Recombinant OPCML fusion proteins optimizing homodimerization of the OPCML D1 domain and methods of use for cancer treatment

Abstract

Presented herein are recombinant OPCML-Fc fusion protein molecules, in which the Fc portion of the fusion protein molecule is only a CH2CH3 portion and lacks a hinge region containing disulfide bridges that prevent dimerization of the Fc portion and optimize dimerization of the D1 domain of OPCML, and methods for their use in cancer treatment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 419,645, filed October 26, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0002] Incorporation of sequence listings This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on October 26, 2023, is named "7071-0104PWO1.xml" and is 20,745 bytes in size. The Sequence Listing contained in this .XML file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to the field of medicine, and more particularly to the field of cancer, and in particular to the role of OPCML in cancer prognosis, theragnosis, treatment, and research. [Background technology]

[0004] Cancer is a leading cause of death worldwide, resulting in 7.9 million deaths in 2007 (approximately 13% of all deaths), with lung, stomach, liver, colon, and breast cancers causing the most deaths each year (World Health Organization; WHO). According to the WHO, cancer deaths are predicted to continue to increase worldwide, with an estimated 12 million deaths in 2030. Therefore, there is a great need for additional and improved methods of treating cancer.

[0005] Opioid-binding protein / cell adhesion molecule-like (OPCML) is a GPI-anchored tumor suppressor protein previously validated as an epigenetic biomarker for ovarian cancer. The OPCML gene shows frequent loss of heterozygosity in human tumors, is inactivated through extensive CpG island methylation in its promoter, and behaves as a tumor suppressor gene in in vivo models (WO 03 / 002,765 and Sellar et al. (2003) Nature Genetics 34(3):337-343). OPCML has also been found to be epigenetically inactivated and downregulated in a broader range of cancers (Reed et al. (2007) Neuropathology and Applied Neurobiology 33(1):77-85; Cui et al. (2008) PLoS ONE 3(8):e2990).

[0006] OPCML acts in the external leaflet of the plasma membrane in lipid rafts, interacting with and inhibiting a specific network of nine receptor tyrosine kinases (RTKs; Antony et al (2021) Cancer Gene Therapy 28(1-2):18-26). Recombinant forms of OPCML have been shown to be effective treatments for HER-positive cancers, including, but not limited to, HER2-positive ovarian cancer, breast cancer, renal cancer, gastrointestinal cancer, brain cancer, lung cancer, nasopharyngeal cancer, endometrial cancer, endometrial serous carcinoma, esophageal cancer, gastric cancer, colon cancer, liver cancer, cervical cancer, prostate cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, and nasal NK-T cell lymphoma, as well as HER-negative cancers (Buza (2021) Arch Pathol Lab Med 145(6):687-691; WO 2011 / 128,701).

[0007] Further improvements to the OPCML molecule will likely be needed to allow for more efficient and effective administration with less pain or potential side effects for patients. Therefore, there is still a need to modify the structure of the OPCML molecule so that it can reach its potential as an independent and effective cancer therapy. Summary of the Invention

[0008] Herein, we describe the design of two OPCML tumor-suppressing biotherapeutic molecules: one in the form of an anchorless OPCML protein (r-OPCML PYTX-001) and one in the form of a fusion protein (rOPCML-Fc PYTX-004) linking recombinant OPCML to a human IgG Fc peptide containing only the CH2CH3 region. The functional effects and mechanisms of action of these two recombinant proteins were evaluated in vitro, and rOPCML-Fc PYTX-004 was evaluated intravenously in vivo, demonstrating its high efficacy when used as a monotherapy treatment. Notably, intravenously administered rOPCML-Fc PYTX-004 therapeutic agent possessed good targeting affinity to a human ovarian cancer patient-derived xenograft (PDX) model. This molecule demonstrated substantial dose-dependent efficacy in vitro and in vivo without compromising safety, making it an attractive biotherapeutic agent for progression to preclinical drug development. [Brief explanation of the drawings]

[0009] [Figure 1A] Schematic diagram of wild-type GPI-anchored OPCML, showing the three IgG-like C2 domains (D1, D2, and D3) and the GPI anchor that attaches OPCML to the cell surface. [Figure 1B] 1 is a schematic diagram of wild-type GPI-anchored OPCML. The sequence of the wild-type human OPCML molecule (SEQ ID NO: 1) is provided. The sequence in bold font is the sequence of the wild-type signal peptide (SEQ ID NO: 2) and the region surrounding the wild-type GPI anchor site (SEQ ID NO: 8), which is underlined.

[0010] [Figure 2A]1 shows the effect of human serum albumin (HSA) on r-OPCML expression in HEK-293 cells. Shown are three different signal peptides in bold font and the first 10 amino acids of human OPCML (underlined); the sequences of wild-type human OPCML (SEQ ID NO: 3), ceruloplasmin (SEQ ID NO: 4), and HSA (SEQ ID NO: 5). [Figure 2B] Figure 1 shows the effect of human serum albumin (HSA) on r-OPCML expression in HEK-293 cells. Western blot expression data for OPCML protein expression in relation to the signal sequences of wild-type OPCML, ceruloplasmin, and HSA. [Figure 2C] Figure 1 shows the effect of human serum albumin (HSA) on r-OPCML expression in HEK-293 cells. Western blotting densitometry bar graph of OPCML protein expression associated with the signal sequences of wild-type OPCML, ceruloplasmin, and HSA.

[0011] [Figure 3A] 1 provides a schematic diagram of a new generation anchorless recombinant OPCML molecule (PYTX-001). 1 is a schematic diagram of a recombinant OPCML (rOPCML) molecule with three OPCML IgG-like domains (D1, D2, and D3) in which the wild-type GPI anchor is replaced by an alternative histidine-tagged sequence. [Figure 3B] 1 provides a schematic diagram of the new generation anchorless recombinant OPCML molecule (PYTX-001). 1 is a schematic diagram of the current soluble recombinant OPCML (r-OPCML) molecule containing a histidine tag, which can penetrate HEK-293 cells and enter the cytoplasm. [Figure 3C] Schematic diagram of the new generation anchorless recombinant OPCML molecule (PYTX-001). Wild-type OPCML signal sequence.

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[0012] [Figure 4A]1 shows the sequence and structure of recombinant OPCML fused to a human IgG F C fragment (rOPCML-Fc) containing only the CH2CH3 region. Residues 1-18 are the human HSA signal peptide (bold font; SEQ ID NO: 7), the underlined residues 19-28 are the first part of the human OPCML protein, residues 29-109 are human OPCML domain 1, residues 110-118 are the natural human OPCML linker sequence, residues 119-202 are human OPCML domain 2, residues 203-205 are the natural human OPCML linker, residues 206-293 are human OPCML domain 3, residues 294-298 are the natural human OPCML linker, residues 299-301 are the artificial linker, residues 302-309 are the human rhinovirus 3C protease cleavage site (LEVLFQGP; SEQ ID NO: 11) used as a linker between OPCML and Fc, residues 310-315 are the artificial linker, and residues 315-525 are the human CH2CH3 IgG. The sequence of rOPCML-Fc (SEQ ID NO: 10) is an Fc fragment (residues 310-525 shown in italics; SEQ ID NO: 13), in which residues 526-528 are an artificial linker and residues 529-534 are a cleavable polyhistidine tag (bold font). [Figure 4B] 1 shows the sequence and structure of recombinant OPCML fused to a human IgG F-C fragment (rOPCML-Fc) containing only the CH2CH3 region. Structural representation of rOPCML-Fc PYTX-004 showing the three OPCML IgG-like domains homodimerized via domain 1 and the Fc portion of the molecule linked to OPCML domain 3.

[0013] [Figure 5A] 1 shows the expression and purification data for PYTX-004. The results of transfection with pcDNA3.1-rOPCML-Fc (pcDNA3.1-PYTX-004). [Figure 5B] Expression and purification data for PYTX-004 are shown. Subcloning results. [Figure 5C] Expression and purification data for PYTX-004 are shown. Results of serum deprivation. [Figure 5D] Figure 1 shows the expression and purification data for PYTX-004. Results of affinity purification: lane 1 is cell culture medium pre-bound; lane 2 is flow-through; lane 3 is wash; lane 4 is protein pre-stained marker; lanes 5-10 are eluates.

[0014] [Figure 6] These results demonstrate that rOPCML-Fc impairs receptor tyrosine kinase (RTK) phosphorylation and inhibits downstream signaling in ovarian cancer cell lines. Cell culture media lacked 10% fetal calf serum (FCS) and PYTX-001 or PYTX-004 protein (indicated by "-") or contained 10% FCS, PYTX-001, or PYTX-004 protein (indicated by "+"). A: Results for cell line SKOV3. B: Results for cell line OVCAR8. Both the anchorless PYTX-001 molecule and the CH2CH3 human IgG Fc-fused PYTX-004 molecule demonstrated equipotency in terms of on-target pharmacokinetics and downstream proximal pharmacokinetics, as well as comparable cancer function assays.

[0015] [Figure 7A] Figure 1 shows the expression and purification of rOPCML-Fc (PYTX-004). SDS-PAGE results: Lane 1 - standard marker; Lane 2 - rOPCML-Fc protein. [Figure 7B] Expression and purification of rOPCML-Fc (PYTX-004). HPLC results.

[0016] [Figure 8A] Figure 1 shows that rOPCML-Fc PYTX-004 significantly inhibited the proliferation of OVCAR3 and SKOV3 cells in vitro. IC50 measurements for cell lines OVCAR3, SKOV3, and PEO1. [Figure 8B] Figure 1 shows that rOPCML-Fc PYTX-004 significantly inhibited the proliferation of OVCAR3 and SKOV3 cells in vitro. Inhibitory ability of rOPCML-Fc PYTX-004 on cancer cell line proliferation.

[0017] [Figure 9A] 1 shows that rOPCML-Fc PYTX-004 inhibited wound healing in OVCAR3 and SKOV3 cells in vitro. Results for OVCAR3. [Figure 9B] 1 shows that rOPCML-Fc PYTX-004 inhibited wound healing in OVCAR3 and SKOV3 cells in vitro. SKOV3 results. [Figure 9C] 1 shows that rOPCML-Fc PYTX-004 inhibited wound healing in OVCAR3 and SKOV3 cells in vitro. [Figure 9D] 1 shows that rOPCML-Fc PYTX-004 inhibited wound healing in OVCAR3 and SKOV3 cells in vitro.

[0018] [Figure 10A] rOPCML-Fc (PYTX-004 construct) inhibited OVCAR3 and SKOV3 cell invasion in vitro. Photomicrographs of OVCAR3 and SKOV3 cells treated with 0.05 or 0.1 mg / ml OPCML-Fc PYTX-004 versus control (NC). [Figure 10B] 1 shows that rOPCML-Fc (PYTX-004 construct) inhibited the invasion of OVCAR3 and SKOV3 cells in vitro. [Figure 10C] 1 shows that rOPCML-Fc (PYTX-004 construct) inhibited the invasion of OVCAR3 and SKOV3 cells in vitro.

[0019] [Figure 11A]Figure 1 shows that rOPCML-Fc (PYTX-004 construct) inhibited ovarian cancer patient-derived organoids (PDO) in vitro. Photomicrographs of OV016X-PDO, OV044-PDO, and OV041-PDO treated with 0.05 or 0.1 mg / ml rOPCML-Fc versus control (NC). [Figure 11B] Graphical representation of the treatment of OV016X-PDO, OV044-PDO, and OV041-PDO shows that rOPCML-Fc (PYTX-004 construct) inhibited ovarian cancer patient-derived organoids (PDO) in vitro.

[0020] [Figure 12A] rOPCML-Fc PYTX-004 protein downregulates pHER2, pAKT, and RAF / MEK / ERK pathways in ovarian cancer cells. Cell culture media lacked EGF and rOPCML-Fc protein (indicated by "-") or contained EGF or rOPCML protein (indicated by "+"). [Figure 12B] rOPCML-Fc PYTX-004 protein downregulates pHER2, pAKT, and RAF / MEK / ERK pathways in ovarian cancer cells. Cell culture media either contained EGF (indicated by "+") and no (indicated by "-") or some concentration of rOPCML-Fc protein.

[0021] [Figure 13] We present the techniques used to develop robust models of primary human ovarian tumors using patient-derived xenografts (PDX) in NDG mice with severe T cell deficiency, B cell deficiency, and NK cell deficiency.

[0022] [Figure 14] 1 shows photomicrograph results of PDX models demonstrating robust uptake rates.

[0023] [Figure 15A] Figure 1 shows that rOPCML-Fc PYTX-004 is active as a single agent applied intravenously in vivo. Effect of rOPCML-Fc PYTX-004 on the growth of human ovarian cancer PDXs in mice. [Figure 15B] Figure 1 shows that OPCML-Fc PYTX-004 is active as a single agent applied intravenously in vivo. Tumor weight after treatment. [Figure 15C] Figure 1 shows that OPCML-Fc PYTX-004 is active as a single agent applied intravenously in vivo. Tumor volume during treatment. [Figure 15D] Figure 1 shows that OPCML-Fc PYTX-004 is active as a single agent applied intravenously in vivo. Animal body weights during treatment.

[0024] [Figure 16A] OPCML-Fc PYTX-004 targets tumor cells and inhibits their proliferation. Photomicrographs of OPCML-Fc PYTX-004 targeting tumor cells within the tumor-rich PDX model microenvironment, using OPCML as a marker. [Figure 16B] Figure 1 shows that OPCML-Fc PYTX-004 targets tumor cells and inhibits their proliferation. Ki-67 was used as a cancer cell marker to demonstrate the inhibition of cancer cells by OPCML-Fc PYTX-004 treatment. DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Such terms are found defined and used in context within a variety of standard reference works, including, by way of example, J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed., 2012; F.M.Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; DL Nelson and M.M.Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; and Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004.

[0026] Before the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims.

[0027] Preferably, terms used herein are consistent with the definitions set out in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W., Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland. Throughout this specification and the appended claims, unless the context otherwise requires, the word "comprise", as well as variations such as "comprises" and "comprising", will be understood to mean the inclusion of the stated members, integers or steps, or group of members, integers or steps, but not the exclusion of any other members, integers or steps, or group of members, integers or steps.

[0028] As used in this disclosure and the appended claims, "and / or" refers to and includes any and all possible combinations of one or more of the listed items, and the absence thereof when interpreted in the alternative ("or"). As used herein, the term "about" encompasses the explicitly recited amount and a deviation of ±10%. For example, a deviation of 5% is encompassed by the term "about." Throughout the text of this specification, several documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, etc.) are cited. Any document cited herein is characterized as "incorporated by reference." In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching recited herein, the text of this specification shall control.

[0029] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to cover non-exclusive inclusions. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or that are inherent in such composition, mixture, process, method, article, or device. Furthermore, unless expressly stated otherwise, "or" refers to an inclusive "or," not an exclusive "or." For example, condition A or B can be satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0030] definition As used herein, the term "affinity chromatography" refers to a specific mode of chromatography in which a ligand attached to a stationary phase interacts with molecules (e.g., amino acids or portions of immunoglobulins) in a mobile phase (sample). That is, the ligand has a specific binding affinity for the molecule to be purified. As understood in the context of this disclosure, affinity chromatography involves adding a protein-containing sample to a stationary phase containing a chromatographic ligand.

[0031] The terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix," as used interchangeably herein, refer to a matrix, e.g., a chromatography matrix, to which an affinity ligand (e.g., an Fc-binding protein or a metal ion) is attached. The ligand (e.g., an Fc-binding protein or a metal ion) is capable of specifically binding to at least a portion of a molecule of interest (e.g., an immunoglobulin or a portion of an amino acid) to be purified or removed from a mixture.

[0032] As used herein, the term "affinity purification" refers to a method of purifying a molecule of interest from a liquid or mixture by binding it to a suitable ligand (e.g., an Fc-binding protein or a metal ion) immobilized on a matrix. This results in the removal of all other components of the liquid or mixture except the molecule of interest. In a further step, the bound molecule of interest is eluted in purified form.

[0033] As used herein, the term "amino acid sequence identity" refers to a quantitative comparison of the identities (or differences) of the amino acid sequences of two or more peptides or proteins.

[0034] As used herein, the term "anion exchange chromatography" refers to the removal of one or more contaminants from a sample that bind to a positively charged resin. Examples of such contaminants include, but are not limited to, DNA, host cell proteins, endotoxins, and viruses. Bound molecules are eluted with an anion gradient.

[0035] As used herein, the term "artificial" refers to something that does not occur in nature. That is, the term refers to something that has been created or modified by humans. For example, a polypeptide or polynucleotide sequence that has been created or intentionally modified by humans (e.g., in a laboratory, by genetic engineering, by shuffling, or by chemical reaction, etc.) is artificial.

[0036] The term "binding" as used herein refers to the act or process by which one molecule selectively or stoichiometrically binds to another molecule through non-covalent forces. "Specific binding" means that at least a portion of the molecule of interest binds strongly to the ligand for which it is specific compared to its binding to another non-specific target.

[0037] The term "binding activity" refers to the measured interaction between a molecule of interest and a ligand. For example, binding activity can be measured and determined for a molecule of interest when the ligand is bound to a matrix, i.e., via an immobilized binding protein or metal ion.

[0038] As used herein, the term "capture chromatography" refers to the removal of a molecule of interest from a sample. Examples of capture chromatography include, but are not limited to, the use of ligands such as protein A to capture Fc-containing molecules or nickel to capture histidine-tagged molecules. Often, capture chromatography relies on an immobilized capture ligand.

[0039] As used herein, the term "chemotherapeutic agent" refers to any agent approved for use as chemotherapy for cancer. Examples include, but are not limited to, all-trans retinoic acid, actimide, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hirasuzumab ... Examples of chemotherapy agents include droxyurea, idarubicin, irinotecan, lenalidomide, leucovorin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, Revlimid, temozolomide, teniposide, thioguanine, thiotepa, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine. The chemotherapy agents used in the combinations described herein may themselves be combinations of various chemotherapy agents. Suitable combinations include a combination of 5-fluorouracil (5-FU), leucovorin, and oxaliplatin (which may be referred to as FOLFOX), or a combination of irinotecan, 5-FU, and leucovorin (which may be referred to as IFL).

[0040] The term "chromatography" refers to a separation technique that uses a mobile phase and a stationary phase to separate one type of molecule from other molecules (e.g., contaminants) in a sample. The liquid mobile phase contains a mixture of molecules and transports them across or through a stationary phase (such as a solid matrix). Differential interactions of various molecules in the mobile phase with the stationary phase allow the molecules in the mobile phase to be separated.

[0041] The term "conjugate" as used herein relates to a molecule comprising or consisting essentially of at least a first protein chemically bound to another substance, such as a second protein or non-proteinaceous moiety.

[0042] As used herein, the term "deletion" or "amino acid deletion" refers to the removal of at least one amino acid at a particular position in a parent polypeptide sequence.

[0043] As used herein, the term "disulfide bond" or "disulfide bridge" or "SS-bond" refers to a functional group present in some proteins, representing the covalent bonding of two thiol groups, usually cysteines, linking residues either within the same polypeptide chain or between two polypeptide chains.

[0044] As used herein, the term "Fab" refers to the portion of an immunoglobulin molecule consisting of one intact light chain linked by a disulfide bond to the N-terminal portion of an adjacent heavy chain. Two Fab fragments are obtained from each IgG antibody molecule, and each fragment contains one antigen-binding site.

[0045] The terms "Fc-binding protein" or "immunoglobulin-binding protein" or "Ig-binding protein" are used to refer to proteins that can specifically bind to the Fc region of an immunoglobulin and / or to the Fc region of a fusion protein. Examples of Fc-binding proteins include, but are not limited to, Protein A, Protein G, Protein L, Protein Z, etc. (See, e.g., Choe et al. (2016) Materials 9:994).

[0046] As used herein, an "Fc binding protein" or "Ig binding protein" is capable of binding to whole immunoglobulins as well as to immunoglobulin fragments containing the Fc region, fusion proteins containing at least the Fc region of an immunoglobulin, and conjugates containing at least the Fc region of an immunoglobulin, due to specific binding to the Fc region.

[0047] As used herein, the term "Fc polypeptide," "Fc fragment," or "Fc region" refers to a crystallizable protein fragment of an immunoglobulin molecule, such as that obtained from human IgG, a 50 kDa protein consisting of the C-terminal halves of two heavy chains linked by two disulfide bonds.

[0048] The term "fused" means that the components are linked by a peptide bond, either directly or via a peptide linker.

[0049] The term "fusion protein" refers to a protein comprising at least a first protein genetically linked to at least a second protein. Fusion proteins can be created by joining two or more genes that originally encoded separate proteins. Thus, a fusion protein can comprise multimers of the same or different proteins expressed as a single linear polypeptide.

[0050] The term "hinge region" as used herein refers to a stretch of amino acids located between the Fab and Fc portions of an immunoglobulin molecule that contains a variable number of disulfide bonds: 2 for IgG1 and IgG4, 4 for IgG2, and 11 for IgG3.

[0051] As used herein, a subject is "in need of" a treatment if such subject would benefit biologically, medically, or in terms of quality of life from such treatment.

[0052] The term "insertion" or "amino acid insertion" refers to the addition of an amino acid to a parent polypeptide sequence.

[0053] As used herein, "immunoglobulin" can include, but is not necessarily limited to, mammalian IgG, e.g., human IgG1, human IgG2, human IgG4, mouse IgG, rat IgG, goat IgG, bovine IgG, guinea pig IgG, rabbit IgG; human IgM, human IgA; and immunoglobulin fragments containing the Fc region.

[0054] As used herein, the term "linker" refers in its broadest sense to a molecule that covalently links at least two other molecules. For example, a "linker" is a moiety that connects an Fc region to at least one additional peptide, protein, or protein domain. In some embodiments, a "linker" is a peptide linker, which can be one single amino acid or a peptide comprising two or more amino acids.

[0055] The term "modification" or "amino acid modification" refers to the replacement, deletion, or insertion of an amino acid at a specific position in a parent polypeptide sequence. Given the known genetic code and recombinant and synthetic DNA techniques, one skilled in the art can readily construct DNA encoding amino acid variants.

[0056] As used herein, the term "operably linked" refers to the juxtaposition of two or more components (e.g., sequence elements) that allows for the normal function of both components and the potential for at least one of the components to mediate a function exerted on at least one of the other components.

[0057] As used herein, the term "parent" in "parent signal peptide," "parent protein," or "parent domain" refers to a region that is subsequently modified to generate a variant of the parent signal peptide, parent protein, or parent domain. The parent signal peptide, parent protein, or parent domain can be a naturally occurring domain (e.g., SEQ ID NO: 2), an artificial domain (e.g., but not limited to, SEQ ID NO: 9), or a variant or engineered (modified) version of a naturally occurring domain (e.g., SEQ ID NO: 13). The term "parent" is used interchangeably with the term "native" herein and is used in the same manner as described above for "parent."

[0058] As used herein, the term "percent (%) amino acid sequence identity" or "percent identical" or "percent identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0059] To determine sequence identity, the sequence of the query protein is aligned with the sequence of the reference protein. Methods for alignment are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide to a reference amino acid sequence, the SIM Local similarity program is preferably used (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol. 12: 337-357), which is freely available (see also http: / / www.expasy.org / tools / sim-prot.html). For multiple alignment analysis, ClustalW is preferably used (Thompson et al. (1994) Nucleic Acids Res., 22(22): 4673-4680). Preferably, when calculating sequence identity percentage, the default parameters of the SIM Local similarity program or ClustalW are used.

[0060] In the present disclosure, unless otherwise stated, the degree of sequence identity is generally calculated based on the full length of unmodified sequence.At given position, each amino acid of query sequence that is different from reference amino acid sequence is counted as one difference.Then, the sum of differences is related to the length of reference sequence to obtain the percentage of non-identity.The quantitative percentage of identity is calculated as 100 minus the percentage of non-identity.

[0061] As used herein, the term "polishing chromatography" refers to an additional chromatographic technique that is performed after at least one initial purification step and is typically optimized to minimize levels of product aggregates such as HCPs, DNA, and residual Protein A. Chromatographic methods can include, but are not limited to, cation exchange, mixed-mode (i.e., methods that use multiple forms of interaction between the stationary phase and the analyte to achieve separation), or hydrophobic interaction chromatography.

[0062] The terms "protein" and "polypeptide" refer to any linear molecular chain of two or more amino acids linked by peptide bonds, and do not refer to a specific length of the product. Thus, "peptide," "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-translational modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, proteolytic cleavage, modification with unnatural amino acids, and similar modifications known in the art. Thus, an Fc region comprising two or more protein domains also falls within the definition of the term "protein" or "polypeptide."

[0063] As used herein, the term "size exclusion chromatography" or "molecular sieve chromatography" refers to the separation of molecules in a solution based on their size by filtration through a gel or matrix, such as spherical beads, containing pores of a specific size distribution.

[0064] The terms "solid support" or "solid matrix" are used interchangeably herein for the stationary phase matrix in chromatography.

[0065] As used herein, the term "subject" refers to an animal. Typically, an animal is a mammal. A subject also refers to, for example, a primate (e.g., a human, male or female), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. In some embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

[0066] The term "substitution" or "amino acid substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution G46C refers to an Fc binding protein in which the glycine at position 46 has been replaced with a cysteine. In the above example, 46C refers to the cysteine ​​at position 46. For purposes herein, multiple substitutions are typically separated by a slash. For example, A1I / S11A / K35R / A46C refers to a variant containing the combination of substitutions A1I, S11A, K35R, and A46C.

[0067] The term "therapeutically effective amount" refers to an amount of a substance that induces a biological or medical response in a subject, such as a reduction or inhibition of enzymatic or protein activity, or that improves symptoms, alleviates a condition, or slows or delays disease progression. In one non-limiting aspect, the term "therapeutically effective amount" refers to an amount of a disclosed protein that, when administered to a subject, is sufficient to achieve an immunomodulatory effect that at least partially alleviates, inhibits, prevents, and / or improves a cancerous condition.

[0068] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer, in one aspect, to ameliorating the disease or disorder (i.e., delaying, halting, or reducing the onset of the disease or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including one that may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another aspect, "treat," "treating," or "treatment" refers to delaying the onset or development or progression of the disease or disorder.

[0069] As used herein, the term "variant" includes an amino acid sequence that differs from another amino acid sequence by at least one amino acid substitution, deletion, or insertion. These modifications may be produced by genetic engineering or by chemical synthesis or reactions performed by humans. Variants may include, but are not limited to, peptides, polypeptides, or protein domains, such as the Fc domain.

[0070] Consideration OPCML is a member of the IgLON family, which consists of five cell adhesion molecules: OPCML (also known as OBCAM; IgLON1), NTM (also known as HNT, NTRI, and CEPU-1; IgLON2), LSAMP (also known as LAMP; IgLON3), NEGR1 (also known as KILON; MGC46680; Ntra; IgLON4), and IgLON5. They are members of the immunoglobulin (Ig) superfamily of cell adhesion molecules and are the most abundant glycosylphosphatidylinositol-anchored proteins expressed in neurons (Struyk et al. (1995) J Neurosci 15:2141-2156), supporting neuronal growth and neuronal connectivity. IgLON proteins contain three immunoglobulin (Ig)-like C2 domains, Ig1, Ig2, and Ig3 (also known as D1, D2, and D3, respectively), followed by a glycosylphosphatidylinositol-anchored protein with six or seven potential glycosylation sites (Itoh et al. (2008) Biochemistry 47:10132-10154). IgLON family members are found in many species, including humans, apes, rodents, amphibians, fish, birds, and insects (Kubic et al. (2018), Evolutionary Bioinformatics Online 14:1-10 and references identified).

[0071] As described above and shown in Figure 1, the wild-type OPCML molecule consists of three IgG-like domains, typically referred to as domain 1 (D1), domain 2 (D2), and domain 3 (D3). The molecule contains a 345-amino acid signal peptide that directs the protein to the outside of the plasma membrane, where it homodimerizes via D1 and is anchored to the outer leaflet of the plasma membrane via a GPI anchor at asparagine 322 (Figure 1A). With regard to post-translational modifications, N-linked glycosylation can occur at residues 44, 70, 140, 285, 293, and 306. Disulfide bonds exist between residues 57 and 115; 157 and 202; and 244 and 296. A lipidation signal occurs at residue 322, the GPI-anchored amidated asparagine.

[0072] Removal of the GPI anchor region allows the OPCML protein to be secreted into the extracellular environment, where it can accumulate and be isolated. Previous studies have shown that replacing the native OPCML signal peptide with the human serum albumin (HSA) signal peptide increases OPCML expression (Kober et al. (2013) Biotechnology Bioengineering 110(4):1164-73). Consequently, replacing the native OPCML signal peptide with an alternative signal peptide increased the concentration of anchorless OPCML molecules in the external environment.

[0073] Constructs expressing various recombinant OPCML molecules have been generated. Expression vectors are DNA molecules used to transfer and express foreign genetic material in cells. Such vectors contain a promoter sequence recognized by the host organism operably linked to a gene encoding the protein to be expressed. The term "promoter" refers to a minimal DNA sequence sufficient to direct transcription of the DNA sequence to which it is operably linked. The term "promoter" also encompasses promoter elements sufficient for promoter-dependent gene expression that can be controlled for cell-type specific expression. Such elements can be located in the 5' or 3' regions of the native gene.

[0074] An expression vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon may be used to express foreign genetic material from an expression vector. Suitable vectors include plasmids, binary vectors, phages, phagemids, viral vectors, and artificial chromosomes (e.g., yeast artificial chromosomes or bacterial artificial chromosomes). Some expression vectors may further include one or more reporter genes encoding at least one reporter protein. An example of a reporter protein is green fluorescent protein (GFP). Other expression vectors lack a reporter gene.

[0075] Various methods are available for the integration of exogenous nucleic acid molecules, such as the use of CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 technology, TALEN (transcription activator-like effector nuclease)-based methods, or ZFN (zinc finger nuclease)-based methods.

[0076] Homologous recombination (HR) constructs for inserting exogenous nucleic acid molecules into target sites can be designed by those skilled in the art. The construct typically contains a first homologous arm homologous to a sequence upstream of the target site and a second homologous arm homologous to a sequence downstream of the target site. Each homologous arm can contain, for example, 200 to 1,500 nucleotides (e.g., 200 to 250, 200 to 400, 250 to 500, 300 to 500, 400 to 600, 450 to 650, 500 to 800, 550 to 750, 650 to 900, 800 to 1,000, 950 to 1,200, or 1,000 to 1,500 nucleotides). The HR construct can further contain multiple cloning sites between the two homologous arms so that genes to be inserted into the genome can be ligated into the construct. Alternatively, an HR construct containing a gene flanked by two homologous sequences can be constructed using techniques known in the art, such as PCR. The HR construct can be used in a TALEN system or a CRISPR / Cas9 system to insert a nucleic acid molecule into the genome of a host cell.

[0077] Both TALEN and CRISPR / Cas9 methods work by introducing double-stranded DNA breaks into the genome at target sites. Based on the selected site, a HR construct can be designed and constructed that contains the nucleic acid molecule to be inserted into the target site.

[0078] CRISPR / Cas9 requires a gRNA specific for a target site and the endonuclease Cas9. The target site is unique compared to the rest of the genome and can be any sequence (approximately 20 nucleotides) immediately upstream of a protospacer adjacent motif (PAM). When the Cas9 / gRNA complex binds to the target site, Cas9 cleaves the DNA. Those skilled in the art will be able to design CRISPR / Cas9 constructs that target the target site.

[0079] TALEN utilizes chimeric nucleases containing an artificial DNA-binding domain of a transcription activator-like effector (TALE) protein and the catalytic domain of the restriction endonuclease Fokl. Because the DNA recognition code of TALE proteins has been deciphered, artificial DNA-binding domains can be designed to recognize any DNA sequence. To minimize off-site effects, the TALEN method can use a pair of chimeric nucleases, each recognizing sequences on either side of a double-stranded DNA break site. Those skilled in the art will be able to design TALEN constructs to target a selected site.

[0080] Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. The zinc finger domain can be engineered (modified) to target specific desired DNA sequences, allowing the zinc finger nuclease to target unique sequences within complex genomes. Each ZF set is linked to a cleavage domain that must dimerize to cleave DNA. Cleavage of the intended target gene can result in the destruction of its coding sequence by imprecise repair via non-homologous end joining. When a homologous donor DNA is introduced along with the ZFN, it can be integrated into the target by homologous recombination.

[0081] Molecular biology techniques suitable for expressing polypeptides in cells are well known in the art. For further details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press, or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, (1995, and periodic supplements).

[0082] Suitable host cells for cloning the DNA in the vector or for expressing the DNA in the vector include prokaryotic, yeast, or other eukaryotic cells. Examples of useful mammalian host cell lines include, but are not limited to, mouse L cells (LM[TK-], ATCC #CRL-2648), SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2) and human embryonic kidney (HEK) cells.

[0083] Generally speaking, one skilled in the art is fully capable of constructing vectors and designing protocols for recombinant gene expression. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, signal peptide sequences, marker genes, and other sequences, as appropriate.

[0084] Alternatively, the sequence of the desired protein to be expressed by the construct can be sent to various organizations that generate expression vectors and verify the encoded protein sequence therein, such as Invitrogen Life Technologies (Carlsbad, CA), System Biosystems (Palo Alto, CA), and PackGene (Zurich, Switzerland), to name just a few.

[0085] A recombinant OPCML (rOPCML) molecule was generated. To maximize secretion of the recombinant molecule, the native signal peptide and native linker preceding the OPCML protein were replaced with alternative signal peptides (i.e., the ceruloplasmin signal peptide and the human serum albumin (HSA) signal peptide). However, any signal peptide that allows the protein to translocate across the plasma membrane and out of the cell can be used.

[0086] The sequence surrounding the GPI anchor site was also replaced with a sequence containing a cleavable histidine tag, eliminating the GPI anchor, allowing for purification using a nickel column.

[0087] The construct was then transfected into a host cell according to methods well established in the art. "Transfection" of a cell with a transgene of interest (typically meaning an expression cassette containing the transgene) can result in a transfected host cell (or the same, a transformed host cell), which has integrated the transgene into its chromosome. The transgene can be integrated into the chromosome once or several times. That is, when the construct is introduced into a host cell, the exogenous nucleotide sequence is integrated into the genome of the cell.

[0088] Constructs containing various signal sequences were tested in vitro in HEK-293 cells to identify the most efficient signal peptide (Figure 2), with the HSA signal peptide selected. HEK-293 cells were used, but any suitable mammalian cell line can be used, including, but not limited to, those listed above, or PER.C6, CAP™, HT-1080, NSO mouse myeloma, SP2 / 0 mouse hybridoma cells, C127 mouse mammary gland cells, YB2 / 0 rat myeloma, etc.

[0089] The resulting recombinant HSA signal peptide construct, PYTX-001, had the sequence shown in Figure 3. This resulted in a soluble recombinant OPCML protein molecule (rOPCML), i.e., anchorless rOPCML was able to cross the plasma membrane of HEK-293 cells into the culture medium and remain in solution.

[0090] Fusion to the IgG Fc region is a well-established strategy for extending the half-life of therapeutic proteins (Unverdorben et al. (2016) Mabs 8(1):120-128). Often, the hinge region of human IgG, the stretch of heavy chain between the Fab and Fc portions of the molecule, is included along with the Fc portion (Rath et al. (2015) Crit Rev Biotechnol 35(2):235-254; Strohl (2015) Biodrugs 29(4):215-239). The hinge region is known to be involved in dimerization via disulfide bonds at cysteine ​​residues located within the hinge region. Because OPCML dimerizes via its D1 domain, we eliminated the hinge region and used only the CH2CH3 Fc portion to eliminate potential interference or destabilization due to native OPCML dimerization and hinge region dimerization.

[0091] A new expression construct, PYTX-004, was generated containing the HSA signal peptide inserted 5' into rOPCML D1 and the sequence surrounding the GPI anchor site removed. This GPI-anchorless rOPCML molecule was then fused to an Fc molecule containing only the CH2CH3 region. Figure 4 shows the sequence of the resulting rOPCML-Fc protein.

[0092] Stable transfection of the construct into suitable cells was performed according to methods well established in the art. Suitable cells include those listed above. Single-cell cloning was then performed to identify the most productive single-cell clones and select them for further development and scale-up production (Figure 5). To eliminate the possibility that serum in the cell culture medium may affect protein purification, the transfected cells were subjected to a serum reduction scheme (Figure 5C).

[0093] The rOPCML-Fc protein derived from PYTX-004 was then purified. Purification can be achieved by any number of methods, including, but not limited to, affinity purification, affinity chromatography, anion exchange chromatography, capture chromatography, polishing chromatography, and / or size exclusion chromatography. Often, capture chromatography using Protein A immobilized on a matrix is ​​used to capture Fc-containing molecules, or nickel, cobalt, or copper immobilized on a matrix is ​​used to capture His-tagged molecules. The rOPCML-Fc PYTX-004 protein was purified to high purity (FIGS. 5D and 7) and then characterized.

[0094] To determine whether the fused CH2CH3 Fc region affects the signaling activity of rOPCML-Fc, its activity was compared with that of the rOPCML protein (PYTX-001 construct). Here, ovarian cancer cell lines were treated with fetal calf serum (FCS), and rOPCML or rOPCML-Fc proteins were purified. Figure 6 shows the results demonstrating equipotency of on-target pharmacokinetics and downstream proximal pharmacokinetics.

[0095] The ability of rOPCML-Fc protein to induce significant signaling, along with its effect on ovarian cancer cells, was further investigated. Three different cell lines, OVCAR3, SKOV3, and PEO1, were selected to evaluate the IC of rOPCML-Fc. 50 As can be seen in Figure 8A, rOPCML-Fc had IC values ​​of 0.379 mg / ml for OVCAR3, 0.519 mg / ml for SKOV3, and 0.612 mg / ml for PEO1. 50 The ability of various concentrations of rOPCML-Fc to inhibit OVCAR3 and SKOV3 cell proliferation over periods of 48, 96, and 144 hours was also determined (FIG. 8B).

[0096] The effect of rOPCML-Fc on wound healing was also determined in vitro in OVCAR3 and SKOV3 cells using a gap closure assay (Birtley et al. (2019) Nature Communications 10:3134). As can be seen in Figures 9A and 9B, administration of 0.05 or 0.1 mg / ml of rOPCML-Fc inhibited the ability of OVCAR3 and SKOV3 cells to invade and close the gap within 24 hours of rOPCML-Fc administration. Figures 9C and 9D present this data in graphical form, showing that while both concentrations had a positive inhibitory effect, the difference between the control and 0.1 mg / ml rOPCML-Fc administration was highly significant in both cell types.

[0097] Similarly, we determined the ability of rOPCML-Fc to inhibit the invasion of OVCAR3 and SKOV3 cells. Using the invasion assay described by Birtley et al. (Birtley et al. (2019) Nature Communications 10:3134), we applied either 0.05 or 0.1 mg / ml of rOPCML-Fc. Figure 10A shows the results in micrographs, while Figures 10B and 10C show the data in graphical format for OVCAR3 and SKOV3, respectively. Again, both concentrations had a positive inhibitory effect, but the difference between the control and 0.1 mg / ml rOPCML-Fc treatment was highly significant for both cell types.

[0098] Taken together, the results in Figures 8, 9 and 10 demonstrate that rOPCML-Fc potently inhibits invasion and migration in OVCAR3 and SKOV3 cells.

[0099] We investigated the effects of rOPCML-Fc protein on ovarian cancer patient-derived organoids (PDOs). Organoids derived from ovarian tumor tissue obtained during initial surgery from patients diagnosed with ovarian serous carcinoma were used. Organoid tissue-derived cells were treated with 0.1 mg / ml or 0.8 mg / ml of rOPCML-Fc. The results are shown in Figure 11A. Cells were treated with rOPCML-Fc at concentrations of 0 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.4 mg / ml, or 0.8 mg / ml. The results are shown in Figure 11B. Treatment of OV016X PDOs with 0.4 mg / ml or 0.8 mg / ml of rOPCML-Fc and OV044 PDOs with 0.8 mg / ml of rOPCML-Fc resulted in a statistically significant decrease in cell viability and proliferation activity. Treatment with 0.8 mg / ml rOPCML-Fc in OV041 PDO resulted in a highly statistically significant decrease in viability / proliferative activity.

[0100] Additional signal transduction studies were performed using cells stimulated with EGF, where rOPCML-Fc downregulated pHER2 and the RAF / MEK, ERK pathways in ovarian cancer cells (Figure 12).

[0101] To determine the efficacy and safety of intravenous administration of rOPCML-Fc, we developed a robust model of primary human ovarian tumors, summarized in Figure 13. Here, primary human ovarian tumor cells were introduced into NDG mice with severe T cell, B cell, and NK cell deficiencies to generate patient-derived xenograft (PDX) models. Figure 14 shows the results of the ovarian cancer marker PAX-8 for the P0, P1, and P2 generations. Strong uptake rates were observed even in the P1 (61.9%) and P2 (75.0%) generations, demonstrating the generation of robust PDX models.

[0102] Using this model, two experimental groups were established for rOPCML-Fc administration: a low-dose group (5 mg / kg) and a high-dose group (10 mg / ml). Intravenous injections into the tail vein were administered every two days for 28 days. Figures 15A-C show that administration of rOPCML-Fc alone was sufficient to inhibit human ovarian PDX in a dose-dependent manner at both 5 mg / kg and 10 mg / kg levels, reducing tumor weight by 77.3% and 93.0%, respectively. Importantly, intravenous administration of rOPCML-Fc protein did not affect the daily activity or body weight of mice (Figure 15D).

[0103] Immunohistochemistry was used to identify cells targeted by rOPCML-Fc (Figure 16). Using wild-type OPCML and Ki-67 as markers, rOPCML-Fc was found to target tumor cells in the PDX model tumor environment without aggregating in normal mouse organs, and inhibited cancer cell growth in the model.

[0104] The cancer to be treated may be ovarian cancer, breast cancer, renal cancer, gastrointestinal cancer, stomach cancer, colon cancer, liver cancer, cervical cancer, prostate cancer, endometrial, endometrial serous carcinoma, esophageal, any type of glioma, lung cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, nasopharyngeal, and nasal NK / T lymphoma.

[0105] Administration of rOPCML-Fc protein to patients in need thereof is most easily accomplished by intravenous injection, although other types of administration are possible, such as intraperitoneal, intrapleural, subcutaneous, intraventricular, and intramuscular injection. Furthermore, it can be inhaled / nebulized or applied topically to the skin, or injected intervenously into an artery for the blood supply of tumor deposits.

[0106] In some embodiments, pharmaceutical compositions comprising rOPCML-Fc protein and a pharmaceutically acceptable carrier are useful. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active rOPCML-Fc component, its use in therapeutic or pharmaceutical compositions is contemplated.

[0107] The pharmaceutical composition can be prepared according to conventional mixing, granulating, or coating methods, and can contain about 0.1% to about 99%, about 5% to about 90%, or about 1% to about 20% of the rOPCML-Fc protein by weight or volume.

[0108] In one embodiment, rOPCML-Fc is administered in a therapeutically effective amount in combination with one or more therapeutic agents (pharmaceutical combinations) or therapies, e.g., non-drug therapies. For example, synergistic effects may occur with other antiproliferative, anticancer, immunomodulatory, or anti-inflammatory agents. In particular, receptor tyrosine kinase inhibitors may be co-administered with rOPCML-Fc. When the compounds of the present invention are administered in combination with other therapies, the dosage of the co-administered compounds will vary depending on the type of co-drug used, the specific drug used, the condition being treated, etc.

[0109] Combination therapy includes the administration of a compound of the present invention in combination with one or more other biologically active components, including, but not limited to, a second and different anti-neoplastic agent or agent targeting DNA repair, and non-drug therapies, including, but not limited to, surgery or radiation therapy. For example, rOPCML-Fc protein can be used in combination with other pharmaceutically active compounds, preferably biologics or compounds that can enhance the effects of rOPCML-Fc protein. rOPCML-Fc protein can be administered simultaneously or sequentially with other drug therapies or treatment modalities, for example, as a single preparation or separate preparations. Generally, combination therapy contemplates the administration of two or more biologics or drugs in a single cycle or during a course of treatment.

[0110] In one embodiment, the enhancement of cancer chemotherapy treatment in a mammal undergoing treatment with an anti-cancer agent is achieved by co-administering an effective amount of rOPCML-Fc protein to the mammal. In a specific embodiment, the anti-cancer agent is a DNA damaging agent. The DNA damaging agent can be any suitable DNA damaging agent. Non-limiting examples of suitable DNA damaging agents include DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, tenyposide, triethylenethiophosphoramide, and etoposide. The DNA damaging agent can also be radiation, or a biotherapeutic agent such as an antibody.

[0111] In one aspect, a therapeutically effective dose is about 0.01 mg to about 5,000 mg of rOPCML-Fc protein per day. Accordingly, an rOPCML-Fc protein or rOPCML-Fc pharmaceutical composition should provide a dosage of rOPCML-Fc of about 0.01 mg to about 5,000 mg. In certain aspects, pharmaceutical unit dosage forms are prepared to provide about 1 mg to about 2,000 mg, about 10 mg to about 1,000 mg, about 20 mg to about 500 mg, or about 25 mg to about 250 mg of the essential rOPCML-Fc active ingredient or combination of essential ingredients per unit dosage form. In certain embodiments, pharmaceutical unit dosage forms are prepared to provide about 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1,000 mg, or 2,000 mg of the essential rOPCML-Fc active ingredient.

[0112] The following examples are intended to further illustrate certain aspects of the present disclosure and are not intended to limit the scope in any way. [Example]

[0113] Example 1 - Determining the efficiency of r-OPCML signal peptide Constructs for recombinant OPCML (r-OPCML) molecules were prepared by sending the sequences to Invitrogen for generation of expression vectors. To ensure that anchorless recombinant OPCML could cross the plasma membrane of HEK-293 cells and enter the cytoplasm, all constructs excluded the last 30 amino acids of the wild-type OPCML protein sequence, including the GPI anchor. Briefly, the encoded amino acid sequences for the desired signal peptide, all three OPCML Ig domains (D1, D2, and D3, OPCML residues 36–316), the human rhinovirus 3C protease cleavage site (LEVLFQGP; SEQ ID NO: 11), and the His tag were placed into the PCDNA3.1™ expression vector (THERMOFISHER®). The amino acid sequences of the native OPCML signal peptide sequence, the native linker sequence, and the first 10 amino acids of the OPCML protein (SEQ ID NO: 3), the ceruloplasmin signal peptide sequence, and the first 10 amino acids of the OPCML protein (SEQ ID NO: 4), and the human serum albumin (HSA) signal peptide and the first 10 amino acids of the OPCML protein (SEQ ID NO: 5) are shown in Figure 2.

[0114] After receiving the expression vector construct, HEK-293 cells were cultured in DMEM high-glycemic culture medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37°C and 5% CO. Transfection was achieved using the EFFECTENE® Transfection Reagent System (QIAGEN®) with 1 μg / μl of pcDNA3.1-rOPCML plasmid according to the manufacturer's instructions.

[0115] A single stable transgenic cell line was selected for each construct and cultured in DMEM high-glycemic culture medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37°C and 5% CO. Culture medium supernatants were filtered using a 0.45 μm organic filter membrane and then purified via Ni-NTA resin (QIAGEN®) according to the manufacturer's instructions.

[0116] The purified protein was soluble. The extent to which the signal peptide sequence enabled the expressed recombinant OPCML protein to be imported into the culture medium was determined by separating the purified protein using an 8% SDS-PAGE gel, transferring it to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane (Millipore Corporation), blocking it, and incubating it with polyclonal goat anti-human OPCML (OPCML; R&D Systems). Densitometry was then performed using a Sapphire FL Biomolecular Imager for Western densitometry and Image J software (Azure Biosystems). Figures 2B and 2C show the results. Protein expression from the construct containing the native OPCML signal peptide was significantly lower than that observed with the ceruloplasmin signal peptide construct and the HSA signal peptide construct (PYTX-001). Consequently, the HSA signal peptide showed the strongest activity, and therefore the PYTX-001 construct was used for subsequent experiments. The entire amino acid sequence of the PYTX-001 protein (SEQ ID NO: 6) is shown in Figure 3C.

[0117] Example 2 - rOPCML-FC molecule PYTX-004 A construct for a recombinant OPCML (r-OPCML) molecule containing a human IgG Fc fragment (accession 3AGV_A; SEQ ID NO: 12) containing only the CH2CH3 region was prepared by sending the desired amino acid sequence (SEQ ID NO: 10) to Invitrogen for generation of an expression vector. Here, the amino acid sequence of the human rhinovirus 3C protease cleavage site (LEVLFQGP; SEQ ID NO: 11) was inserted along with a linker and the wild-type human IgG Fc region (SEQ ID NO: 13). The wild-type human IgG Fc region (SEQ ID NO: 13) contains only the CH2CH3 region of human IgG Fc and lacks the 23-amino acid hinge region, which contains a site for disulfide pairing. This prevented Fc dimerization and its potential interference with the dimerization of the OPCML D1 domain. In addition, a His tag was inserted at the C-terminus of the Fc sequence. This resulted in the rOPCML-Fc construct (PYTX-004; Figure 4).

[0118] After receiving the construct, HEK-293 cells were cultured at 37°C and 5% CO2 in DMEM high-glycemic culture medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Transfection was achieved using the EFFECTENE® Transfection Reagent System (QIAGEN®) with 1 μg / μl of pcDNA3.1-rOPCML-Fc plasmid according to the manufacturer's instructions. Controls did not contain the pPCDNA3.1-rOPCML-Fc plasmid (PYTX-004 construct). Cells were cultured at 37°C and 5% CO2 in DMEM high-glycemic culture medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. After 48 hours, the medium was replaced and fresh medium containing 800 μg / ml G418 was added. Further culture was performed using G418 medium.

[0119] Fourteen days after transfection, cells transfected with rOPCML-Fc showed colony growth, whereas the control did not (Figure 5A). Individual cells were isolated and grown in culture medium for 3 days, and the relative content of rOPCML-Fc protein in the culture medium supernatant of each clone was detected using an ELISA assay (Figure 5B). The clone with the highest rOPCML-Fc protein content in the culture supernatant (18G9) was used for all further experiments.

[0120] Because serum in the cell culture medium can affect subsequent protein purification, we performed serum-free culture to gradually reduce the serum dependency of the cells. After the cells reached 100% confluence, the amount of serum in the culture medium was reduced, resulting in a decrease in confluence to approximately 80% in medium containing 5% serum and approximately 50% in serum-free medium (Figure 5C).

[0121] The rOPCML-Fc clone was cultured, and 1 L of the culture medium supernatant was filtered using a 0.45 μm organic filter membrane and then purified via Ni-NTA resin (QIAGEN®) according to the manufacturer's instructions. Briefly, the cell culture medium was applied to the column and eluted with 200 mM imidazole elution buffer. For SDS-PAGE detection, samples of the cell culture medium before purification, the effluent after purification, the wash, and the eluate from various tubes were collected. The following samples were tested: cell culture medium pre-bound, effluent, wash, and six separate samples of purified rOPCML-Fc protein were collected / eluted consecutively.

[0122] Samples were separated using 8% SDS-PAGE gels, transferred to nitrocellulose or polyvinylidene fluoride (PVDF) membranes (Millipore Corporation), blocked, and incubated overnight at 4°C with polyclonal goat anti-human OBCAM (OPCML; R&D Systems) at a 1:1000 dilution as the primary antibody. After washing, the membranes were incubated with HRP-conjugated anti-goat antibody at a 1:5000 dilution as the secondary antibody. After a final wash, proteins were detected using Immobilon Western Chemiluminescent HRP Substrate System (Millipore) and GE Healthcare Amersham "Hyperfilm" ECL film with a Kodak SRX2000 (Rochester, NY, USA) developer. The results are shown in Figure 5D. Here, more than 95% of the rOPCML-Fc protein still exhibited two forms after purification: one polymeric form with a molecular weight of 160 kDa and the other a monomeric form with a molecular weight of 80 kDa. The lack of signal for the effluent after purification indicated that the binding efficiency reached 100%.

[0123] The purified protein was soluble, and expression of rOPCML-Fc protein was determined by Western blotting. Here, purified protein samples were separated using an 8% SDS-PAGE gel, transferred to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane (Millipore Corporation), blocked, and incubated overnight at 4°C with a polyclonal goat anti-human OBCAM (OPCML; R&D Systems) primary antibody at a 1:1000 dilution. After washing, the membrane was incubated with a 1:5000 dilution of HRP-conjugated anti-goat secondary antibody. After a final wash, the protein was detected using Immobilon Western Chemiluminescent HRP Substrate System (Millipore) and GE Healthcare Amersham "Hyperfilm" ECL film with a Kodak SRX2000 (Rochester, NY, USA) developer. HPLC was performed using a Perkin Elmer LC 300 HPLC system according to the manufacturer's instructions. The results are shown in Figures 7A and 7B.

[0124] Example 3 - Evaluation of signaling potency of rOPCML-Fc (PYTX-004 construct) To confirm that the Fc portion of rOPCML-Fc does not adversely affect its signaling capacity / potency, we performed a Western blot comparing the activity of rOPCML (PYTX-001 construct) with that of rOPCML-Fc (PYTX-004 construct). Logarithmic growth phase SKOV3 and OVCAR8 cells were used. Cells were serum-starved and synchronized by culturing in medium without fetal bovine serum for 24 hours. The medium was replaced with medium containing 10% fetal bovine serum (FCS) and 40 μg / ml of rOPCML (PYTX-001 construct) protein or 40 μg / ml of rOPCML-Fc (PYTX-004 construct) protein. Controls consisted of the addition of medium containing only 10% FCS or unmodified medium. Proteins were extracted after 30 minutes.

[0125] Protein samples were separated on 8% SDS-PAGE gels, transferred to nitrocellulose or PVDF membranes, blocked, and incubated with primary antibodies at a 1:1000 dilution overnight at 4°C. The primary antibodies used were: phosph-HER2 / ErbB2 (T1248) (rabbit, Cell Signaling Technology); Total-HER2 (rabbit, Cell Signaling Technology); human phospho-Axl (Y779) (rabbit, R&D Systems); phospho-AKT (S473) (rabbit, Cell Signaling Technology); Pan-AKT (rabbit, Cell Signaling Technology); phospho-ERK1 / 2 (T202 / Y204) (rabbit, Cell Signaling Technology); Total-ERK (rabbit, Cell Signaling Technology); Calnexin (rabbit, Cell Signaling Technology); and OPCML (goat, R&D Systems).

[0126] After washing, the membrane was incubated with a 1:5000 dilution of HRP-conjugated secondary antibody (i.e., anti-rabbit or anti-goat). After a final wash, proteins were detected using Immobilon Western Chemiluminescent HRP Substrate System (Millipore) and GE Healthcare Amersham "Hyperfilm" ECL film with a Kodak SRX2000 (Rochester, NY, USA) developer. The results are shown in Figure 6. The results demonstrate similarity between rOPCML and rOPCML-Fc proteins with respect to phospho-AXL-Y779, phospho-AKT-S473, and phospho-ERK-1 / 2-T202-Y204. Both the r-OPCML protein (PYTX-001 construct) and the rOPCML-Fc protein (PYTX-004 construct) appeared to be essentially equipotent in terms of on-target and downstream pharmacokinetics.

[0127] Example 4 - IC of rOPCML-Fc fusion protein (PYTX-004) 50 test Three different cell lines were selected to determine the IC50 value of the rOPCM-Fc fusion protein: OVCAR3, SKOV3, and PEO1. Briefly, logarithmically growing cells were washed with PBS buffer and treated with 0.25% trypsin-EDTA for 1-2 minutes at 37°C and 5% CO2, followed by treatment with medium containing 10% fetal bovine serum. The cells were isolated, diluted, and further subcultured at 37°C and 5% CO2. rOPCM-Fc protein was added at concentrations of 0.0 mg / ml, 0.025 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, and 0.8 mg / ml. Cells were cultured in 96-well plates for 6 days, and then each well was treated with 100 μl of medium containing 10% CCK8 reagent. After incubation at 37°C and 5% CO for 1.5–2 hours, absorbance was measured at A450 nm. The results are shown in Figure 8A, which shows IC50 values ​​of 0.379 for OVCAR-3 cells, 0.519 for SKOV3 cells, and 0.612 for PEO1.

[0128] The ability of various concentrations of rOPCML-Fc protein (PYTX-004 construct) to inhibit cell proliferation of OVCAR3 and SKOV3 cells was also determined. Cells were prepared as described above. rOPCML-Fc protein at concentrations of 0.8 mg / ml, 0.4 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.025 mg / ml, and 0.0 mg / ml was added to the cell cultures for periods of 48, 96, and 144 hours (Figure 8B).

[0129] Example 5 - Effect of rOPCML-Fc protein (PYTX-004 construct) on wound healing To determine the effect of rOPCML-Fc protein (PYTX-004 construct) on wound healing, an in vitro gap closure assay was performed using OVCAR3 and SKOV3 cells as described by Birtley et al. (2019, Nature Communications 10:3134). Briefly, cell lines were seeded at 100% confluence onto culture inserts in 24-well plates and serum-starved overnight. The inserts were removed to create a 500 μm gap, and cells were stimulated with 0.05 mg / ml or 0.1 mg / ml of rOPCML-Fc protein. Control cells were unstimulated. After 24 hours, cells were visualized under a microscope (Figures 9A and 9B).

[0130] Both concentrations inhibited the ability of OVCAR3 and SKOV3 cells to invade and close the gap within 24 hours of rOPCML-Fc protein application. Figures 9C and 9D show the results in graphical form. Notably, administration of 0.1 mg / ml rOPCML-Fc was highly significant for both cell types compared to the control.

[0131] Example 6 - Effect of rOPCML-Fc protein (PYTX-004 construct) on the invasive potential of OVCAR3 and SKOV3 cells The ability of rOPCML-Fc to inhibit OVCAR3 and SKOV3 cells was evaluated using the invasion assay described by Birtley et al. (2019, Nature Communications 10:3134). Fertilized chicken eggs were prepared as described in Birtley et al. On ED9, grafts were prepared by suspending 10 OVCAR3 or SKOV3 cells in 100 μl of Matrigel (BD Biosciences). rOPCML-Fc protein at concentrations of 0.0 mg / ml, 0.05 mg / ml, or 0.1 mg / ml was added to the grafts, which were then deposited onto the membrane, and the window was recoated and sealed.

[0132] The results shown in Figures 10A-C indicate that rOPCML-Fc protein had a positive inhibitory effect at both concentrations, although the difference between the control and 0.1 mg / ml rOPCML-Fc protein treatment was highly significant.

[0133] Example 7 - Effect of rOPCML-Fc protein (PYTX-004 construct) on ovarian cancer patient-derived organoids (PDO) To further characterize the inhibitory potential of rOPCML-Fc, experiments were performed using ovarian cancer patient-derived organoids (PDO). Here, patient-derived organoids from ovarian cancer patients were obtained from the PDO tissue sample library, constructed by Beijing Keyu Technology Co., Ltd. and numbered OV016X-PDO, OV009-PDO, OV044-PDO, OV041-PDO, and OV024-PDO. All organoids were derived from ovarian tumor tissue obtained during initial surgery from patients who had been pathologically diagnosed with high-grade serous carcinoma of the ovary but were not treated.

[0134] Briefly, organoid tissue was collected and digested with TRYPLE™ digestion solution containing 10 μM Y-27632 at 37°C for 15 minutes with shaking. The digestion was stopped by adding Advanced-DMEM / F12 medium containing 5% fetal bovine serum (FBS), followed by centrifugation at 1000 rpm for 5 minutes. Cells were resuspended in GAS-Ad-ES medium, diluted to 8 × 104 cells / ml, mixed with Matrigel at a 1:1 ratio, and 0.5 ml was added to each well of a 24-well plate. The cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes to solidify, after which GAS-Ad-ES medium was added and incubation continued. Fresh medium was added every 3 days. Experiments were performed after 3–4 passages.

[0135] To determine the effect of rOPCML-Fc on PDO, 1.6 × 10 cells / ml in GAS-Ad-ES medium were mixed with Matrigel at a 1:1 ratio and added at 50 μl per well to a low-adhesion 96-well plate. After incubating the cells for 30 minutes at 37°C in a 5% CO2 incubator to allow solidification, 40 μl of GAS-Ad-ES medium was added per well and incubation continued at 37°C for 2 days. The cells were then divided into six concentration groups, each with three replicate wells, and each well was supplemented with 30 μl of GAS-Ad-Es medium containing either 0.05, 0.1, 0.2, 0.4, or 0.8 mg / ml of rOPCML-Fc. Control wells were supplemented with 60 μl of GAS-Ad-Es medium. Plates were then incubated at 37°C in a 5% CO2 incubator for 2 days, after which wells were supplemented with 30 μl of the appropriate rOPCML-Fc mixture or GAS-Ad-Es medium for control wells. Cells were further cultured at 37°C in a 5% CO2 incubator for 2 days.

[0136] Chemiluminescence was then measured to assess cell viability. Here, 10 ml of CELLTITER_GLO® buffer was added to one vial of CELLTITER_GLO® buffer substrate and mixed thoroughly. 50 μl of the resulting CELLTITER_GLO® mixture was added to each well, and the microplate was gently mixed on a shaker for 2 minutes. The plate was incubated at room temperature in the dark for 10 minutes and then placed in a multifunction microplate reader to detect luminescence values. The results are shown in Figures 11A and 11B. Figures 11A and 11B show a statistically significant decrease in cell viability / proliferative activity for administration of 0.4 and 0.8 mg / ml of rOPCML-Fc in OV016X PDO, a statistically significant decrease in viability / proliferative activity for administration of 0.8 mg / ml of rOPCML-Fc in OV044 PDO, and a highly statistically significant decrease in viability / proliferative activity for administration of 0.8 mg / ml of rOPCML-Fc in OV041 PDO.

[0137] Example 8 - Effect of rOPCML-Fc on signaling pathways The signaling ability of rOPCML-Fc was investigated, and the results are shown in Figure 12. Here, logarithmic growth phase OVCAR3 cells were used. Serum starvation and synchronization of the cells were achieved by culturing them in medium without fetal bovine serum for 24 hours. The medium was replaced with medium containing 50 ng / ml EGF and 0.1 mg / ml rOPCML-Fc (PYTX-004 construct) protein. Controls consisted of the addition of medium containing 50 ng / ml EGF alone or unmodified medium. Proteins were extracted after 30 minutes.

[0138] Protein samples were separated on 8% SDS-PAGE gels, transferred to nitrocellulose or PVDF membranes, blocked, and incubated overnight at 4°C with primary antibodies at a 1:1000 dilution. The primary antibodies used were: phosph-HER2 / ErbB2 (T1248) (rabbit, Cell Signaling Technology); Total-HER2 (rabbit, Cell Signaling Technology); human phospho-Axl (Y779) (rabbit, R&D Systems); phospho-AKT (S473) (rabbit, Cell Signaling Technology); p-AKT (rabbit, Cell Signaling Technology), GAPDH (rabbit, Cell Signaling Technology), p-EGFR (rabbit, Cell Signaling Technology), p-RAF (rabbit, Cell Signaling Technology); p-MEK (rabbit, Cell Signaling Technology); and p-ERK (rabbit, Abcam).

[0139] After washing, the membrane was incubated with a 1:5000 dilution of HRP-conjugated secondary antibody (i.e., anti-rabbit). After a final wash, proteins were detected using Immobilon Western Chemiluminescent HRP Substrate System (Millipore) and GE Healthcare Amersham "Hyperfilm" ECL film with a Kodak SRX2000 (Rochester, NY, USA) developer. The results are shown in Figure 12. rOPCML-Fc downregulated pHER2 and the RAF / MEK / ERK pathway in ovarian cancer cells.

[0140] Example 9 - Construction of PDX models of NDG mouse ovarian cancer A flowchart of the PDX model generation is shown in Figure 13. Here, omental metastases from a patient with high-grade serous carcinoma of the ovary were subcutaneously inoculated into the dorsum of seven NDG mice, each at three sites on their backs. Transplanted tumor growth was observed in the dorsum of seven mice, a total of 13 / 21. Successful modeling was achieved in each site, representing the P1 generation transplanted tumors, with a success rate of 61.9%. A total of 12 NDG mice were inoculated with P2 generation transplanted tumors. One week after inoculation, three mice died of disease. The tumors in the dorsum of the remaining nine mice all grew well, resulting in a 75% success rate. After tumor formation, tumor tissues from the NDG mice were sectioned and observed. The transplanted tumors were round or nodular, had solid flesh on the cut surface, and abundant capillaries on the surface.

[0141] The original tumor tissue, P1-generation xenograft tumors, and P2-generation xenograft tumors were stained with HE and observed under a light microscope (Figure 14). The histological morphology of the P1-generation and P2-generation xenograft tumors was very similar to that of the original tumor tissue, with no polarized cellular arrangement, large nuclei, or deep staining. Compared with the original tumor tissue, the P1-generation and P2-generation xenograft tumors were less differentiated and more malignant. To determine whether the xenograft tumor tissues retained the molecular phenotypic characteristics of the original tumor tissue, PAX-8 immunohistochemical staining was performed on the original tumor tissue, P1-generation xenograft tumors, and P2-generation xenograft tumors. PAX-8 was positively expressed in the original tumor tissue, P1-generation xenograft tumors, and P2-generation xenograft tumor specimens.

[0142] Example 10 - Intravenous application of rOPCML-Fc (PYTX-004 construct) P2 generation mice were used. The transplanted tumor volume was 100 mm 3 When the tumor volume reached 1000 mg / kg, the animals were divided into three groups. The experimental group received 5 mg / kg or 10 mg / kg of rOPCML-Fc fusion protein according to the body weight of the NDG mice, while the control group received the same volume of phosphate-buffered saline (PBS). Both groups were injected into the tail vein every other day. The long and short diameters of the tumor volume on the back of the NDG mice were measured every four days. The animals were sacrificed on the 28th day of treatment, and growth curves of the transplanted tumors were plotted. Treatment was initiated when the tumor volume before treatment was essentially the same. The tumor volume in the control group showed a gradual increase, while the volumes in the two rOPCML-Fc fusion protein-treated groups gradually decreased. A clear dose-dependent and statistically significant decrease in tumor volume (p = 0.010, p = 0.002) was evident in the treated animals.

[0143] The tumors in each group were removed and weighed (Figure 15A). The results showed that the mean tumor weight in the 5 mg / kg rOPCML-Fc fusion protein group was 18.13 ± 8.64 mg, representing a 77.3% inhibition rate, compared with 79.87 ± 51.81 mg in the control group. The mean tumor weight in the 10 mg / kg rOPCML-Fc fusion protein group was 5.53 ± 1.26 mg, representing a 93.0% tumor weight reduction rate (Figures 15B-D).

[0144] Example 11 - Immunohistochemical analysis of rOPCML-Fc protein after treatment of P2 generation transplanted tumors Ki-67 is an antigen associated with proliferating cells and is used to label cells during the proliferation cycle. Cells positive for this label have a high proliferation rate and low tissue differentiation potential. Immunohistochemical methods were used to detect Ki-67 expression and binding of rOPCML-Fc protein to P2-generation transplanted tumors after administration.

[0145] The results showed that Ki-67 nuclear staining significantly decreased Ki-67 expression in the 5 mg / kg and 10 mg / kg rOPCML-Fc protein-treated groups compared with the control group (Figure 16B), indicating that its proliferative activity was inhibited.

[0146] Simultaneously, immunohistochemical testing was performed to detect the binding activity of rOPCML-Fc protein to the transplanted tumors. The results showed that in the 5 mg / kg and 10 mg / kg rOPCML-Fc protein treatment groups, rOPCML-Fc specifically targeted cancer cells within the transplanted tumors, but the content of mesenchymal cells was low (Figure 16A).

Claims

1. A polypeptide molecule comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

6.

2. The polypeptide molecule of claim 1, wherein the sequence identity is at least 95%.

3. A pharmaceutical composition comprising the polypeptide molecule of claim 1 or claim 2 and a pharmaceutically acceptable carrier.

4. 4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier is suitable for intravenous administration.

5. A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% identity to SEQ ID NO:

6.

6. The nucleic acid molecule of claim 5, wherein the sequence identity is at least 95%.

7. (a) a first nucleic acid having a regulatory sequence that directs transcription and / or translation; (b) a second nucleic acid having the nucleotide sequence of claim 5 or claim 6; and A vector construct comprising: A vector construct, wherein the first and second nucleic acids are operably linked.

8. A host cell comprising the nucleic acid molecule of claim 5 or claim 6.

9. A host cell comprising the vector construct of claim 7.

10. 1. A method for introducing nucleic acid into a host cell, comprising: (a) providing a nucleic acid molecule according to claim 5 or claim 6; and (b) contacting said nucleic acid with said host cell under conditions that allow insertion of said nucleic acid into said host cell.

11. A method of transfecting a host cell, comprising contacting the host cell with the vector construct of claim 7.

12. A method for treating a disease or condition, comprising administering to a patient in need thereof an effective amount of a polypeptide molecule according to claim 1 or claim 2.

13. 13. The method of claim 12, wherein the disease or condition is a HER2-positive cancer.

14. 13. The method of claim 12, wherein the disease or condition is a HER-negative cancer.

15. 13. The method of claim 12, wherein the disease or condition is a cancer selected from the group consisting of ovarian cancer, breast cancer, renal cancer, gastrointestinal cancer, stomach cancer, colon cancer, liver cancer, cervical cancer, prostate cancer, endometrial, endometrial serous carcinoma, esophageal, any type of glioma, lung cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, nasopharyngeal, and nasal NK / T lymphoma.

16. 16. The method of claim 15, wherein the cancer is ovarian cancer.

17. 13. The method of claim 12, wherein the polypeptide molecule is administered intravenously.

18. 10. A method of treating a disease or condition, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 3.

19. 19. The method of claim 18, wherein the disease or condition is a HER2-positive cancer.

20. 19. The method of claim 18, wherein the disease or condition is a HER-negative cancer.

21. 19. The method of claim 18, wherein the disease or condition is a cancer selected from the group consisting of ovarian cancer, breast cancer, renal cancer, gastrointestinal cancer, stomach cancer, colon cancer, liver cancer, cervical cancer, prostate cancer, endometrial, endometrial serous carcinoma, esophageal, any type of glioma, lung cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, nasopharyngeal, and nasal NK / T lymphoma.

22. 22. The method of claim 21, wherein the cancer is ovarian cancer.

23. 20. The method of claim 18, wherein the polypeptide molecule is administered intravenously.

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