previously reported) and to E. cadherin (previously unreported). Notably, the loss of PTGFRN expression also occurs concurrently with an increase in autophagy (a regulated form of cell death). These findings collectively lead us to propose that PTGFRN overexpression influences cell phenotype and promotes metastatic features.

[0219] Example 2. Effects of PTFGRN expression on the proteomic characteristics of A431 cells and determination of the PTGFRN interactome. A. Materials and Methods 1. Cell lines All cell lines used in this study were obtained from the American Type Culture Collection (ATCC, Manassas, VA). A431 (CRL-1555) was cultured in Dulbecco's Modified Eagle Medium (DMEM) / Ham's F12 medium (DMEM / F12 1:1 mixture) supplemented with 50 μg / ml gentamicin and 5% FBS, and kept in an incubator at 37°C and 5% CO2.

[0220] 2. Silencing / Overexpression of PTGFRN and Clonal Screening A431 cells were stably transfected with two different human PTGFRN shRNAs (Fenics Bio, HSH321177-100) to silence PTGFRN expression (shRNA #1 sequence: TAGCCTTAAGAATGAATATGAA (SEQ ID NO: 209); shRNA #2 sequence: GTGGTATGTTTTGCTTTCCTAA (SEQ ID NO: 210), and a scrambled shRNA as a control. Transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher, L3000015) according to the manufacturer's instructions. After transfection, the mixed cells were sorted using a Hana single-cell sorter (Namocell, #NI004) and aliquoted into 96-well plates. These single-cell clones were then expanded, and clones with the lowest PTGFRN expression were screened by flow cytometry and Western blotting.

[0221] 3. Co-immunoprecipitation All immunoprecipitations (IPs) were performed in triplicate. A431 cells were lysed with 1% Brij-O10 lysis buffer containing a mixture of protease inhibitors, and the lysis buffer was pre-cleaned overnight at 4°C with 100 µL of Protein G agarose beads (Protein Mods). The next day, aliquots of the lysis buffer (250 µg each) were incubated overnight at 4°C with 10 µg of control human IgG or our self-prepared anti-PTGFRN human monoclonal antibody 8C7. The next day, Protein G agarose beads were added to the lysis buffer and mixed for 2 hours. After washing five times with 1% Brij lysis buffer, 2X SDS+DTT sample buffer was added to the beads. All immunoprecipitated proteins were then digested using an S-trap microcolumn (ProtiFi, NY). The peptides eluted from the S-column were dried, reconstituted with 0.1% formic acid, and peptide concentrations were determined using a BCA assay kit (Thermo Fisher Scientific, A53225).

[0222] 4. Mass spectrometry-based proteomics analysis Cell lysis and protein digestion were performed as previously described (Weldemariam et al., Proteomics [Internet]. July 24, 2023; n / a(n / a):2300022). Briefly, samples were lysed in a lysis buffer containing 5% sodium dodecyl sulfate (Sigma, L4509) and 50 mM triethylamine bicarbonate (1 M, pH 8.0) (Sigma, 7408). Proteins were extracted and separated on a nanoACQUITY ultra-high performance liquid chromatography (UHPLC) column (BEH130 C18, 1.7 µm, 75 µm x 200 mm; Waters Corporation, Milford, MA, USA) using a linear acetonitrile gradient (3%–40%) over 185 minutes with the addition of 0.1% formic acid. Separation was then performed using a nanoACQUITY UHPLC system (Waters Corporation, Milford, MA, USA), followed by analysis on a mass spectrometer coupled with an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, San Jose, CA, USA). Full scans were performed at a resolution of 240,000 m / z, with precursor fragmentation using high-energy collisional dissociation at a resolution of 35% and a maximum cycle time of 3 seconds. MS / MS raw files were processed using Proteome Discoverer (PD, version 2.5.0.400, Thermo Fisher Scientific) and the Sequest HT search engine, targeting the UniProt human reference proteome (version 2022.04, 20292 entries). Search parameters were set as follows: cysteine ​​carbamoyl methylation modification as static modification (+57.021 Da), methionine residue oxidation modification as dynamic modification (+15.995 Da), precursor mass tolerance of 20 ppm, and fragment mass tolerance of 0.5 Da. Trypsin was used as the digestive enzyme with a maximum of two missed cleavages. The minimum and maximum peptide lengths were set to 6 and 144 amino acids, respectively. Label-free quantification was performed using the Minora feature detector embedded in the PD bioinformatics platform (Palomba et al., J Proteome Res [Internet]. 2021 / 05 / 26. 20(7):3497–507). To obtain high-confidence results, protein identification was filtered to a 1% false detection rate (FDR) at both the peptide spectrum matching (PSM) and protein levels. The FDR was calculated using the Percolator algorithm embedded in PD.Next, the exported protein abundance values ​​were analyzed and visualized using Perseus software (version 1.6.14.0) (Tyanova et al., NatMethods [Internet]. 2016;13(9):731–40). To ensure high confidence in the statistical analysis, the data were further filtered to retain only proteins for which no missing values ​​were found in any of the biological samples. The quantitative protein data were log2 transformed and then normalized using median centering. A two-tailed Student's t-test was used to compare the two groups (p<0.05) to determine whether there was a significant difference between each treatment group and the control group. Metascape (http: / / metascape.org) was used to perform functional and signaling pathway enrichment analysis on differentially expressed proteins (DEPs) identified under different conditions, as described above (Weldemariam et al.). supra ; Zhou et al., Nat Commun [Internet]. 2019;10(1):1523). Once differentially expressed proteins were identified, bioinformatics pathway analysis was used to further infer the perturbed pathways. IngenuityPathway Analysis (IPA) was used to predict classical pathways and upstream regulators, and Z-scores were provided for overlapping pathways from two databases (Krämer et al., Bioinformatics [Internet]. 15 Feb 2014;30(4):523–30; Huang et al., Health Phys [Internet]. 2020;119(5)).

[0223] B. Result 1. Effects of PTGFRN expression on the proteomic characteristics of A431 cells This study aimed to investigate the effects of PTGFRN gene knockdown on the proteome of A431 cells. To this end, we performed mass spectrometry analysis on A431 cells transfected with shRNA to suppress PTGFRN expression (A431 shRNA) and control A431 cells transfected with disordered shRNA (cultured under 2D conditions). Proteomics analysis revealed 5680 proteomes identified with a 1% FDR after data filtering as described in the Methods section. Of these proteomes, 3455 proteomes were quantified in all 12 samples with no missing values. All proteins whose expression was significantly increased or decreased after PTGFRN gene knockdown were evaluated. Using this list of regulatory proteins, we performed pathway analysis using Metascape or IPA analysis to determine which cellular functions and pathways were most affected by decreased PTGFRN expression.

[0224] PTGFRN knockdown most significantly downregulated biological processes or pathways involved in the synthesis of multiple metabolites, metabolic precursors, and energy, including those involved in the respiratory electron transport chain. Other significantly inhibited pathways include anterograde transport from the endoplasmic reticulum to the Golgi apparatus, ribosome biosynthesis, exosome function, NADH oxidation, and the VEGFA-VEGFR2 signaling pathway. PTGFRN knockdown also led to enhanced activity of other pathways, many of which are involved in immune system signaling, such as cytokine signaling, interferon signaling, and neutrophil degranulation. The heatmap in Figure 11A shows the top 20 cellular processes whose activity was enhanced after PTGFRN knockdown via shRNA transfection. Figure 11B The table shows the top 20 cellular processes whose activity is reduced after PTGFRN knockdown. Table 16 lists the top 10 proteins whose expression is upregulated after PTGFRN knockdown, and Table 17 lists the top 10 pathways represented by these proteins. Table 18 lists the top 10 proteins whose expression is downregulated after PTGFRN knockdown, and Table 19 describes the pathways most affected by these downregulated proteins.

[0225] Table 16 10 proteins most significantly upregulated after PTGFRN knockdown

[0226] Table 17 The 10 most significantly upregulated cellular pathways after PTGFRN knockdown

[0227] Table 18 10 proteins most significantly downregulated after PTGFRN knockdown

[0228] Table 19 10 cellular pathways most significantly downregulated after PTGFRN knockdown

[0229] 2. PTGFRN interactome analysis In addition to studying the impact of PTGFRN expression regulation, mass spectrometry was used to detect PTGFRN-related proteins. This involved co-precipitation with anti-PTGFRN antibody and comparison with co-immunoprecipitation with control IgG. Based on these proteins co-immunoprecipitated with PTGFRN, pathway analysis was performed to identify which pathways contain proteins that directly bind to or are associated with PTGFRN.

[0230] To date, RNA metabolism is the most frequently involved cellular process in relation to PTGFRN. Many protein components in the VEGFA-VEGFR2 signaling pathway, translational regulation, and ribonucleoprotein complex biosynthesis pathway also co-precipitate with PTGFRN. Figure 12 Heatmaps show the 20 processes with the highest protein composition after co-immunoprecipitation with PTGFRN, indicating the presence of protein interactions. Table 20 lists the 10 most abundant proteins besides PTGFRN after co-immunoprecipitation with PTGFRN, while Table 21 lists the 10 pathways associated with these co-immunoprecipitated proteins.

[0231] Table 20 The 10 most abundant proteins in PTGFRN and 8C7 co-immunoprecipitation

[0232] Table 21 The top 10 cellular pathways represented by PTGFRN and 8C7 co-immunoprecipitation

[0233] 3. Discussion The mass spectrometry analysis presented in this paper provides new data on the effects of PTGFRN expression on proteomic features and related proteins. Firstly, the cellular processes resulting from PTGFRN knockdown and reduced expression of its protein members appear to validate previously reported findings on PTGFRN, namely that PTGFRN knockdown inhibits VEGF-induced angiogenesis, while PTGFRN overexpression is associated with metastasis-like features (Karhemo, J. Proteomics, 2021, 77:87-100; Aguila et al., Cancer Lett., 2019, 462 (April): 33-42; Guilman et al., Br. J. Cancer, 2011, 104(3): 496-504). The inability of cells to biosynthesize metabolic precursors, transport proteins for appropriate post-translational modifications, or even generate normal levels of ATP through NADH oxidation and the electron transport chain can lead to decreased cell proliferation. Furthermore, low proliferation capacity in individual cells severely limits the number of clones that can be formed. Secondly, although cell migration pathways are not listed in the databases used to analyze proteomics data, some proteins involved in cell migration and invasion do exist, and their expression is reduced after PTGFRN knockdown (data not shown). This article shows that after PTGFRN knockdown, the expression of NUDT1, DKC1, RBBP7, and RSF-1 (all included in chromosome maintenance pathways) and LRRC59 (VEGFA-VEGFR2 signaling pathway) is reduced. These proteins have all been found to be involved in and influence the migration ability of cancer cells (Ou et al., Aging, April 27, 2020, 12(8): 7363-79; Miao et al., Invest New Drugs [Internet], 2019; 37(6):1177–86; Yu et al., Oncol. Lett., Dec 2018; 16(6):7204–11; Zhang et al., Virchows Arch [Internet], 2017; 470(5):553–60; Li et al., Onco Targets Ther [Internet], July 3, 2020; 13:6453–63). Enhanced proliferation, migration, and colony formation abilities are all hallmarks of metastatic cancer cells (Fares et al., Signal Transduct Target Ther [Internet]. 2020; 5(1):28).

[0234] Analysis of proteins that directly bind to / form complexes with PTGFRN and those co-immunoprecipitated with PTGFRN using anti-PTGFRN antibodies revealed that the pathways interacting most strongly with PTGFRN are RNA metabolic pathways. In fact, nine of the top 20 protein pathways that bind to PTGFRN are directly or indirectly involved in RNA processing and / or translation. Proteins involved in ribosome subunit formation (RPL3, RPS8), RNA polymerase (POLR2H), precursor mRNA intron removal (SNRPB2), and translation initiation factor (EIF2a) are among the most abundant proteins co-immunoprecipitated with PTGFRN. This is interesting because these proteins are usually found in the cytoplasm, near the endoplasmic reticulum (Reid et al., J Biol Chem [Internet]. 2011 / 12 / 23. 2012 Feb 17; 287(8):5518–27), while PTGFRN is most commonly found in the plasma membrane. This suggests that PTGFRN acts as a membrane anchor, serving as part of a large scaffold that stabilizes ribosomes, various polymerases, and / or translation factors, enabling them to process RNA correctly. PTGFRN may also function simply as a transport protein, ensuring these components reach their desired subcellular locations from the Golgi apparatus, without actually contributing to its overall function. Alternatively, these results may point to an intracellular form of PTGFRN that functions differently from its membrane counterpart. It remains unclear whether these different functions are due to different isoforms resulting from alternative splicing, different glycosylation patterns, or whether the subcellular localization of PTGFRN affects its overall function. PTGFRN is known to have three putative splicing sites, which could potentially lead to isoforms with different functions (Cunningham et al., Nucleic Acids Res [Internet]. Jan 7, 2022; 50(D1):D988–95).

[0235] The second most numerous pathway co-immunoprecipitated with PTGFRN was the VEGFA-VEGFR2 signaling pathway. Interestingly, this pathway was also significantly reduced upon PTGFRN knockdown. Notably, the only protein downregulated by PTGFRN knockdown and co-precipitated with PTGFRN in our co-immunoprecipitate was LMAN1, which has been shown to regulate protein folding, transport from the endoplasmic reticulum to the Golgi apparatus, and other organelles (26). This further supports the hypothesis that PTGFRN appears to be highly involved in RNA processing and translation, as well as the transport of newly translated proteins to the Golgi apparatus and other cellular locations. These results appear to confirm the findings published by Colin et al., which demonstrated that transfection of cells with truncated forms of PTGFRN leads to reduced angiogenesis, thereby inhibiting tumor formation (Br J Cancer. 2011;105(7):1002–11). In summary, this analysis provides proteomics information supporting previously published observations linking PTGFRN to VEGF-induced angiogenesis and further establishing the relationship between PTGFRN expression and cancer metastasis. Furthermore, PTGFRN appears to be primarily associated with proteins involved in mRNA and protein translation / transportation, revealing other mechanisms by which PTGFRN influences cell proliferation and migration and promotes cancer metastasis. In summary, mass spectrometry analysis provides new insights into the impact of PTGFRN expression on the A431 cell proteome. Previously published findings, such as the role of PTGFRN in the VEGF signaling pathway, have also been confirmed. Interactomics analysis also provides more information on PTGFRN binding chaperones, showing that PTGFRN is associated with proteins involved in mRNA / protein translation and processing. This information provides pathways through which PTGFRN expression may influence cancer cell phenotypes and how it is associated with previously reported metastatic-like features.

[0236] Example 3. Development and functional characterization of candidate antibodies Tc mice producing human (Hu) antibodies (TC-mAb mice) stably maintain a mouse-derived engineered chromosome in a mouse Ig knockout background, containing complete human Ig heavy chain and κ chain loci (Moriwaki et al., Exp. Cell. Res. 390(2): 111914). Transchromosomal (TC) mice carrying mini-chromosomes containing human immunoglobulin (Ig) loci can be used to develop fully human therapeutic monoclonal antibodies (Abs) after immunization with target antigens. In this study, TC-mAb mice were inoculated with recombinant human PTGFRN extracellular domain protein (PTGFRNECD) according to a previously validated inoculation protocol.

[0237] Thirty days later, the serum titer of anti-PTGFRN in immunized mice was examined by enzyme-linked immunosorbent assay (ELISA). The serum was incubated with human PTGFRN ECD immobilized in a 96-well plate, followed by incubation with HRP-conjugated goat anti-human Fc secondary antibody, and then tetramethylbenzidine (TMB), a peroxidase substrate. The absorbance of each well was read at 650 nm using a microplate reader. B cells were collected from the spleen and lymph nodes of mice with the highest anti-PTGFRN titers and fused with mouse myeloma HL-1 cells via electroporation. The fused hybridoma cells were seeded as single cells in semi-solid hybridoma medium in 10 cm tissue culture dishes. Eleven days later, 1248 monoclonal hybridoma cells were picked from the semi-solid medium dishes and transferred to 96-well plates (one clone per well), also seeded in hybridoma medium. Four days later, the hybridoma clones were preliminarily screened using the PTGFRN-ECD ELISA method. The first 246 positive clones were selected and transferred to 48-well plates. The supernatant was used for confirmatory screening by ELISA. ELISA was performed by comparing the binding of Hek-293A cells (HEK-PTG) overexpressing human PTGFRN cDNA with HEK-293A cells (PTGFRN-negative cells) using flow cytometry. 207 clones were confirmed to be strongly positive in both ELISA and flow cytometry. These clones were transferred in duplicate to 6 (6)-well plates and cryopreserved in liquid nitrogen under suitable culture conditions for long-term storage. Culture medium containing secreted human anti-PTGFRN immunoglobulin was collected and preserved for subsequent evaluation and screening of target hybridomas. Various characteristics of the anti-PTGFRN antibodies produced by these selected hybridoma clones were evaluated. The binding of the antibodies to the cell surface of HEK293 cells (HEK-PTG) and HEK293A cells (PTGFRN-negative) overexpressing PTGFRN was detected by flow cytometry. Candidate anti-PTGFRN antibodies that showed significant binding to the surface of HEK-PTG cells but weak or no binding to HEK293A cells were then screened for further development. Internalization of the candidate antibodies in the HEK-PTG cell line was measured using HEK cells as a negative control. Subsequently, the binding and internalization of the candidate antibodies with known naturally expressing PTGFRN human cancer cells (including A431 cells, DAOY cells, and MSTO-211H cells) were measured.

[0238] Functional assays were performed on antibodies with cytotoxic payloads, both in vitro and in vivo, to determine whether internalized antibodies could deliver the cytotoxic payload to cells and inhibit their survival or stimulate cell death. For the in vitro cytotoxicity assay of the indirect antibody-drug conjugates, PTGFRN-positive cancer cells were incubated for three days with a candidate anti-PTGFRN antibody and a Fab anti-human IgG Fc-Duocarmycin DM antibody with a cleavable linker. The negative control group consisted of cells incubated with human IgG and the Fab anti-human IgG-Duocarmycin conjugate. After three days, cells were lysed, and cell proliferation was measured using the Cell-Glo assay (Promega). The candidate antibodies screened by this indirect assay were then evaluated as direct Duocarmycin antibody-drug conjugates using in vitro cytotoxicity assays and in vivo experiments in a mouse xenograft model. The methods described below were used to perform these assays.

[0239] For cell surface flow binding, cells suspended in a V-shaped substrate were incubated with fully human anti-PTGFRN antibody in a medium containing 0.5% bovine serum albumin at 4°C for 1 hour, followed by incubation with goat anti-human immunoglobulin conjugated with a fluorescent dye at 4°C for 1 hour. After washing the cells, the fluorescence intensity was measured by Intellicyt to determine flow binding.

[0240] For internalization studies using pHab probes, conjugate goat anti-human IgG (H+L) secondary antibody to the pH probe (Promega) according to the manufacturer's instructions. Premix the anti-PTGFRN antibody with the pHab-labeled goat anti-human antibody (antibody combination) at 22-25°C for 30 minutes. Add 2x10 5 A431 cells were incubated with the antibody at 37°C for 20 hours. After incubation, the cells were washed twice with cold PBS and resuspended in PBS. Fluorescence intensity was measured using flow cytometry in the Mean FL2-H channel. Using this assay, the fluorescence intensity value increased if the antibody was internalized.

[0241] More than 1,000 (1,248) monoclonal hybridomas producing candidate anti-PTGFRN antibodies were screened for further characterization. Preliminary and confirmatory EIAs against PTGFRN-ECDs revealed 208 strongly positive candidate antibodies in this initial cohort of 1,248. The binding of HEK-PTG to HEK-293A was compared by flow cytometry, followed by internalization, ultimately resulting in 17 clones (see Table 22). At least four hybridomas were screened after testing the activity of the candidate antibodies against natural human cancer cell lines. Inhibition of PTGFRN expression via siRNA or shRNA expression specifically inhibited the flow cytometry binding of these antibodies.

[0242] Table 22 Streaming cytometry binding and internalization data of 17 selected clones

[0243] Data showed that certain fully human anti-PTGFRN antibodies can induce the internalization of PTGFRN (including 4F8, 6B2, 8C7, and 12D8) expressed on the cell surface. Prolonged (six (6) hours) exposure of A431 cells (expressing high levels of PTGFRN) to anti-PTGFRN antibodies not conjugated with cytotoxic drugs was found to inhibit landmark biological events of metastasis, such as migration. These data suggest that anti-PTGFRN antibodies can have therapeutic efficacy even when not used as antibody-drug conjugates. In summary, A431 cells were plated in duplicate in collagen-coated six (6)-well plates in DME-F12 medium supplemented with 5% fetal bovine serum (FBS). Cells were then treated with 10 µg / ml of 8C7 antibody (anti-PTGFRN internalization antibody), 3G3 antibody (anti-PTGFRN non-internalization antibody), or human IgG (as a control). After incubation with the corresponding antibody for 6 hours, cells were digested with PBS-EDTA, and their migration ability was determined by the following method: 7.5 x 10⁻⁶ cells were incubated with the corresponding antibody. 4 Cells were plated in duplicate in collagen-treated Transwell chambers. The upper chamber was cultured in DME-F12 medium containing 0.1% bovine serum albumin, and the lower chamber in DME-F12 medium containing 5% fetal bovine serum as a migration inducer. Cells were cultured for 20 hours. After 20 hours, the cells in the lower chamber were stained with crystal violet and counted using standard methods known in the art (Table 23; each antibody was included at 10 µg / ml).

[0244] Table 23 migrate hIgG 8C7 3G3 A431 cell migration 4000 1014 4238 % control 100 25 106

[0245] Data showed that incubation with the 8C7 antibody inhibited 75% of the migration of A431 cells, while incubation with 3G3 (a non-internalized anti-PTGFRN antibody) had no inhibitory effect on migration.

[0246] Figure 13 Additional data related to the 8C7 antibody are presented. To evaluate the effectiveness of the anti-PTGFRN antibody 8C7 as a naked antibody, A431 cells were incubated with non-immune human IgG and anti-PTGFRN antibody 3G3 (which binds to PTGFRN but is not internalized). All antibodies were measured at a concentration of 10 μg / ml. After incubation, cells were isolated and their migration ability was assessed by transwell assay for 5 hours. See Table 24 and... Figure 13 As shown, cells incubated in the presence of the 8C7 antibody exhibited a 67% lower migration inhibition rate compared to hIgG and a 75% lower rate compared to cells treated with 3G3. This indicates that 8C7 can neutralize the biological effects of PTGFRN even as a naked antibody not conjugated to the adapter payload. This neutralizing effect of the 8C7 antibody may be related to its ability to internalize PTGFRN.

[0247] Table 24 After incubation with hIgG, 3G3, or 8C7 antibodies for 6 hours, the number of migrating cells was determined by transwell assay.

[0248] In order to generate Figure 13 The data shown indicate that A431 cells were pre-incubated for 6 hours with 10 μg / ml hIgG, non-internalized anti-PTGFRN antibody 3G3, or internalized antibody 8C7, before cell isolation and migration assessment using a transwell assay. Exposure of A431 cells to 10 μg / ml of internalized anti-PTGFRN 8C7 antibody for 6 hours resulted in a 70% inhibition of cell migration; however, incubation with non-internalized anti-PTGFRN antibody 3G3 did not show any inhibitory effect on A431 cell migration compared to the negative control human IgG. These results suggest that the 8C7 antibody has a neutralizing effect even without conjugation to the adapter-cytotoxic payload. Incubation with the internalized antibody led to the internalization of PTGFRN on the cell surface, thereby inhibiting cell migration. Tables 17, 18, and... Figure 13 Data from the study indicate that internalized anti-PTGFRN antibody (8C7) has an impact on the biological characteristics of cancer without binding to payloads such as antibody drug conjugates.

[0249] Furthermore, the study found that antibody 4F8 could inhibit the migration of 56% of the human dermatoma cell line MSTO-211H-PTG (which, like A431, overexpresses PTGFRN). The data are summarized in Table 25 below: Table 25 migrate 3G3 4F8 MSTO-PTG cell migration 13,846 6115 % control 100% 44%

[0250] The 8C7 antibody was further tested by flow cytometry and additional internalization studies. Cell lines used in these studies were obtained from the American Type Culture Collection (ATCC, Manassas, VA). A431 (CRL-1555), DAOY (HTB-186), MSTO-211H (CRL-2081), and MDA-MB-231 (CRM-HTB-26) cells were plated in Dulbecco's Modified Eagle Medium (DMEM) / Ham's F12 medium (DMEM / F12 1:1 mixture) supplemented with 50 μg / ml gentamicin and 5% fetal bovine serum (FBS) and maintained in a 37°C, 5% CO2 incubator. Cells were digested and collected with PBS-5mM EDTA for flow cytometry-based assays. 5 x 10⁶ cells were plated. 5 Cells were incubated with increasing concentrations of human IgG or fully human anti-PTGFRN antibody 8C7 in DMEM medium containing 1% BSA at 4°C for 1 hour. Afterwards, the cells were washed three times with cold PBS and incubated for 1 hour in DMEM + 1% BSA medium containing 20 μg / mL goat anti-human IgG-Alexa Fluor 647 (Jackson Immunoresearch #109-605-088). Subsequently, the cells were washed three times with cold PBS, resuspended in PBS, and binding was measured using an Intellicyt flow cytometer (Intellicyt HTFC screening system). For flow cytometry binding analysis, cells were separated and collected using PBS-5mM EDTA. 5 x 10⁸ cells were then... 5Cells were incubated with increasing concentrations of human IgG or fully human anti-PTGFRN antibody 8C7 in DMEM medium containing 1% BSA at 4°C for 1 hour. Afterwards, the cells were washed three times with cold PBS and incubated for 1 hour in DMEM medium containing 20 μg / mL goat anti-human IgG-Alexa Fluor 647 (Jackson Immunoresearch #109-605-088) at 4°C. Subsequently, the cells were washed three times with cold PBS, resuspended in PBS, and binding was measured using an Intellicyt flow cytometer (Intellicyt HTFC screening system). For immunofluorescence analysis, chambered coverslips (Thermo Fisher #155380) were coated overnight at 4°C with sterile deionized water containing 40 µg / mL type II rat collagen (Corning #354236). The following day, aspirate the collagen solution from the coverslip and air dry at room temperature for 2 hours. Add 1x10 5 Cells were seeded on coverslips and cultured overnight in a humidified incubator at 37°C and 5% CO2 to allow for adhesion. The following day, the cells were washed once with PBS and then incubated for 1 hour at 4°C with 1 µg / mL of 8C7 antibody (directly conjugated to Alexa Fluor 647) diluted in DMEM with 1% BSA. After binding incubation, two coverslips were returned to the 37°C incubator to initiate internalization, followed by incubation for 3 and 5 hours. At each time point, the coverslips were washed three times with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. After washing three more times with PBS, the cells were mounted with ProLong™ glass anti-quenching mounting medium (Thermo Fisher #P36980) and observed using a Nikon A1 point scanning laser confocal microscope (NIS-Elements, 60X). After observing the difference in PTGFRN expression between normal and cancerous tissues, we developed a fully human anti-PTGFRN antibody capable of internalizing the target protein. The advantage of directly developing fully human antibodies is that we can skip the humanization process required for mouse-derived antibodies. By inoculating humanized TC mice with the extracellular domain of PTGFRN, a monoclonal hybridoma library is generated and screened to select fully human antibodies that bind to PTGFRN on the cell surface, have internalization ability, and high affinity.

[0251] Among the developed fully human anti-PTGFRN antibodies, anti-PTGFRN antibody 8C7 was selected due to its internalization properties, and its K+ was determined using the Octet Red96 bilayer interferometry (BLI) assay. D 10 -12M.8C7 antibody is an IgG1 that is considered a promising candidate for ADC development (Baah et al., Molecules. 2021;26(10)). Figure 14 (A) Control shRNA A431 cells, (B) PTGFRN shRNA A431 clone cells, (C) Control shRNA DAOY cells, (D) PTGFRN shRNA DAOY clone cells, (E) Control MSTO-211H cells, (F) PTGFRN-overexpressing MSTO-211H clone cells, and (G) MDA-MB-231 cells) showed that 8C7 could bind to the cell surface of A431, DAOY, and MSTO-211H cells. However, after transfection with shRNA to knock down PTGFRN expression, the binding of 8C7 in A431 and DAOY cells was significantly weakened. Furthermore, after transfection of MSTO-211H cells with human PTGFRN cDNA to overexpress PTGFRN, the binding level of 8C7 increased. Finally, flow cytometry analysis showed that MDA-MB-231 cells did not bind to 8C7 compared to the negative control antibody, and PTGFRN in MDA-MB-231 cells was negative at both 8C7 concentrations used.

[0252] Immunofluorescence assays were performed to evaluate the internalization of PTGFRN after 8C7 binding. As shown in Figure 15, at t=0, most of the 8C7 fluorescence was localized at the cell membrane and intercellular junctions. After incubation at 37°C for 3 hours, 8C7 immunofluorescence was observed to be intracellular, with some signal still present on the cell membrane. After incubation at 37°C for 5 hours, 8C7 fluorescence was mainly located intracellularly, with almost no signal detected on the cell surface, confirming that PTGFRN was internalized after binding to the 8C7 antibody. Since 8C7 is a PTGFRN internalization antibody, its ability to deliver a cytotoxic payload to cells was then determined.

[0253] In vitro cytotoxicity assays used to characterize candidate antibodies include assays for inhibition of cell proliferation and survival using indirect antibody-drug conjugate combinations (Marquez et al. (2021) "Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizing target in cancer cells for antibody-drug conjugate development." PLoS One 16(1): e0246197). These assays are briefly described below.

[0254] For the in vitro assay of cytotoxicity of indirect antibody-drug conjugates, PTGFRN-positive cancer cells (A431 cells) were incubated for three days with candidate anti-PTGFRN antibodies (e.g., 4F8, 6B2, 8C7, and 12D8) and Fab anti-human IgG Fc-Duocarmycin DM antibody containing a cleavable linker. The negative control group consisted of cells incubated with human IgG and the Fab anti-human IgG-duocarmycin conjugate. After three days, cells were lysed, and cell proliferation was measured using a Cell-Glo assay (Promega). Figure 16 Indirect assay data for 8C7, 4F8, and 12D8 antibodies were summarized. For example... Figure 16 As shown, the three antibodies evaluated (8C7, 4F8 and 12D8) demonstrated the ability to inhibit the proliferation and survival (equivalent to survival rate) of A431 cells in a dose-dependent manner, with 8C7 exhibiting the strongest potency.

[0255] To investigate the direct effects of anti-PTGFRN antibody-drug conjugates, we prepared duocarmycin conjugates of candidate anti-PTGFRN antibodies (e.g., 8C7, 4F8, 6B2, and 12D8) using the following method. Briefly, the candidate antibodies were conjugated to a Mc-Vc-PAB-duocarmycin (VcDuo) linker payload (MedChem Express HY-128904) via cysteine ​​residues. The candidate antibodies were dissolved in 25 mM NaCl + 25 mM borate + 1 mM DTPA (pH: 8) and treated with tris(2-carboxyethyl)phosphine (TCEP) at 37 °C for 30–45 min, then cooled on ice. The candidate antibody solution in 10% N,N-dimethylacetamide (DMA) was incubated with Vc-Duocarmycin (MedChem Express HY-128904) (1 equivalent M - SH: 1.2 equivalent M payload) on ice for 1 h. Add 20 times the excess of cysteine ​​hydrochloride (1 M stock solution) and incubate at room temperature for 30 minutes to terminate the reaction, followed by buffer exchange with phosphate-buffered saline (PBS). This prepares the antibody-drug conjugate (ADC). Purification can then be performed, for example, by hydrophobic chromatography using a butyl agarose column. The resulting ADC can then be buffer-exchanged in PBS, filtered sterilely, aliquoted into individual tubes, and stored frozen at -80°C until use.

[0256] Among the cytotoxic payloads evaluated, duocarmycin in combination with anti-PTGFRN antibodies effectively inhibited the proliferation and survival of PTGFRN-expressing cells. However, other payloads were also evaluated and found to be effective. Therefore, antibody-duocarmycin drug conjugates (ADCs) were prepared using 4F8, 6B2, and 8C7 anti-PTGFRN antibodies. The effects of these ADCs on three cell lines were evaluated in vitro. The cell lines evaluated included the human epidermal-like cancer cell line A431, the breast cancer cell line MDA-MB-468 2E12 (moderately expressing PTGFRN), and the MDA-MB-468 4C5 cell line (overexpressing PTGFRN via cDNA transfection). The doses of 8C7-ADC or 4F8-ADC were increased from 0.1 nM to 10 nM (8C7-ADC at 0.1 nM, 1 nM, and 10 nM) and 20 nM (4F8-ADC at 0.1 nM, 1 nM, 10 nM, and 20 nM), and their effects on the viability of three cell lines were determined. Data are as follows: Figure 17 As shown in the figure, 8C7-ADC at concentrations ≥ 1 nM inhibited the cell survival of A431 (blue bars) and MDA-4C5 (gray bars), which highly express PTGFRN, while the effect on the low PTGFRN-expressing MDA-2E12 cell line was relatively small at a concentration of 10 nM. 4F8-ADC at concentrations ≥ 1 nM strongly inhibited the survival of MDA-4C5 cells, had a smaller effect on A431 cells, but at a concentration of 20 nM, the survival rate of A431 cells was inhibited by 40%, while this effect was not observed in the low PTGFRN-expressing MDA-2E12 cell line. As described above, a 6B2duocarmycin conjugate was also prepared and tested in vitro on A431 cells. Figure 18 The effects of 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM and 20 nM 6B2-ADC on A431 cell proliferation were shown. Figure 17-18 Data show that ADCs have different efficacies in cells with different PTGFRN expression levels, and that they have different uses, which can be used to develop anti-PTGFRN DC drugs.

[0257] Figure 19 illustrates another study on 8C7-ADC. In these studies, the ability of 8C7-ADC to inhibit the proliferation of PTGFRN-expressing cell lines (e.g., epidermoid carcinoma (A431), biphasic mesothelioma (MSTO-211H), and pediatric medulloblastoma (DAOY)) was evaluated in vitro. In addition to these PTGFRN-positive cancers, we also tested the ADC with the previously identified PTGFRN-negative triple-negative breast cancer cell line MDA-MB-231 (22). As shown in Figure 5, 8C7-ADC treatment showed dose-dependent inhibition of proliferation in all PTGFRN-expressing cell lines. Specifically, at the highest tested dose of 10 nM, A431 proliferation was reduced by 95%, DAOY proliferation by 80%, and MSTO-211H proliferation by approximately 40%. This degree of inhibition appears to be related to the PTGFRN levels in these three cell lines. The expression level was correlated. Compared with the A431 and DAOY cell lines with high PTGFRN expression, the proliferation inhibition of MSTO-211H cells with low PTGFRN expression was reduced, but still significant. The PTGFRN-negative cancer cell line MDA-MB-231 did not show growth inhibition when treated with 8C7-ADC, even at the highest evaluable concentration, indicating that 8C7-ADC is specific for PTGFRN.

[0258] The efficacy of the prepared ADC was also tested in vivo using a mouse xenograft model. Three cell lines were used to evaluate the effect of the fully human anti-PTGFRN ADC on tumor growth: 1) A431 human epidermoid carcinoma; 2) DAOY human medulloblastoma; and 3) MSTO-211H human dermatoma. Depending on the cell line, cells (0.5 x 10⁻⁶) were used to evaluate the effect of the ADC on tumor growth. 6 Up to 2x10 6 (One cell per mouse) was subcutaneously injected into female athymic nude mice. When the tumor became prominent and reached 50-100 mm³, the mice were randomly assigned to experimental groups (n=8 per group). The antibody-drug conjugate was injected intraperitoneally weekly, while the control group received an isotope-conjugated human antibody directly conjugated to duocarmycin using the method described above. Tumor size was measured weekly using calipers, and tumor volume was calculated accordingly. At the end of the experiment, the mice were euthanized, the tumors were collected, and body weight, tumor weight, and organ weight were measured. Figure 20A -F displays the relevant data for the 8C7 ADC. Figure 20A -B shows data using A431 squamous cell carcinoma cells. Figure 20C-D shows data using DAOY medulloblastoma cells. Figures 20E-F show data using MSTO-211H mesothelioma cells. Left panels A, C, and E show the effect of 8C7-Duocarmycin ADC on tumor volume compared to the hIgG-duocarmycin ADC control group. Right panels B, D, and F show the body weight and organ weight of mice in each experimental group. The data show that intraperitoneal injection (ip) of the fully human anti-PTGFRN 8C7-duocarmycin antibody-drug conjugate (ADC) dose-dependently inhibited tumor growth in the three tested cell lines. At concentrations of 1 mg / kg (DAOY) and 4 mg / kg (A431), 8C7ADC resulted in a tumor volume inhibition rate >90%. At a dose of 4 mg / kg, the tumor growth inhibition rate of MSTO-211H cells was 70%. Tests on body weight, organ weight, and tumor weight showed that treatment with 8C7-ADC only affected tumor weight, without affecting body weight or the weight of the liver, heart, lungs, and kidneys, indicating that anti-PTGFRN ADC has no off-target toxicity.

[0259] The binding affinity of two fully human antibodies (4F8 and 8C7) to PTGFRN was determined using the Octet method. Data are shown in Table 26.

[0260] Table 26 Affinity assay of selected antibodies with respect to Kon, Koff, chi2, and R2 values Antibody Concentration (nM) response Kd (M) Ka (1 / Ms) kdis (1 / 2) <![CDATA[Full X 2 ]]> <![CDATA[Full R 2 ]]> 4F8 100 0.7627 5.24E-09 4.73E+04 2.48E-04 0.0165 0.9986 8C7 100 0.7611 <1.0E-12 3.51E+04 <1.0E-07 0.0035 0.9998

[0261] Studies have found that 4F8 and 8C7 antibodies exhibit excellent Kd measurements, ranging from up to 10. -12 M to 5.2 x 10 - 9 M is suitable for drug development.

[0262] In preclinical studies, assessing the impact of antibody therapy on patient-derived xenografts (PDXs) is considered crucial and valuable because PDX tumors are freshly extracted from patients, eliminating the need for cell line establishment and thus closely mimicking in vivo tumors. Since PDX tumors do not undergo cell line establishment, they avoid the potential loss of phenotypic diversity in the primary tumor. First, Western blot analysis was used to measure PTGFRN expression in different head and neck patient-derived tumor samples (PDX1, PDX2, PDX3, PDX4, PDX5, PDX6, and PDX7) to identify samples expressing PTGFRN (using A431 cell lysate as a positive control). Figure 21As shown, six of the seven PDX samples evaluated (PDX1, PDX2, PDX3, PDX4, PDX5, and PDX7) expressed PTGFRN. After normalization using GAPDH expression as an internal control, the tumor sample PDX1 showed the highest expression level. Based on this data and the growth rate of these tumors, PDX1 was selected as a candidate PDX tumor model to evaluate the role of 8C7-ADC in vivo.

[0263] Figure 22 and Figure 23 This study demonstrates the effect of 8C7-ADC on the growth of patient-derived head and neck tumors in NRG mice (xenograft) compared to isotype control ADC. Head and neck tumors JZ0628 were implanted into female NRG mice. When the tumor volume reached 100 mm³, the mice were randomly assigned to two experimental groups. The first group received weekly intraperitoneal injections of the isotype control duocarmycin ADC for 44 days, while the second group received 8C7-duocarmycin ADC. Figure 22 (Left figure) shows that tumors in the control group grew steadily, while tumors in the 8C7 treatment group grew slowly (p<0.05). Compared with the isotype control group, the tumor doubling time in the 8C7-ADC treatment group mice was slowed by 73% ( Figure 22 (See right figure) (p<0.0001). Figure 23 This study demonstrated the long-term effects of 8C7-ADC on the growth of patient-derived head and neck tumors after treatment cessation. On day 44, 8C7 treatment was discontinued in the 8C7-ADC group, and mice were observed for another 15 days to determine tumor growth. Figure 23 The results showed that even after treatment was discontinued, tumors in the 8C7-ADC group did not begin to grow (comparison of square and circle symbols), indicating that 8C7-ADC has durable efficacy. Even mesothelioma cell lines with low PTGFRN expression showed dose-dependent tumor growth inhibition after 8C7-ADC treatment, while the PTGFRN-negative cell line MDA-MB-231 did not respond to 8C7-ADC. No difference in tumor growth was observed between the IgG-negative control ADC group and the 8C7-ADC group. Figure 24The results indicate that PTGFRN is an excellent therapeutic target, highly expressed in various human cancers, but expressed at very low levels or not at all in normal tissues. PTGFRN is associated with characteristics of several aggressive cancers, such as enhanced cell migration, clonal formation, proliferation under low serum conditions, and the formation of spheroids under three-dimensional culture conditions. 8C7 has been shown to inhibit the migration of A431 cells. In summary, these results suggest that PTGFRN may be a valuable target for novel antibody-drug conjugates (ADCs), as PTGFRN-negative cells are unresponsive to 8C7-ADCs, while tumors with low PTGFRN expression (e.g., MSTO-211H tumors) still show statistically significant responses to 8C7-ADCs. Tumors with high PTGFRN expression (e.g., A431 and DAOY tumors) show significant tumor growth inhibition against 8C7-ADCs, demonstrating their therapeutic efficacy. This data indicates that the fully human anti-PTGFRN internalization antibodies (e.g., 8C7) disclosed in this paper demonstrate that these antibodies (e.g., preferably 8C7-ADC) inhibit tumor formation in a dose-dependent manner, and most importantly, inhibit the formation of patient-derived tumors in the head and neck region.

[0264] Examples 1-3 of this paper demonstrate that PTGFRN is overexpressed in cancer, while it is expressed negatively or at low levels in healthy tissues. PTGFRN silencing in epidermoid carcinoma A431 cells leads to suppression of hallmark features of cancer invasiveness, such as migration, low-serum proliferation, colony formation, and 3D globule formation. This disclosure provides fully human antibodies prepared by immunizing humanized transgenic mice with human PTGFRN. Several functional anti-PTGFRN antibodies have been developed and screened by combining the use of TC mice with proprietary immunization methods. These antibodies have a Kd of 10. -12 M to 10 -9 Between M and M. This article shows that the screened antibody 8C7 can be rapidly internalized and forms an antibody-drug conjugate (ADC) through cysteine ​​conjugation with Val-Cit-Duocarmycin. The 8C7-ADC inhibits the in vitro proliferation of A431 cells in a dose-dependent manner. The 8C7-ADC inhibits A431 tumor formation and exhibits cytoseptic inhibitory effects on the growth of head and neck PDX tumors. These data suggest that PTGFRN is a therapeutic target for a variety of cancers, including head and neck cancers, and that the fully human antibody approach disclosed in this article is highly effective for screening antibodies with suitable properties for therapeutic development.

[0265] Example 4. Polynucleotide and amino acid sequences of candidate antibodies A. Sample Preparation Hybridoma cell lines for each antibody were thawed and cultured. After cell counting, reverse transcription was performed to convert RNA into cDNA. Multiple PCR reactions were conducted using multiple pairs of mouse heavy and light chain-specific primers to amplify the variable region sequences of the heavy and light chains for each antibody. The PCR products were loaded onto agarose gels and electrophoresed to confirm the generation of correct PCR bands. The PCR products were then mixed and sequenced individually using a Miseq sequencer. NGS sequencing data for each PCR product were processed using BCR analysis software.

[0266] As described below, the nucleotide and amino acid sequences of four fully human antibodies generated from hybridoma clones 4F8, 6B2, 8C7, and 12D8 were determined. DNA sequence data for all constructs were analyzed, and common sequences for the heavy and light chains were determined. These common sequences were compared with known variable region sequences to rule out human error and / or process contamination. The common sequences were then analyzed to verify whether they encode effective immunoglobulins. The amino acid sequences of the complementarity-determining regions (CDRs) of 4F8, 6B2, 8C7, and 12D8 were determined according to the Kabat and Chothia method, as shown below (each is indicated in bold and underline). CDRs were also determined using an alternative method, as shown in Figures 6-13 and Tables 4-7. The 4F8 antibody was determined to be an IgG4 Kappa antibody. The 6B2, 8C7, and 12D8 antibodies were determined to be IgG1 Kappa antibodies.

[0267] A1. Antibody 4F8 (AGRD014-4F8-IgG Variable Heavy Chain (V) H )) 4F8 V H Amino acid sequences (Kabat (K) CDRs are underlined) CDR1-H1(K) CDR-H2(K) EVQLVESGEGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS GISWDSGRIGY CDR-H2(K) CDR-H3(K) ADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DRGGGNWNYYYYGMDV WGQGTT VTVSS (SEQ ID NO: 189) 4F8 V H Preferred DNA sequence (Kabat CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGAAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTGAT GATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCA GGTATTAGTTGGGATAGTGGTCGCATAGGCTAT GCGGACTCTGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GATCGG GGCGGTGGTAACTGGAACTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACG GTCACCGTCTCCTCAG (SEQ ID NO: 197) 4F8 V H CDR1-H1(K) coding sequence: GATTATGCCATGCAC (SEQ ID NO: 209) 4F8 V H CDR-H2(K) coding sequence: GGTATTAGTTGGGATAGTGGTCGCATAGGCTATGCGGACTCTGTGAAGGGC (SEQ ID NO: 210) 4F8 V H CDR-H3(K) coding sequence: GATCGGGGCGGTGGTAACTGGAACTACTACTACTACGGTATGGACGTC (SEQ ID NO: 211) 4F8 V H Amino acid sequences (Chothia (Ch) CDRs are underlined) CDR-H1 (Ch) CDR-H2(Ch) EVQLVESGEGLVQPGRSLRLSCAAS GFTFDDY AMHWVRQAPGKGLEWVSGI SWDSGR IGY CDR-H3(Ch) ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DRGGGNWNYYYYGMDV WGQGTT VTVSS (SEQ ID NO: 189) 1. 4F8 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGAAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCT GGATTCACCTTTGATGATTAT GCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATT AGTTGGGATAGTGGTCGC ATAGGCTAT GCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GATCGG GGCGGTGGTAACTGGAACTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACG GTCACCGTCTCCTCAG (SEQ ID NO: 198) 4F8 V H CDR-H1 (Ch) coding sequence: GGATTCACCTTTGATGATTAT (SEQ ID NO: 212) 4F8 V H CDR-H2 (Ch) coding sequence: AGTTGGGATAGTGGTCGC (SEQ ID NO: 213) 4F8 V H CDR-H2 (Ch) coding sequence: GATCGGGGCGGTGGTAACTGGAACTACTACTACTACGGTATGGACGTC (SEQ ID NO: 214) B1. Antibody 4F8 (AGRD014-4F8-IgK variable light chain (V) L )) 4F8 V L Amino acid sequences (K / Ch CDRs are underlined) CDR-L1 (K / Ch) CDR-L2(K / Ch) DIQMTQSPSSLSASVGDRVTITC RASQGISNYLA WFQQKPGKAPKSLIY AASSLQS GVPS CDR-L3(K / Ch) KFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSFPFT FGPGTKVDIK (SEQ ID NO: 190) 4F8 V L DNA sequence (K / Ch) gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcacc atcacttgt cgggcgagtcagggcattagcaattatttagcc tggtttcagcagaaacca gggaaagcccctaagtccctgatctat gctgcatccagtttgcaaagt ggggtcccatca aagttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct gaagattttgcaacttattactgc caacagtatatagtttccccttcact ttcggccct gggaccaaagtggatatcaaac (SEQ ID NO: 199) 4F8 V L CDR-L1 (K / Ch) coding sequence: CGGGCGAGTCAGGGCATTAGCAATTATTTAGCC (SEQID NO: 215) 4F8 V L CDR-L2 (K / Ch) coding sequence: GCTGCATCCAGTTTGCAAAGT (SEQ ID NO: 216) 4F8 V L CDR-L3 (K / Ch) coding sequence: CAACAGTATAATAGTTTCCCCTTCACT (SEQ ID NO:217) A2. Antibody 6B2 (AGRD014-6B2-IgG variable heavy chain (V H ) ) 6B2 V H Amino acid sequences (Kabat (K) CDRs are underlined) CDR-H1(K) CDR-H2(K) QLQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWD WIRQPPGKGLEWIG TIYYGGSTY CDR-H2(K) CDR-H3(K) YNPSLKS RVTISVDTSKNQFSLKLNSVTAADTAVYYCAR GNSYGLDY WGQGTLVTVSS (SEQ IDNO: 191) 6B2 V H Preferred DNA sequence (Kabat CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCATCAGC TAKETTACTACTGGGAC TGGATCCGC CAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGG ACTATCTATTATGGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGT CGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTC TCTCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA GGG AACAGCTATGGCCTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:200) 6B2 V H CDR-H1 (K) coding sequence: AGTAGTAGTTACTACTGGGAC (SEQ ID NO: 218) 6B2 V H CDR-H2 (K) coding sequence: ACTATCTATTATGGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGT (SEQ ID NO: 219) 6B2 V H CDR-H3 (K) coding sequence: GGGAACAGCTATGGCCTTGACTAC (SEQ ID NO: 220) 6B2 V H Amino acid sequences (Chothia (Ch) CDRs are underlined) CDR1-H1(Ch) CDR2-H2(Ch) QLQLQESGPGLVKPSETLSLTCTVS GGSISSSSY YWDWIRQPPGKGLEWIGTI YYGGS TY CDR3-H3(Ch) YNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCAR GNSYGLDY WGQGTLVTVSS (SEQ IDNO: 191) 6B2 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCT GGTGGCTCCATCAGCAGTAGTTAC TACTGGGACTGGATCCGC CAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGACTATC FATHERGGTGGGAGC ACCTAC TACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTC TCTCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA GGG AACAGCTATGGCCTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:201) 6B2 V H CDR-H1 (Ch) coding sequence: GGTGGCTCCATCAGCAGTAGTAGTTAC (SEQ ID NO:221) 6B2 V H CDR-H2 (Ch) coding sequence: TATTATGGTGGGAGC (SEQ ID NO: 222) 6B2 V H CDR-H3 (Ch) coding sequence: GGGAACAGCTATGGCCTTGACTAC (SEQ ID NO: 223) B2. Antibody 6B2 (AGRD014-6B2-IgK variable light chain (V L )) 6B2 V L Amino acid sequences (Kabat (K) / Chotia (Ch) CDRs are underlined) CDR-L1(K / Ch) CDR-L2(K / Ch) DVVMTQSPLSLPVTLGQPASISC RSSQSLVHSDGNMYLN WFQQRPGQSPRRLIY KVSNRDS CDR-L3(K / Ch) GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC MQSTHWPPVT FGQGTKLEIK (SEQ ID NO: 192) 6B2 V L Preferred DNA sequence (Kabat CDR coding sequence is underlined) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGC AGGTCAAGTCAAAGCCTCGTACACAGTGATGGAAACATGTACTTGAAT TGG TTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGTCTAATTTAT AAGGTTTCTAACCGGGAC TCT GGGGTCCCAGACAGATTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGGATTTATTACTGC ATGCAAAGTACACACTGGCCT CCCGTCACT TTTGGCCAGGGGACCAAGCTGGAGATCAAAC (SEQ ID NO: 202) 6B2 V L CDR-H1 (K / Ch) coding sequence: AGGTCAAGTCAAAGCCTCGTACACAGTGATGGAAACATGTACTTGAAT (SEQ ID NO: 224) 6B2 V L CDR-H2 (K / Ch) coding sequence: AAGGTTTCTAACCGGGACTCT (SEQ ID NO: 225) 6B2 V L CDR-H3 (K / Ch) coding sequence: ATGCAAAGTACACACTGGCCTCCCGTCACT (SEQ IDNO: 226) A3. Antibody 8C7 (AGRD014-8C7-IgG variable heavy chain (V H )) 8C7 V H Amino acid sequences (Kabat CDRs are underlined) CDR-H1(K) CDR-H2(K) QLQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWG WIRQPPGKGLEWIG SIYYGGSTY CDR-H2(K) CDR-H3(K) YNPSLKS RVTISVDTSTNQFSLKLNSVTAADTAVYYCAR QGLGSFDC WGQGTLVTVSS (SEQ IDNO: 193) 8C7 V H Preferred DNA sequence (Kabat CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCATCAGC TATTACTACTGGGGC TGGATCCGC CAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATCTATTATGGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGT CGAGTCACCATATCCGTAGACACGTCCACGAACCAGTTC TCCCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA CAA GGGCTGGGGTCCTTTGACTGC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:203) 8C7 V H CDR-H1 (K) Encoding: AGTAGTTACTACTGGGGC (SEQ ID NO: 227) 8C7 V H CDR-H2 (K) motif: AGTATCTATTATGGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGT (SEQ ID NO: 228) 8C7 V H CDR-H3 (K) sequence: CAAGGGCTGGGGTCCTTTGACTGC (SEQ ID NO:229) 8C7 V H Amino acid sequences (Chothia (Ch) CDRs are underlined) CDRH1(E) CDRH2(E) QLQLQESGPGLVKPSETLSLTCTVS GGSISSSSY YWGWIRQPPGKGLEWIGSI YYGGS TY CDRH3(Ch) YNPSLKSRVTISVDTSTNQFSLKLNSVTAADTAVYYCAR QGLGSFDC WGQGTLVTVSS (SEQ IDNO: 193) 8C7 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCT GGTGGCTCCATCAGCAGTAGTTAC TACTGGGGCTGGATCCGC CAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGAGTATC FATHERGGTGGGAGC ACCTAC TACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCACGAACCAGTTC TCCCTGAAGCTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGA CAA GGGCTGGGGTCCTTTGACTGC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:204) 8C7 V H CDR-H1 (Ch) coding sequence: GGTGGCTCCATCAGCAGTAGTAGTTAC (SEQ ID NO:230) 8C7 V H CDR-H2 (Ch) coding sequence: TATTATGGTGGGAGC (SEQ ID NO: 231) 8C7 V H CDR-H3 (Ch) coding sequence: CAAGGGCTGGGGTCCTTTGACTGC (SEQ ID NO: 232) B3. Antibody 8C7 (AGRD014-8C7-IgK variable light chain (V L )) 8C7 V L Amino acid sequences (Kabat (K) and Chothia (Ch) CDRs are underlined) CDR-L1(K / Ch) CDR-L2(K / Ch) DVVMTQSPLSLPVTLGQPASISC RSSQSLVHSDGNTYLN WFQQRPGQSPRRLIY KVSNRDS CDR-L3(K / Ch) GVPDRFSGSGSGTDFTLKISGVEAEDVGIYYC MQGTHWPPLT FGGGTKVEIK (SEQ ID NO: 194) 8C7 V L Preferred DNA sequence (Kabat(K) / Chothia(Ch) CDR coding sequence is underlined) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGC AGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAACACCTACTTGAAT TGG TTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTAT AAGGTTTCTAACCGGGAC TCT GGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCGGGGTCGAGGCTGAGGATGTTGGAATTTATTACTGC ATGCAAGGTACACACTGGCCT CCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAAC (SEQ ID NO: 205) 8C7 V L CDR-L1 (K / Ch) coding sequence: AGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAACACCTACTTGAAT (SEQ ID NO: 233) 8C7 V L CDR-L2 (K / Ch) coding sequence: AAGGTTTCTAACCGGGACTCT (SEQ ID NO: 234) 8C7 V L CDR-L3 (K / Ch) coding sequence: ATGCAAGGTACACACTGGCCTCCGCTCACT (SEQ IDNO: 235) A4. Antibody 12D8 (12D8-IgG variable heavy chain (V) H )) 12D8 V H Amino acid sequence (Kabat (K) CDR amino acid sequence is underlined) CDR-H1(K) CDR-H2(K) EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS GISWSSGSLGYEDSVKG RFTIS CDR-H3(K) RDNAKKTLYLQMNSLRAEDTALYYCAK DMGFGDFLYYYGMDVWGQGTTVTVSS (SEQ ID NO:195) 12D8 V H Preferred DNA sequence (Kabat CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGAT GATTATGCCATGCAC TGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCA GGTATTAGCTGGAGTAGTGGTAGCTTAGGCTATGAGGACTCTGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAAAACCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GA TATGGGGTTCGGGGACTTCCTCTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 206) 12D8 V H CDR-H1 (K) coding sequence: GATTATGCCATGCAC (SEQ ID NO: 236) 12D8 V H CDR-H2 (K) coding sequence: GGTATTAGCTGGAGTAGTGGTAGCTTAGGCTATGAGGACTCTGTGAAGGGC (SEQ ID NO: 237) 12D8 V H CDR-H3 (K) coding sequence: GATATGGGGTTCGGGGACTTCCTCTACTACTACGGTATGGACGTC (SEQ ID NO: 238) 12D8 V H Amino acid sequence (Chothia (Ch) CDR amino acid sequence is underlined) CDR-H1(Ch) CDR-H2(Ch) EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDY AMHWVRQAPGKGLEWVSGI SWSSGS LGYEDSVK CDR-H3(Ch) GRFTISRDNAKKTLYLQMNSLRAEDTALYYCAK DMGFGDFLYYYGMDV WGQGTTVTVSS (SEQ IDNO. 195) 12D8 V H Preferred DNA sequence (Chothia (Ch) CDR coding sequence is underlined) GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTGATGATTAT GCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATT AGCTGGAGTAGTGGTAGC TTAGGCTATGAGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAAACCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAA GA TATGGGGTTCGGGGACTTCCTCTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 207) 12D8 V H CDR-H1 (Ch) coding sequence: GGATTCACCTTTGATGATTAT (SEQ ID NO: 239) 12D8 V H CDR-H2 (Ch) coding sequence: AGCTGGAGTAGTGGTAGC (SEQ ID NO: 240) 12D8 V H CDR-H3 (Ch) coding sequence: GATATGGGGTTCGGGGACTTCCTCTACTACTACGGTATGGACGTC (SEQ ID NO: 241) B4. Antibody 12D8 (AGRD014-12D8-IgK variable light chain (V) L )) 12D8 V L Amino acid sequences (Kabat (K) and Chothia (Ch) CDRs are underlined) CDR-L1(K / Ch) CDR-L2(K / Ch) EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLT WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTD CDR-L3(K / Ch) FTLTIRRLEPEDFAVYYC QQYGDSPPWT FGQGTKVEIK (SEQ ID NO: 196) 12D8 V L DNA sequences (Kabat and Chothia CDR sequences are underlined) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGC AGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAACC TGGTACCAGCAGAAA CCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCCAGCAGGGCCACT GGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGGAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGT CAGCAGTATGGTGACTCACCTCCGTGGACG TTC GGCCAAGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 208) 12D8 V L CDR-L1 (K / Ch) encoded sequence: AGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAACC (SEQ ID NO: 242) 12D8 V L CDR-L2 (K / Ch) encoded sequence: GGTGCATCCAGCAGGGCCACT (SEQ ID NO: 243) 12D8 V L CDR-L3 (K / Ch) encoding sequence: CAGCAGTATGGTGACTCACCTCCGTGGACG (SEQ ID NO: 244) The top image shows the sequence alignment results of the heavy and light chain variable regions of the 4F8, 6B2, 8C7, and 12D8 antibodies, with the CDR regions highlighted. The bottom image shows the sequence alignment results of the light and heavy chains of the 4F8, 6B2, 8C7, and 12D8 antibodies, with the CDR regions highlighted. The amino acid sequences of CDR1, CDR2, and CDR3 are labeled and underlined. The variable weights (V) of the 4F8, 6B2, 8C7, and 12D8 antibodies are compared below. H The chain, in which the CDR amino acid sequence determined by the Kabat method is underlined: The following compares the variable lightness (V) of antibodies 4F8, 6B2, 8C7, and 12D8. L The chain, in which the CDR amino acid sequence determined by the Kabat method is underlined: Figures 25 - 32 Table 1 provides alternative (Alt) CDRs for the 4F8, 6B2, 8C7, and 12D8 antibodies. In preferred embodiments, this disclosure considers any CDR shown herein, or that derived from the V of the corresponding antibody by any other method known in the art. H and V L Any CDR determined by amino acid sequence analysis.

[0268] This article also provides other advantages of the reagent and its use, which will be understood by those skilled in the art.

[0269] While certain embodiments have been described based on preferred embodiments, those skilled in the art will understand that various variations and modifications can be made. Therefore, the appended claims are intended to cover all equivalent variations falling within the scope of the following claims.

Claims

1. An isolated antibody or its antigen-binding fragment, comprising: a) Specifically, the heavy chain variable regions containing complementarity-determining region (CDR) sequences SEQ ID NO: 1, 2 and 3, and the light chain variable regions containing CDR sequences SEQ ID NO: 7, 8 and 9; b) Specifically, the heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NO: 4, 5 and 6, and the light chain variable regions containing the CDR sequences SEQ ID NO: 7, 8 and 9; c) Specifically, the heavy chain variable regions containing complementarity-determining region (CDR) sequences SEQ ID NO: 4, 5 and 6, and the light chain variable regions containing CDR sequences SEQ ID NO: 7, 8 and 9; d) The heavy chain variable region of SEQ ID NO: 189 and the light chain variable region of SEQ ID NO: 190; e) Specifically, the heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NO: 18, 19 and 20, and the light chain variable regions containing the CDR sequences SEQ ID NO: 24, 25 and 26; f) Specifically, heavy chain variable regions containing complementarity-determining region (CDR) sequences SEQ ID NO: 21, 22 and 23, and light chain variable regions containing CDR sequences SEQ ID NO: 24, 25 and 26; g) Each of the heavy chain variable regions containing complementarity-determining region (CDR) sequences SEQ ID NO: 27, 28 and 29 or 30, and the light chain variable regions containing CDR sequences SEQ ID NO: 31 or 32, and 33, and 34 or 35; h) The heavy chain variable region of SEQ ID NO: 191 and the light chain variable region of SEQ ID NO: 192; i) respectively, the heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NO: 36, 37 and 38, and the light chain variable regions containing the CDR sequences SEQ ID NO: 42, 43 and 44; j) Specifically, the heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NO: 39, 40 and 41, and the light chain variable regions containing the CDR sequences SEQ ID NO: 42, 43 and 44; k) Specifically, heavy chain variable regions comprising complementarity-determining region (CDR) sequences SEQ ID NO: 45, and 46 or 47, and 48 or 49, and light chain variable regions comprising CDR sequences SEQ ID NO: 50, and 51, and 52 or 53; l) The heavy chain variable region of SEQ ID NO: and the light chain variable regions of SEQ ID NO: 193 and 194; m) respectively, the heavy chain variable region containing the complementarity-determining region (CDR) sequences SEQ ID NO: 54, 55 and 56, and the light chain variable region containing the CDR sequences SEQ ID NO: 60, 61 and 62; n) respectively, the heavy chain variable regions containing the complementarity-determining region (CDR) sequences SEQ ID NO: 57, 58 and 59, and the light chain variable regions containing the CDR sequences SEQ ID NO: 60, 61 and 62; o) respectively, the heavy chain variable region containing the complementarity determination region (CDR) sequences SEQ ID NO: 63, and 64 or 65, and 66, and the light chain variable region containing the CDR sequences SEQ ID NO: 67, 68, and 69 or 70; p) The heavy chain variable region of SEQ ID NO: 195 and the light chain variable region of SEQ ID NO: 196; or derivatives of any one of a) - p), wherein the derivative optionally contains one to four amino acid substitutions in at least one CDR, preferably wherein the substitution(s) are one or more conserved amino acid sequences; The antibody or its derivative specifically binds to human prostaglandin F2 receptor inhibitors (PTGFRN).

2. The antibody of claim 1, wherein the antibody binds to cells expressing PTGFRN in vitro and / or in vivo.

3. An antibody that competes with the antibody of claim 1 for binding to PTGFRN on cells.

4. A combination of antibodies according to any one of the preceding claims.

5. The antibody of any of the preceding claims is an isolated monoclonal antibody.

6. The antibody of claim 5, wherein the monoclonal antibody is a human monoclonal antibody.

7. The antibody of any of the preceding claims, wherein the antibody is derived from human antibodies, human IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, human IgE, canine antibodies, canine IgGA, canine IgGB, canine IgGC, canine IgGD, chicken antibodies, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, cat antibodies, goat antibodies, goat IgG, mouse antibodies, mouse IgG, porcine antibodies, rat antibodies, alpaca antibodies, llama antibodies, shark antibodies, and camel antibodies.

8. A derivative of the antibody of any of the preceding claims, optionally selected from F ab F ab2 Fab' single-chain antibody, F v Single-chain antibodies, monospecific antibodies, bispecific antibodies, trimer antibodies, multispecific antibodies, multivalent antibodies, chimeric antibodies, canine-human chimeric antibodies, canine-mouse chimeric antibodies, antibodies containing canine Fc, humanized antibodies, human antibodies, canine-derived antibodies, CDR transplantation antibodies, shark antibodies, and nanobodies.

9. A derivative of the antibody according to any one of the preceding claims, comprising a detectable label for immobilizing and attaching an antibody thereto, optionally wherein the detectable label is selected from fluorescein, DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein, 5-FAM, serotonin, 5-hydroxytryptamine (5-HAT), 6-carboxyfluorescein (6-FAM), FITC, 6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET), 6-carboxy-1,4-dichloro-2',4',5',7'-tetrachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-Dimethoxyfluorescein (6-JOE), Alexa Fluor, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor700, Alexa Fluor 750, BODIPY fluorescent dye, BODIPY 492 / 515, BODIPY 493 / 503, BODIPY 500 / 510、BODIPY 505 / 515、BODIPY 530 / 550, BODIPY 542 / 563, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650-X, BODIPY 650 / 665-X, BODIPY 665 / 676, FL, FL ATP, FI-ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-XSE, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine B, Rhodamine B 200, Rhodamine BB, Rhodamine BG, Rhodamine Bextra, 5-Carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, Rhesine, Rhesine-rhodamine B, Phallicidine, Phalloidine, Rhodamine Red, Rhodamine-2, 6-carboxy-X-rhodamine (ROX), Carboxy-X-rhodamine (5-ROX), Sulforhodamine B and C, Sulforhodamine G Extra, 6-carboxytetramethylrhodamine (TAMRA), Tetramethylrhodamine (TRITC), Rhodamine WT, Texas Red, and Texas Red-X.

10. The antibody of any of the preceding claims, comprising an effector moiety attached thereto, optionally selected from therapeutic agents, cytotoxic agents, abrinogen A chain, anthracyclines, amanita, α-amanita, oliquistatin, monomethyloliquistatin E, monomethyloliquistatin F, calcitrol, camptothecin, comprbetastain, crotonin, novolucretin, curcin, dolalastatin, duocarmycin, DNA alkylating agents, DNA repair inhibitors, duocarmycin, enediyne, ethanotecan or derivatives thereof, DX-8951, exotoxin A chain, Delutecan, diphtheria A chain, enoxamycin, geldmycin, hemiasterlin, ataxia-telangiectasia and Rad3-related kinase inhibitors, bezosertib, indole-benzodiazepine dimer, maytansine, maytansine DM1, maytansine DM4, ozomicin, phenolmycin, pladienolide, phytotoxins, puromycin, pyrrole-benzodiazepine dimer, ricin A chain, splicing inhibitor, taxane, toxins, tubulolysin, tumor-activating prodrugs, topoisomerase inhibitors, vinca alkaloids, radiochemicals, radioisotopes, iodine-131 and yttrium-90.

11. The antibody of claim 10, wherein the cytotoxic agent is duocarmycin.

12. The antibody of claim 10 or 11, wherein an uncuttable or cuttable linker is located between the antibody and the effector moiety, wherein the cuttable linker releases the effector moiety into or within cells.

13. An isolated polynucleotide encoding an antibody of any of the preceding claims; or a combination comprising at least one polynucleotide having at least about 90% identity with any one of the polynucleotides.

14. An expression vector comprising one or more polynucleotides of claim 13.

15. A host cell comprising the isolated polynucleotide of claim 13 and / or the expression vector of claim 14.

16. A composition comprising at least one antibody or a derivative thereof of any one of claims 1-12; at least one isolated polynucleotide of claim 13; or at least one expression vector of claim 14; and / or at least one host cell of claim 15; or a combination thereof; and a pharmaceutically acceptable vector.

17. A method for detecting PTGFRN on cells and / or tissues, the method comprising contacting a test biological sample with an antibody or a derivative thereof of any one of claims 1-13 and detecting the antibody bound to the biological sample or a component thereof.

18. The method of claim 17, further comprising comparing the amount of binding of a test biological sample or a component thereof with the amount of binding of a control biological sample or a component thereof, wherein an increase in the amount bound to the test biological sample or a component thereof relative to the control biological sample or a component thereof indicates the presence of cells expressing PTGFRN in the test biological sample.

19. The method of claim 17 or 18, wherein the biological sample being tested is a mammalian cell, tissue, or biological fluid, optionally selected from blood, urine, plasma, serum, cerebrospinal fluid, saliva, and exosomes.

20. The method of any one of claims 17-19, wherein the method is an in vivo method or an in vitro method.

21. An in vivo method for detecting tumor cells and / or diagnosing cancer, the method comprising administering at least one antibody, combination or derivative of any one of claims 1-13, and detecting at least one antibody bound to tumor cells.

22. The method of claim 21, wherein at least one antibody or derivative comprises at least one detectable marker.

23. The method of claim 22, comprising at least two antibodies and / or derivatives, each comprising at least one detectable marker, wherein the detectable markers of each antibody and / or derivative are the same or different.

24. The method of any one of claims 21-23, wherein the method includes imaging a tumor for targeted therapy of cancer.

25. The method of claim 22, further comprising treating cancer.

26. The method of claim 25, further comprising isolating cancer cells or tissue and determining whether the cancer tissue overexpresses PTGFRN compared to non-cancer cells, optionally wherein PTGFRN expression is determined by measuring the expression of PTGFRN protein and / or RNA encoding PTGFRN.

27. A method for treating cancer, the method comprising administering to a mammal at least one antibody, combination or derivative of any one of claims 1-13.

28. The method of claim 27, wherein at least one antibody or derivative comprises an effector moiety.

29. The method of claim 22, comprising at least two antibodies or derivatives, each comprising at least one effector moiety, wherein the effector moiety of each antibody or derivative may be the same or different.

30. The method of any one of claims 27-29, further comprising isolating cancer cells and determining whether the cancer tissue overexpresses PTGFRN compared to non-cancer cells, optionally wherein PTGFRN expression is determined by measuring the expression of PTGFRN protein and / or RNA encoding PTGFRN.

31. A method for detecting, diagnosing, and treating cancer, the method comprising imaging a tumor containing an antibody, composition, or derivative of any one of claims 1-13 attached thereto, and targeting the treatment of the cancer to the tumor.

32. The method of claim 27, wherein the antibody or a derivative thereof comprises a detectable label and / or effector moiety.

33. The method of claim 31 or 32, further comprising isolating cancer cells and determining whether the cancer tissue overexpresses PTGFRN compared to non-cancer cells, optionally wherein PTGFRN expression is determined by measuring the expression of PTGFRN protein and / or RNA encoding PTGFRN.

34. A method for treating, preventing, and / or improving cancer in mammals, comprising administering to a mammal at least one effective dose of at least one pharmaceutical composition, said at least one pharmaceutical composition comprising at least one antibody and / or derivative of any one of claims 1-13.

35. The method of claim 34, wherein at least one antibody in at least one pharmaceutical composition comprises a cytotoxic effector moiety attached thereto, optionally wherein the effector moiety is selected from therapeutic agents, cytotoxic agents, abrinogen A chain, anthracyclines, amanita, α-amanita, oliquistatin, monomethyloliquistatin E, monomethyloliquistatin F, calcitrol, camptothecin, comprbetastain, crotonin, novolucrin, curcin, dolalastatin, duocarmycin, DNA alkylating agents, DNA repair inhibitors, duocarmycin, enediyne, ethatec or derivatives thereof, DX-8951, exotoxin A Chain, delutecan, diphtheria A chain, enoxacin, geldmycin, hemiasterlin, ataxia-telangiectasia and Rad3-related kinase inhibitors, bezosertib, indole-benzodiazepine dimer, maytansine, maytansine DM1, maytansine DM4, ozomicin, phenolmycin, pladienolide, phytotoxins, puromycin, pyrrole-benzodiazepine dimer, ricin A chain, splicing inhibitor, taxane, toxins, tubulolysin, tumor-activating prodrugs, topoisomerase inhibitors, vinca alkaloids, radiochemicals, radioisotopes, iodine-131 and yttrium-90.

36. The method of claim 35, wherein the cytotoxic drug is duocarmycin.

37. The method of claim 35 or 36, wherein an uncuttable or cuttable linker is located between the antibody and the effector moiety, wherein the cuttable linker releases the effector moiety into or within cells.

38. The method of any one of claims 34-37, wherein the antibody is administered as an antibody-drug conjugate.

39. The method of any one of claims 34-38, comprising administering to a mammal at least two antibodies, wherein at least one antibody is a naked antibody excluding a cytotoxic effect portion thereon, and at least one antibody includes a cytotoxic effect portion thereon.

40. The method of any one of claims 34-38, wherein multiple doses are administered to the animal; and / or, the antibody is administered at a dose of about 1 to 50 mg / kg.

41. The method of any one of claims 34-39, further comprising isolating cancer cells and determining whether the cancer tissue overexpresses PTGFRN compared to non-cancer cells, optionally wherein PTGFRN expression is determined by measuring the expression of PTGFRN protein and / or RNA encoding PTGFRN.

42. The method of any one of claims 34-41, wherein the mammal is a human.

43. The method of any one of claims 34-42, wherein the cancer is selected from head and / or neck cancer, squamous cell carcinoma, epidermoid carcinoma, medulloblastoma, mesothelioma, and hematopoietic system cancers.

44. A kit for detecting the expression of PTGFRN in or on cells, tissues or biological fluids, said kit comprising an antibody or a derivative thereof of any one of claims 1-13 and instructions for use.

45. The kit of claim 41, wherein the antibody or derivative is in lyophilized form.

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