Actinohibin variant polypeptides and related methods

Abstract

Treatment of ovarian cancer (e.g., epithelial ovarian cancer) sensitive to a polypeptide comprising actinohibin or a variant thereof (e.g., AvFc) is described. Methods for killing ovarian cancer cells (e.g., epithelial ovarian cancer cells) are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 156,715, filed March 4, 2021, the entire teachings of which are incorporated herein by reference.

[0002] Incorporation by reference of material in ASCII text files This application is filed concurrently with the following ASCII text file: a) File name: 56001010001PCT_Sequence_Listing_ST25.txt (created on February 23, 2022, size 19,000 bytes) The sequence listing contained in this application is incorporated by reference.

[0003] Government support This invention was made with government support under grant 1R21CA216447-01 from NIH / NCI and R21 / R33 AI088585 from NIH / NIAID. The government has certain rights in this invention. [Background technology]

[0004] Ovarian cancer (OVCA), specifically epithelial ovarian cancer (EOC), is one of the most lethal gynecologic cancers and the fifth leading cause of cancer deaths in women. EOC usually begins as small borderline epithelial tumors on the surface of either the ovaries, fallopian tubes, or the mesothelial lining of the peritoneal cavity. These tumors grow and become well differentiated, then metastasize primarily to the peritoneal cavity, but rarely to the lungs, liver, and brain. According to the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute (NCI), the overall 5-year survival rate in the United States was 48.3% as of 2016, which is primarily driven by the dismal survival rate of late-stage disease (30.5%). The age-adjusted mortality rate and new case rate of OVCA are 6.8 per 100,000 and 10.5 per 100,000 per year, respectively, slightly higher than the next most deadly gynecologic cancer, uterine cancer, which has a mortality rate of 5.0 per 100,000 per year. This translates into an increase of 22,530 new cases and 13,980 deaths in 2016. Although the number of new cases and deaths has been gradually declining over the past two decades, the prognosis of patients remains poor, especially for those with late-stage disease. This is mainly due to the ineffectiveness of population-based screening, the innocuous nature of the symptoms, and the lack of effective second-line therapy for chemotherapy-resistant disease. In general, patients respond very well to first-line treatment, but the majority of women (75%) may experience a recurrence of the disease, which is incurable due to chemotherapy resistance.

[0005] Standard of care for ovarian cancer and its shortcomings. Primary cytoreductive surgery followed by chemotherapy has been the first-line standard of care for EOC for decades. Surgery for advanced disease consists of total abdominal hysterectomy, bilateral salpingo-oophorectomy, and omentectomy, although patients with lower grade disease can opt for fertility-preserving strategies. The majority of patients will also receive chemotherapy consisting of platinum-based drugs, most often carboplatin, and a taxane, such as paclitaxel. Absence of residual disease after primary therapy is the most important prognostic indicator. This is achievable for most patients regardless of disease stage, but nearly all will invariably experience lethal chemotherapy-resistant disease. Treatment options at this stage are limited based on the patient's platinum-free period (the length of time between platinum drug treatments) and subsequent amenability of the disease to secondary cytoreductive surgery, but survival is still low. For patients who have undergone first-line standard of care, greater benefit has been demonstrated with the use of long-term maintenance therapy consisting of chemotherapy drugs or biologics given after freedom from residual disease has been achieved to prolong survival. FDA approval of bevacizumab and poly ADP-ribose polymerase (PARP) inhibitors has expanded the availability of maintenance therapy and improved progression-free survival; however, current clinical data have not demonstrated a significant increase in overall survival, and these drugs are associated with significant adverse events. Furthermore, there are no FDA-approved targeted immunotherapies for EOC, and clinical trials with checkpoint inhibitors are inconclusive. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a great need for novel anti-ovarian cancer drugs, including complementary therapies, to cure the disease, prolong progression, and improve patient survival. [Means for solving the problem]

[0007] The subject matter disclosed herein is based, in part, on the discovery that a fusion protein (AvFc) consisting of Avaren lectin and human IgG1 Fc targets a unique biomarker for epithelial ovarian cancer (EOC). AvFc is selective for oligomannose glycans, which make up a large proportion of the surface of cancer cells. AvFc mediates anti-cancer activity, including antibody-dependent cell-mediated cytotoxicity (ADCC).

[0008] In one aspect, the disclosure provides a method of killing ovarian cancer (OVCA) cells, the method comprising contacting an OVCA cell with a polypeptide comprising actinohibin or a variant thereof.

[0009] In another aspect, the disclosure provides a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in an OVCA cell, the method comprising contacting the OVCA cell with a polypeptide comprising actinohibin or a variant thereof.

[0010] In another aspect, the disclosure provides a method of treating OVCA in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising actinohibin or a variant thereof.

[0011] In another aspect, the disclosure provides for the use of a polypeptide in the manufacture of a medicament for treating OVCA in a subject in need thereof, wherein the polypeptide comprises actinohibin or a variant thereof, and the treatment comprises administering to the subject an effective amount of the polypeptide.

[0012] In another aspect, the disclosure provides a polypeptide for use in treating OVCA in a subject in need thereof, wherein the polypeptide comprises actinohibin or a variant thereof, and the treatment comprises administering to the subject an effective amount of the polypeptide.

[0013] In another aspect, the disclosure provides a method of reducing tumor size in a subject having OVCA, the method comprising administering to the subject an effective amount of a polypeptide comprising actinohibin or a variant thereof, wherein administration of the polypeptide reduces tumor size.

[0014] In another aspect, the disclosure provides for the use of a polypeptide in the manufacture of a medicament for reducing tumor size in a subject having OVCA, wherein the polypeptide comprises actinohibin or a variant thereof, and wherein treating comprises administering to the subject an effective amount of the polypeptide.

[0015] In another aspect, the disclosure provides a polypeptide for use in reducing tumor size in a subject having OVCA, wherein the polypeptide comprises actinohibin or a variant thereof, and wherein treating comprises administering to the subject an effective amount of the polypeptide.

[0016] In another aspect, the disclosure provides a method of treating EOC in a subject in need thereof, the method comprising administering to the subject an effective amount of a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:16.

[0017] In another aspect, the disclosure provides for the use of a polypeptide in the manufacture of a medicament for treating EOC in a subject in need thereof, wherein the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16, and the method comprises administering to the subject an effective amount of the polypeptide.

[0018] In another aspect, the disclosure provides a polypeptide for use in treating EOC in a subject in need thereof, wherein the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16, and the method comprises administering to the subject an effective amount of the polypeptide.

[0019] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments. [Brief description of the drawings]

[0020] [Figure 1A-1D] Figure 1A-1B shows the activity of Avaren-Fc (AvFc) against ovarian cancer (OVCA). Figures 1A-1B are immunohistochemistry of human epithelial ovarian cancer (EOC) tissue sections showing oligomannose-dependent binding of AvFc to human OVCA tissue. Figure 1A shows malignant and adjacent tissue stained with AvFc. The malignant tissue is strongly bound by AvFc, while the adjacent tissue shows little to no staining. AvFc clearly distinguishes the malignant tissue from the normal adjacent tissue, as seen by the level of DAB staining. Figure 1B shows malignant and adjacent tissue stained with the non-glycobinding mutant AvFclec-. No binding is seen in either case, indicating that AvFc binding to human OVCA tissue is oligomannose-dependent. Figure 1C shows AvFc binding to OVCA cell lines A2780, SKOV3, ID8, and ID8-VEGF-DEFB29, as determined by single-color flow cytometry. The Y-axis shows the percentage of FITC+, determined by gating against background fluorescence. Figure ID shows the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of AvFc against OVCA cell lines using a luciferase-based reporter cell line assay. AvFc potently induced ADCC against four cancer cell lines, with the highest activity seen against ID8 and ID8-VEGF-DEFB29 cell lines. [Diagram 2] Figure 1 shows binding of AvFc to mouse and human OVCA cell lines as determined by flow cytometry. Briefly, AvFc was incubated with each cell line at concentrations ranging from 1.3 to 130 nM, and then AvFc was detected with a goat anti-human Fc-FITC conjugate. The non-glycosylated mutant AvFclec- was used as a negative control. [Figure 3A-3B] Representative fluorescence micrographs of AvFc binding to the murine OVCA cell line ID8 (Figure 3A) and the human cell line A2780 (Figure 3B). [Figure 4] Mouse ID8 OVCA challenge model. Female C57bl / 6 mice were challenged intraperitoneally (ip) with 2 million ID8 cells per mouse. Starting 7 days after tumor challenge, mice were treated ip with either 25 mg / kg AvFc or vehicle control every 2 days for 28 days (days 7-35; 15 doses total) and survival was monitored after cessation of treatment until all animals died or reached euthanasia point (body weight >35 g). Survival curves were determined to be significantly different by long rank test (P=0.0048; GraphPad Prism 8 software). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Some aspects of the present disclosure are described below with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are described to provide a complete understanding of the present disclosure. However, one of ordinary skill in the art will readily recognize that the present disclosure can be practiced without one or more specific details, or can be practiced with other methods, protocols, reagents, cell lines, and animals. The present disclosure is not limited by the described order of acts or events, as some acts may occur in different orders and / or simultaneously with other acts or events. Furthermore, not all acts, steps, or events described are required to perform a methodology in accordance with the present disclosure. Many of the techniques and procedures described or referenced herein are well understood by those of ordinary skill in the art and are commonly used with conventional methodology.

[0022] definition It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology may be used.

[0024] When lists are presented, unless otherwise stated, each individual element of that list, and every combination of that list, should be understood to be a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0025] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0026] The conjunction term "and / or" between multiple elements described is understood to include both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any one of these options is understood to be within the meaning and thus meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more options is also understood to be within the meaning and thus meet the requirements of the term "and / or".

[0027] The transitional terms "comprising," "consisting essentially of," and "consisting of" are intended to connote their generally accepted meanings in patent language; i.e., (i) "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that "do not materially affect the basic and novel characteristics" of the claimed invention. Embodiments described with the phrase "comprising" (or its equivalent) also provide as embodiments those described independently with the phrases "consisting of" and "consisting essentially of."

[0028] "About" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless expressly stated otherwise in the examples or elsewhere in the specification in the context of a particular assay, result or embodiment, "about" means within one standard deviation, or within 5%, whichever is greater, as is customary in the art.

[0029] The term "polypeptide", "peptide" or "protein" refers to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A protein, peptide or polypeptide can contain any suitable L- and / or D-amino acids, including common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statins), and uncommon amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl and / or other functional groups on a peptide can be free (e.g., unmodified) or protected by suitable protecting groups. Suitable protecting groups for amino and carboxyl groups, as well as methods for adding or removing protecting groups, are known in the art and are disclosed, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, 1991. Functional groups of a protein, peptide, or polypeptide can also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorogen or hapten) using methods known in the art. A protein, peptide, or polypeptide can optionally contain one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclization modifications), N-methyl-α-amino group substitutions). In addition, a protein, peptide, or polypeptide can be an analog of a known and / or naturally occurring peptide, e.g., a peptide analog having conservative amino acid residue substitutions.

[0030] As used herein, the term "sequence identity" refers to the degree (expressed as a percentage) that two nucleotide sequences or two amino acid sequences have the same residues at the same positions when the sequences are aligned to achieve the maximum level of identity. For sequence alignment and comparison, one sequence is usually designated as a reference sequence to which a test sequence is compared. The sequence identity between a reference sequence and a test sequence is expressed as the percentage of positions where the reference sequence and the test sequence share the same nucleotide or amino acid over the entire length of the reference sequence when the reference sequence and the test sequence are aligned to achieve the maximum level of identity. As an example, if the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the entire length of the reference sequence when aligned to achieve the maximum level of identity, then the two sequences are considered to have 70% sequence identity.

[0031] Alignment of sequences for comparison to achieve maximum identity levels can be readily performed by one skilled in the art using appropriate alignment methods or algorithms. In some cases, the alignment can include gaps introduced to provide maximum identity levels. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally, Ausubel et al., Current Protocols in Molecular Biology).

[0032] When using sequence comparison algorithm, test sequence and reference sequence are inputted into computer, and if necessary, subsequent coordinates are designated, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the designated program parameters.The commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP), available from the National Library of Medicine, National Center for Biotechnology Information, National Institutes of Health, United States (Altschul et al., 1990).

[0033] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" are used interchangeably herein.

[0034] "Prevent," "preventing," "prevention," or "prophylaxis" of a disease or disorder means preventing the disorder from occurring in a subject.

[0035] "Responsive," "responsive," or "potential to respond" refers to any kind of improvement or positive response, whether detectable or undetectable, including alleviation or amelioration of one or more symptoms, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, prevention of spread of disease, delay or slowing of progression of disease, improvement or palliation of disease symptoms, and remission (partial or total).

[0036] "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the present disclosure is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, histidine, sodium chloride, and sucrose can be used to formulate the polypeptides of the present disclosure. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). For parenteral administration, the carrier can include sterile water, and other excipients may be added to enhance solubility or preservation. Injectable suspensions or solutions can also be prepared using aqueous carriers with appropriate additives. Suitable vehicles and formulations, including other human proteins such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, with particular reference to pages 958-989.

[0037] Exemplary embodiments are described below.

[0038] How to Kill Cancer Cells In one aspect, the disclosure provides a method of killing ovarian cancer (OVCA) cells, the method comprising contacting an OVCA cell with a polypeptide comprising actinohibin or a variant thereof.

[0039] In another aspect, the disclosure provides a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in an OVCA cell, the method comprising contacting the OVCA cell with a polypeptide comprising actinohibin or a variant thereof.

[0040] In some embodiments, contacting OVCA cells with the polypeptide results in an induction of ADCC of at least about 50% compared to baseline levels (e.g., before treatment), for example, an induction of ADCC of at least about: 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 11.5-fold, 12-fold, 12.5-fold, 13-fold, 13.5-fold, 14-fold, 14.5-fold, 15-fold, 15.5-fold, 16-fold, 16.5-fold, 17-fold, 17.5-fold, 18-fold, 18.5-fold, 19-fold, 19.5-fold, or 20-fold. In some embodiments, contacting OVCA cells with the polypeptide induces about 1.5-20 fold induction of ADCC compared to baseline levels, e.g., about: 1.5-19.5 fold, 2-19.5 fold, 2-19 fold, 2.5-19 fold, 2.5-18.5 fold, 3-18.5 fold, 3-18 fold, 3.5-18 fold, 3.5-17.5 fold, 4-17.5 fold, 4-17 fold, 4.5-16.5 fold, ... 16-fold, 5.5-16-fold, 5.5-15.5-fold, 6-15.5-fold, 6-15-fold, 6.5-15-fold, 6.5-14.5-fold, 7-14.5-fold, 7-14-fold, 7.5-14-fold, 7.5-13.5-fold, 8-13.5-fold, 8-13-fold, 8.5-13-fold, 8.5-12.5-fold, 9-12.5-fold, 9-12-fold, 9.5-12-fold, 9.5-11.5-fold, 10-11.5-fold, or 10-11-fold ADCC induction occurs.

[0041] Polypeptides In some embodiments, the polypeptide comprises a wild-type actinohibin amino acid sequence (e.g., SEQ ID NO: 1) or a variant thereof. Actinohibin is a carbohydrate-binding protein that exhibits anti-human immunodeficiency virus activity and was first identified and isolated from Streptomyces strain K97-0003 (Chiba et al., Biochem Biophys Res Commun. 282:595-601 (2001)).

[0042] In particular embodiments, the polypeptide comprises a wild-type actinohibin amino acid sequence (eg, SEQ ID NO:1).

[0043] In certain embodiments, a polypeptide comprises a variant of a wild-type actinohibin amino acid sequence (e.g., SEQ ID NO: 1). As used herein, the term "variant" refers to a polypeptide that comprises an amino acid sequence having at least about 70% sequence identity to a reference sequence, i.e., wild-type actinohibin.

[0044] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to at least one sequence set forth in SEQ ID NOs: 1-15. In particular embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to at least one sequence set forth in SEQ ID NOs: 1-13.

[0045] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 1. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.

[0046] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to at least one of the sequences set forth in SEQ ID NOs: 2-15. In particular embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to at least one of the sequences set forth in SEQ ID NOs: 2-13.

[0047] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 2. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0048] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 3. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3.

[0049] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 4. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

[0050] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 5. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0051] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 6. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6.

[0052] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 7. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7.

[0053] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 8. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:8.

[0054] In particular embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 9. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:9.

[0055] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 10. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:10.

[0056] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 11. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:11.

[0057] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 12. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:12.

[0058] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 13. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:13.

[0059] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 14. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:14.

[0060] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 15. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:15.

[0061] In some embodiments, the polypeptide is modified, for example, by "GASDALIE" and / or "GnGn" modifications. "GASDALIE" modifications refer to G236A / S239D / A330L / I332E mutations in the IgG1 Fc domain (see, e.g., Ahmed, AA, et al., Journal of structural biology 194(1):78-89(2016)). "GnGn" modifications refer to Fc glycan modifications to contain primarily terminal GlcNAc residues and lack plant-specific glycans (Strasser, R., et al., Plant Biotechnology Journal 6(4):392-402(2008)).

[0062] In some embodiments, the polypeptide further comprises a fragment crystallizable domain (Fc) of an antibody, a fragment antigen-binding domain (Fab) of an antibody, or a single chain variable fragment (scFv) of an antibody. In certain embodiments, the polypeptide further comprises a Fab. In particular embodiments, the polypeptide further comprises an scFv. In some embodiments, the polypeptide further comprises an Fc. In certain embodiments, the polypeptide comprises Avaren, a high mannose glycan-binding (actinomycete-derived, oligomannose-binding) lectin, and IgG1 Fc (fragment crystallizable region (Fc) of human immunoglobulin G1) ("lectibody" Avaren-Fc (AvFc)).

[0063] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 16. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0064] In certain embodiments, the Fc region of the polypeptide is modified to optimize pharmacokinetic (PK) and / or pharmacodynamic (PD) properties, such as M428L and N434S mutations for extended plasma half-life (MR Gaudinski, et al., PLoS Med. 15 (2018), e1002493).

[0065] In particular embodiments, the polypeptide is conjugated to a cytotoxic chemical, a DNA damaging agent, a radioisotope, or a combination thereof. In some embodiments, the polypeptide is conjugated to a cytotoxic chemical. In certain embodiments, the cytotoxic chemical comprises a tubulin inhibitor (e.g., a maytansinoid or an auristatin). In certain embodiments, the polypeptide is conjugated to a DNA damaging agent. In some embodiments, the DNA damaging agent comprises calicheamicin. In particular embodiments, the polypeptide is conjugated to a radioisotope. In certain embodiments, the radioisotope is lutetium-177.

[0066] In some embodiments, the polypeptide is produced by a plant, hi other embodiments, the polypeptide is produced in a mammalian cell (e.g., a CHO cell).

[0067] In certain embodiments, the polypeptide is an isolated polypeptide. An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that may interfere with diagnostic and / or therapeutic uses of the polypeptide, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. An "isolated" polypeptide encompasses polypeptides that have been isolated to greater degrees of purity, such as polypeptides that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0068] High-mannose glycan epitopes In certain embodiments, the polypeptide is highly selective for (or specifically binds to) malignant cells over non-cancerous or normal healthy cells. The term "specifically binding" or "specifically binds" refers to a preferential interaction, i.e., a significantly higher binding affinity, between a polypeptide and malignant cells compared to normal healthy cells.

[0069] In some embodiments, the binding affinity between the polypeptide and malignant OVCA cells (e.g., epithelial ovarian carcinoma (EOC) cells) is at least about 2-fold higher than normal healthy cells, e.g., at least about: 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10-fold higher. In certain embodiments, the binding affinity between the polypeptide and malignant OVCA cells (e.g., EOC cells) is at least about 10-fold higher than normal healthy cells, e.g., at least about: 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold higher. In particular embodiments, the binding affinity between the polypeptide and malignant OVCA cells (e.g., EOC cells) is about 2-10 times higher than normal healthy cells, e.g., about: 2.5-10, 2.5-9.5, 2.5-9, 3-9, 3-8.5, 3.5-8.5, 3.5-8, 4-8, 4-7.5, 4.5-7, 4.5-6.5, 5-6.5, 5-6 or 5.5-6 times higher. In some embodiments, the binding affinity between the polypeptide and malignant OVCA cells (e.g., EOC cells) is about 10-100 times higher than normal healthy cells, e.g., about: 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 0, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90 or 90-100 times higher.

[0070] In particular embodiments, the EC of a polypeptide of the present disclosure to OVCA cells (e.g., EOC cells) 50 is about 1 to 10 nM, for example, about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 to 9, 2 to 9, 2 to 8, 3 to 8, 3 to 7, 4 to 7, 4 to 6, or 5 to 6 nM.

[0071] In certain embodiments, the polypeptide specifically binds to a high mannose glycan epitope.

[0072] "Epitope" refers to the portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes may be composed of contiguous amino acids and / or discontinuous amino acids that form a conformational space unit. In discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space due to folding of the protein molecule.

[0073] The term "high mannose glycan" refers to asparagine-linked glycans (N-glycans) that contain 5-9 terminal mannose residues attached to a chitobiose (GlcNAc2) core. High mannose glycans are formed in the endoplasmic reticulum of eukaryotic cells and are attached to newly synthesized nascent polypeptides that contain an asparagine-X-serine / threonine sequence (where X can be any amino acid except proline). These glycans are then typically processed as the nascent polypeptide undergoes a secondary pathway through the Golgi apparatus, maturing into complex glycans that contain fewer mannose residues. As a result, few high mannose glycans remain attached to proteins that appear on the surface of healthy normal cells. However, abnormally high levels of high mannose glycans are often found on cell surface and secreted proteins produced by malignant cells.

[0074] Non-limiting examples of high mannose glycans include Man9GlcNAc2 (Man9), Man8GlcNAc2 (Man8), Man7GlcNAc2 (Man7), Man6GlcNAc2 (Man6), and Man5GlcNAc2 (Man5).

[0075] In some embodiments, the high mannose glycan epitope is OVCA-associated (eg, EOC-associated).

[0076] In particular embodiments, the high mannose glycan epitope comprises one or more terminal α1,2 linked mannose residues.

[0077] In particular embodiments, the polypeptide specifically binds to two or more high mannose type glycan epitopes, for example, three, four, five or more high mannose type glycan epitopes.

[0078] Highly glycosylated proteins In some embodiments, the polypeptide specifically binds to a hyperglycosylated protein.

[0079] In certain embodiments, the hyperglycosylated protein comprises at least about 10 N-glycosylation sites, such as at least about: 11, 12, 13, 14, 15, 16, 17, or 18 N-glycosylation sites. In particular embodiments, the hyperglycosylated protein comprises about 13-16 N-glycosylation sites.

[0080] In some embodiments, the polypeptide specifically binds to more than one hyperglycosylated protein, for example, 2, 3, 4, 5 or more hyperglycosylated proteins.

[0081] cancer cells In some embodiments, the OVCA cells are EOC cells.

[0082] In some embodiments, the OVCA cells (e.g., EOC cells) are in vitro cells. In certain embodiments, the OVCA cells (e.g., EOC cells) are ex vivo cells.

[0083] In some embodiments, the OVCA cell (e.g., EOC cell) is a cell of a subject (e.g., a human patient) described herein. In certain embodiments, the OVCA cell (e.g., EOC cell) is a mammalian cell, such as a cell from a dog, cat, mouse, rat, hamster, guinea pig, horse, pig, sheep, cow, chimpanzee, macaque, cynomolgus monkey, or human. In some embodiments, the OVCA cell (e.g., EOC cell) is a mouse cell. In some embodiments, the OVCA cell (e.g., EOC cell) is a primate cell. In particular embodiments, the OVCA cell (e.g., EOC cell) is a human cell.

[0084] In some embodiments, the OVCA cells (e.g., EOC cells) are cells from an adult human patient, e.g., 18-75 years of age, 18 years of age or older, or 40 years of age or older. In certain embodiments, the OVCA cells (e.g., EOC cells) are cells from a younger human patient. In some embodiments, the OVCA cells (e.g., EOC cells) are cells from a human patient who is 18 years of age or younger and / or 12 years of age or older. In particular embodiments, the OVCA cells (e.g., EOC cells) are cells from a pediatric human patient.

[0085] In some embodiments, the OVCA cells (e.g., EOC cells) are cells of a subject newly diagnosed with OVCA (e.g., EOC). In certain embodiments, the OVCA cells (e.g., EOC cells) are cells of a subject who has been diagnosed with OVCA (e.g., EOC) for at least about one month, at least about one year, at least about five years, or at least about ten years.

[0086] In certain embodiments, the OVCA cells (e.g., EOC cells) are cells of a subject (e.g., a human patient) who has not been previously treated. In particular embodiments, the OVCA cells (e.g., EOC cells) are cells of a subject who has previously undergone one or more anti-cancer therapies. In some embodiments, the OVCA cells (e.g., EOC cells) are cells of a subject with recurrent and / or refractory ovarian cancer.

[0087] In certain embodiments, OVCA cells (e.g., EOC cells) are characterized by abnormal surface accumulation of high mannose glycans. In some embodiments, cell surface high mannose glycans in OVCA cells (e.g., EOC cells) of the present disclosure are at least about 2-fold higher than in normal cells, e.g., at least about: 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold higher than in normal cells. In particular embodiments, about 2-10-fold higher than in normal cells, e.g., about: 2.5-10, 2.5-9.5, 2.5-9, 3-9, 3-8.5, 3.5-8.5, 3.5-8, 4-8, 4-7.5, 4.5-7, 4.5-6.5, 5-6.5, 5-6, or 5.5-6-fold higher than in normal cells.

[0088] In some embodiments, the OVCA cells (e.g., EOC cells) express a protein with an abnormal accumulation of high mannose glycans on its cell surface. In particular embodiments, the OVCA cells (e.g., EOC cells) are characterized by one or more tumor-associated glycobimarkers. In certain embodiments, the OVCA cells (e.g., EOC cells) are characterized by two or more tumor-associated glycobimarkers.

[0089] Treatment methods In another aspect, the disclosure provides a method of treating OVCA in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising actinohibin or a variant thereof.

[0090] In another aspect, the disclosure provides for the use of a polypeptide in the manufacture of a medicament for treating OVCA in a subject in need thereof, wherein the polypeptide comprises actinohibin or a variant thereof, and the treatment comprises administering to the subject an effective amount of the polypeptide.

[0091] In another aspect, the disclosure provides a polypeptide for use in treating OVCA in a subject in need thereof, wherein the polypeptide comprises actinohibin or a variant thereof, and the treatment comprises administering to the subject an effective amount of the polypeptide.

[0092] In another aspect, the disclosure provides a method of reducing tumor size in a subject having OVCA, the method comprising administering to the subject an effective amount of a polypeptide comprising actinohibin or a variant thereof, wherein administration of the polypeptide reduces tumor size.

[0093] In another aspect, the disclosure provides for the use of a polypeptide in the manufacture of a medicament for reducing tumor size in a subject having OVCA, wherein the polypeptide comprises actinohibin or a variant thereof, and wherein treating comprises administering to the subject an effective amount of the polypeptide.

[0094] In another aspect, the disclosure provides a polypeptide for use in reducing tumor size in a subject having OVCA, wherein the polypeptide comprises actinohibin or a variant thereof, and wherein treating comprises administering to the subject an effective amount of the polypeptide.

[0095] The polypeptide comprising actinohibin or a variant thereof can be any one of the polypeptides described herein.

[0096] In another aspect, the disclosure provides a method of treating OVCA (e.g., EOC) in a subject in need thereof, the method comprising administering to the subject an effective amount of a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:16.

[0097] In another aspect, the disclosure provides for the use of a polypeptide in the manufacture of a medicament for treating OVCA (e.g., EOC) in a subject in need thereof, wherein the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16, and the method comprises administering to the subject an effective amount of the polypeptide.

[0098] In another aspect, the disclosure provides a polypeptide for use in treating OVCA (e.g., EOC) in a subject in need thereof, wherein the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16, and the method comprises administering to the subject an effective amount of the polypeptide.

[0099] composition In some embodiments, the polypeptides described herein are provided in a composition, such as a pharmaceutical composition.

[0100] In some embodiments, the composition (e.g., pharmaceutical composition) further comprises one or more pharma- ceutically acceptable carriers, excipients, stabilizers, diluents, or tonifiers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharma- ceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Non-limiting examples of pharma- ceutically acceptable carriers, excipients, stabilizers, diluents, or isotonicity agents include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrins); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); non-ionic surfactants (e.g., Tween), PLURONICS, and the like. TM and polyethylene glycol (PEG).

[0101] In some embodiments, the compositions (e.g., pharmaceutical compositions) of the present disclosure are formulated for an appropriate administration schedule and route of administration. Non-limiting examples of routes of administration include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical. In some embodiments, the compositions (e.g., pharmaceutical compositions) of the present disclosure are stored in the form of an aqueous solution or a dried formulation (e.g., lyophilized).

[0102] In some embodiments, the composition is formulated to be administered by infusion (e.g., intravenous infusion) or injection (e.g., intramuscular, subcutaneous, intraperitoneal, or intratumoral injection). In certain embodiments, the composition is formulated to be administered by intravenous infusion. In some embodiments, the composition is formulated to be administered by intramuscular injection. In particular embodiments, the composition is formulated to be administered by subcutaneous injection. In some embodiments, the composition is formulated to be administered by intraperitoneal injection. In certain embodiments, the composition is formulated to be administered by intratumoral injection.

[0103] In some embodiments, the composition is formulated to be administered with one or more additional therapeutic agents as a combination therapy, non-limiting examples of which include chimeric antigen receptor (CAR)-expressing T cells (CAR-T cells), CAR-expressing natural killer cells (CAR-NK cells), CAR-expressing macrophages (CAR-M cells), chemotherapeutic agents, immune checkpoint inhibitors, T cell redirectors, radiation therapy, surgery, and standard of care drugs.

[0104] In certain embodiments, the surgery comprises a total abdominal hysterectomy, a bilateral salpingo-oophorectomy, or an omentectomy.

[0105] In some embodiments, the chemotherapeutic agent comprises cisplatin.

[0106] In certain embodiments, the chemotherapy includes a platinum-based drug (eg, carboplatin) and a taxane (eg, paclitaxel).

[0107] "Concurrent administration," "administration with," "administration in combination with," "in combination with," and the like are intended to encompass the administration of selected therapies or drugs to a single patient, and include treatment regimens in which the therapies or drugs are administered by the same or different routes of administration or at the same or different times.

[0108] Pharmaceutical compositions (or pharmaceutical combinations), which refer to the product resulting from combining a polypeptide comprising actinohibin or a variant thereof with one or more additional therapeutic agents, include both fixed and non-fixed combinations.

[0109] A "fixed combination" refers to a single pharmaceutical composition comprising two or more compounds, e.g., a polypeptide comprising actinohibin or a variant thereof and one or more additional therapeutic agents, which are administered simultaneously in the form of a single entity or dosage form. In some embodiments, pharmaceutical compositions comprising a polypeptide comprising actinohibin or a variant thereof and one or more additional therapeutic agents are provided as a fixed combination.

[0110] A "non-fixed combination" refers to a pharmaceutical composition comprising one or more compounds, each of which is a separate entity, e.g., a polypeptide comprising actinohibin or a variant thereof and one or more additional therapeutic agents, administered simultaneously, in parallel, or sequentially without any specific time limit, where such administration provides an effective level of the two or more compounds in the subject's body. In some embodiments, a pharmaceutical composition comprising a polypeptide comprising actinohibin or a variant thereof and one or more additional therapeutic agents is provided as a non-fixed combination.

[0111] In some embodiments, a polypeptide (e.g., AvFc) is systemically administered to a subject at about 10-50 mg / kg, e.g., about: 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg, or about: 10-45 mg / kg, 15-45 mg / kg, 15-40 mg / kg, 20-40 mg / kg, 20-35 mg / kg, 25-35 mg / kg, or 25-30 mg / kg. In certain embodiments, a polypeptide (e.g., AvFc) is administered systemically to a subject at about 10-50 mg / kg about every 2-7 days (e.g., about every 2, 3, 4, 5, 6, or 7 days) for about 2-10 weeks (e.g., about every 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or about every 2-9, 3-9, 3-8, 4-8, 4-7, 5-7, or 5-6 weeks). In particular embodiments, a polypeptide (e.g., AvFc) is administered systemically to a subject at about 25 mg / kg every other day (Q2D) for 14 or 20 days (for a total of 8 or 11 administrations, respectively). In some embodiments, a polypeptide (e.g., AvFc) is administered systemically to a subject at about 10-50 mg / kg every 7 days (Q7D) for 1-2 months.

[0112] In some embodiments, the polypeptide or pharmaceutical composition (e.g., AvFc) of the present disclosure is administered in combination with a second therapeutic agent. In certain embodiments, the second therapeutic agent is an antibody (e.g., a monoclonal antibody (mAb)). The antibody can target aspects of the cancer itself (e.g., tumor-associated antigens) or physiological / immunological processes (e.g., checkpoint inhibitors that prevent or delay T-cell anergy and apoptosis), or target endogenous VEGF to inhibit angiogenesis in tumors (e.g., bevacizumab). In particular embodiments, the second therapeutic agent comprises an adoptive immunotherapy agent, an antibody-drug conjugate, a chemotherapeutic agent, an immune checkpoint inhibitor, or a PARP inhibitor, or a combination thereof. Non-limiting examples of chemotherapy combinations include platinum-based drugs (e.g., carboplatin) and taxanes (e.g., paclitaxel). Non-limiting examples of immune checkpoint inhibitors include pembrolizumab (KEYTUDA®, Merck), durvalumab (IMFINZI®, Mediimmune / AstraZeneca), dostallimab (Tesaro), avelumab (Bavencio®, Merck KGaA / Pfizer), atezolizumab (TECENTRIQ®, Genentech / Roche), and nivolumab (OPDIVO®, Bristol-Myers Squibb). Non-limiting examples of PARP inhibitors include niraparib (ZEJULA®, Tesaro), olaparib (LYNPARZA®, AstraZeneca), and rucaparib (Rubraca®, Clovis Oncology). A non-limiting example of an antibody-drug conjugate is mirvetuximab soravtansine, which targets human folate receptor 1 (FOLR1). Non-limiting examples of adoptive immunotherapy are, for example, chimeric antigen receptor T cells (CAR-T cells) that target FOLR1, or cytokine-induced killer cells (CIK cells).

[0113] Ovarian cancer "Cancer" refers to an abnormal growth of cells that tend to proliferate in an uncontrolled manner and, in some cases, metastasize (spread) to other areas of the patient's body.

[0114] In some embodiments, the OVCA is EOC.

[0115] In some embodiments, OVCA (e.g., EOC) is characterized by one or more tumor-associated glycobimarkers. In certain embodiments, OVCA (e.g., EOC) is characterized by two or more tumor-associated glycobimarkers.

[0116] In certain embodiments, OVCA (e.g., EOC) is characterized by abnormal cell surface accumulation of high mannose glycans. In some embodiments, cell surface high mannose glycans in OVCA cells (e.g., EOC cells) of the present disclosure are about 2-10 fold higher than in normal cells.

[0117] In some embodiments, OVCA (eg, EOC) is characterized by cell surface expression of proteins accompanied by abnormal accumulation of high mannose glycans.

[0118] In particular embodiments, the OVCA (eg, EOC) is chemotherapy resistant.

[0119] subject The term "subject" refers to an animal (e.g., a mammal). In some embodiments, the subject is a mammal. In certain embodiments, the subject is a mammal selected from the group consisting of dog, cat, mouse, rat, hamster, guinea pig, horse, pig, sheep, cow, chimpanzee, macaque, cynomolgus monkey, and human. In some embodiments, the subject is a primate. In particular embodiments, the subject is a human.

[0120] The term "subject in need thereof" refers to a mammalian subject, preferably a human, diagnosed with or suspected of having a disease (e.g., OVCA, such as EOC) to which a polypeptide will or has been administered in accordance with the methods of the invention. A "subject in need thereof" includes subjects who already have an unwanted physiological change or disease and subjects prone to having a physiological change or disease.

[0121] Diagnosis may be performed by any method or technique known in the art. One of skill in the art will understand that a subject to be treated according to the present disclosure may have undergone standard testing or may, without testing, have been identified as being at risk due to the presence of one or more risk factors associated with the disease or condition.

[0122] In some embodiments, the subject is an adult patient. In certain embodiments, the subject is a juvenile patient. In particular embodiments, the subject is a pediatric patient.

[0123] In some embodiments, the subject is between 18 and 75 years of age. In certain embodiments, the subject is 40 years of age or older, e.g., at least: 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years of age.

[0124] In certain embodiments, the subject is 18 years of age or older, e.g., 18 to less than 40 years of age, 18 to less than 45 years of age, 18 to less than 50 years of age, 18 to less than 55 years of age, 18 to less than 60 years of age, 18 to less than 65 years of age, 18 to less than 70 years of age, 18 to less than 75 years of age, 40 to less than 75 years of age, 45 to less than 75 years of age, 50 to less than 75 years of age, 55 to less than 75 years of age, 60 to less than 75 years of age, 65 to less than 75 years of age, 40 years of age or older, 45 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, or 75 years of age or older.

[0125] In some embodiments, the subject is 18 years of age or younger, e.g., 0-18 years of age, 0-12 years of age, 0-16 years of age, 0-17 years of age, 2-12 years of age, 2-16 years of age, 2-17 years of age, 2-18 years of age, 3-12 years of age, 3-16 years of age, 3-17 years of age, 3-18 years of age, 4-12 years of age, 4-16 years of age, 4-17 years of age, 4-18 years of age, 6-12 years of age, 6-16 years of age, 6-17 years of age, 6-18 years of age, 9-12 years of age, 9-16 years of age, 9-17 years of age, 9-18 years of age, 12-16 years of age, 12-17 years of age, or 12-18 years of age.

[0126] In some embodiments, the subject is 12 years of age or older.

[0127] In certain embodiments, the subject is 2 years of age or older, e.g., at least: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years of age or older. In some embodiments, the subject is 4 years of age or older. In some embodiments, the subject is 5 years of age or older. In some embodiments, the subject is 6 years of age or older.

[0128] In some embodiments, the subject has been diagnosed with OVCA (e.g., EOC) for at least about 1 month, e.g., at least about: 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 30 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[0129] In a particular embodiment, the subject is newly diagnosed with OVCA (e.g., EOC). "Newly diagnosed" refers to a subject who has been diagnosed with OVCA (e.g., EOC) but has not yet received treatment for OVCA.

[0130] In certain embodiments, the subject is treatment naive.

[0131] In some embodiments, the subject has undergone one or more prior anti-cancer therapies. In particular embodiments, the prior anti-cancer therapies include surgery, one or more chemotherapeutic agents, checkpoint inhibitors, targeted anti-cancer therapies or kinase inhibitors, or any combination thereof. In certain embodiments, the surgery includes total abdominal hysterectomy, bilateral salpingo-oophorectomy, or omentectomy. In some embodiments, the chemotherapy includes platinum-based drugs (e.g., carboplatin) and taxanes (e.g., paclitaxel).

[0132] A major problem in current EOC management is the recurrence of chemotherapy-resistant tumors. In particular embodiments, the subject has undergone first-line therapy and has achieved a state of no residual disease.

[0133] In certain embodiments, a polypeptide of the present disclosure (e.g., AvFc) is administered as a supplement to, or in place of, existing maintenance therapy in the absence of disease recurrence. For example, the polypeptide (e.g., AvFc) is formulated in a solution for intravenous (iv) infusion or intraperitoneal (ip) injection in an outpatient setting and given to the patient after completion of primary therapy. Administration of the drug in an outpatient setting may allow for close monitoring of the patient.

[0134] In some embodiments, a polypeptide of the disclosure (e.g., AvFc) is administered as a supplement to, or instead of, an existing second line therapy in the event of disease recurrence. For example, the polypeptide (e.g., AvFc) is administered initially in an inpatient setting, followed by outpatient care for several weeks depending on the patient's condition, followed by maintenance therapy in an outpatient setting as needed.

[0135] In particular embodiments, the subject has relapsed or is refractory to treatment with one or more prior anti-cancer therapies. "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment prior to or at the start of treatment, or a refractory disease may become resistant during treatment. "Recurrent" refers to a recurrence of a disease or signs and symptoms of a disease after a period of improvement following prior treatment with a therapy. In some embodiments, the subject has recurrent ovarian cancer. In certain embodiments, the subject has refractory ovarian cancer. In particular embodiments, the subject has recurrent and refractory ovarian cancer.

[0136] treatment "Treat," "treating," or "treatment" of a disease or disorder such as OVCA refers to achieving one or more of the following: reducing the severity and / or duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who previously exhibits symptoms of the disorder.

[0137] A "therapeutically effective amount," "effective amount," or "effective dosage" is an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., treating, curing, inhibiting, or ameliorating a physiological response or condition). The maximum therapeutic effect does not necessarily occur in a single administration, but may occur only after a series of administrations. Thus, a therapeutically effective amount may be administered in one or more administrations. A therapeutically effective amount may vary according to factors such as the condition, age, and weight of the mammal, the mode of administration, and the ability of the therapy or combination of therapies to elicit a desired response in an individual.

[0138] In some embodiments, a therapeutically effective amount of a polypeptide is an amount sufficient to induce a cytotoxic effect, which in certain embodiments includes one or more Fc-mediated cytotoxic effects (e.g., ADCC).

[0139] In particular embodiments, the cytotoxic effect (e.g., ADCC) is induced (e.g., increased) by at least about 10%, e.g., by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the cytotoxic effect (e.g., ADCC) is induced by about 1-90%, e.g., by about: 1-85%, 5-85%, 5-80%, 10-80%, 10-75%, 15-75%, 15-70%, 20-70%, 20-65%, 25-65%, 25-60%, 30-60%, 30-55%, 35-55%, 35-50%, or 40-50%.

[0140] In particular embodiments, the cytotoxic effect (e.g., ADCC) is induced by at least about 100%, e.g., by at least about: 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold. In some embodiments, the cytotoxic effect (e.g., ADCC) is induced by about 1-15-fold, e.g., by about: 1-14-fold, 1.5-14-fold, 1.5-13-fold, 2-13-fold, 2-12-fold, 2.5-12-fold, 2.5-11-fold, 3-11-fold, 3-10-fold, 3.5-10-fold, 3.5-9-fold, 4-9-fold, 4-8-fold, 4.5-8-fold, 4.5-7-fold, 5-7-fold, or 5-6-fold.

[0141] In some embodiments, a therapeutically effective amount of a polypeptide is an amount sufficient to inhibit (e.g., slow and / or reduce) tumor growth. In certain embodiments, tumor growth is inhibited by at least about 10%, e.g., at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, tumor growth is inhibited by about 1-90%, e.g., about: 1-85%, 5-85%, 5-80%, 10-80%, 10-75%, 15-75%, 15-70%, 20-70%, 20-65%, 25-65%, 25-60%, 30-60%, 30-55%, 35-55%, 35-50%, or 40-50%.

[0142] In particular embodiments, a therapeutically effective amount is an amount sufficient to significantly reduce tumor burden, improve survival (e.g., prolong survival and / or increase the likelihood of survival), or both.

[0143] "Survival" refers to a patient remaining alive and can be estimated by the Kaplan-Meier method. "Survival" includes progression-free survival (PFS) and overall survival (OS).

[0144] "PFS" refers to the time from treatment to first disease progression or death. PFS can be assessed, for example, by Response Evaluation Criteria in Solid Tumors (RECIST). In some embodiments, PFS is extended by at least about 1 month, for example, at least about: 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, or 24 months, compared to a control.

[0145] "OS" refers to a patient remaining alive for a period of time, such as about: 1, 1.5, 2, 3, 4, 5, or 10 years from the start of treatment or from initial diagnosis. In some embodiments, OS is extended by at least about 1 month, e.g., at least about: 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, or 24 months, compared to a control.

[0146] In some embodiments, a therapeutically effective amount of a polypeptide is an amount sufficient to achieve freedom from residual disease (including any abdominal metastases), prevent recurrence of disease, or both.

[0147] diagnosis In certain embodiments, the method of treatment further comprises determining whether a biological sample from a subject in need thereof is characterized by a high mannose glycan epitope.

[0148] "Diagnosing" or "diagnosis" refers to a method of determining whether a subject suffers from a given disease or condition, or may develop a given disease or condition in the future, or is likely to respond to treatment for a previously diagnosed disease or condition, i.e., to stratify a patient population for likelihood of responding to treatment. Diagnosis is usually performed by a physician based on general guidelines for the disease to be diagnosed, or other criteria that indicate that the subject is likely to respond to a particular treatment.

[0149] In some embodiments, the method further comprises: a) providing a biological sample from a subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; Includes.

[0150] In some embodiments, the biological sample comprises an ovarian biopsy (e.g., an ovarian tumor biopsy). In certain embodiments, the biological sample comprises a blood sample.

[0151] Although example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the scope of the embodiments encompassed by the claims.

[0152] Embodiment SUMMARY OF THE DISCLOSURE A method of treating ovarian cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising an actinohibin variant.

[0153] The method of clause 1, wherein the ovarian cancer is epithelial ovarian cancer.

[0154] The method of clause 1 or 2, wherein the subject has undergone first-line therapy and has achieved no residual disease.

[0155] The method of clause 1 or 2, wherein the subject has recurrent and refractory ovarian cancer.

[0156] The method of clause 1 or 2, wherein the ovarian cancer is chemotherapy resistant.

[0157] 6. The method of any one of clauses 1-5, wherein the therapeutically effective amount is an amount sufficient to significantly reduce tumor burden, improve progression-free survival, improve overall survival, or a combination thereof.

[0158] 7. The method of any one of clauses 1-6, wherein the polypeptide is formulated for intravenous (IV) administration.

[0159] The method of any one of clauses 1-7, wherein the polypeptide is administered in combination with a second therapeutic agent.

[0160] 9. The method of any one of clauses 1-8, wherein the subject is a human.

[0161] The method of clause 9, wherein the polypeptide is administered in an outpatient clinical setting.

[0162] The method of clause 9, wherein the polypeptide is administered in a clinical inpatient setting.

[0163] 20. A method of killing ovarian cancer cells, comprising contacting the ovarian cancer cells with a polypeptide that comprises an actinohibin variant.

[0164] The method of clause 12, wherein the ovarian cancer cells are epithelial ovarian cancer cells.

[0165] 14. The method of clause 12 or 13, wherein the ovarian cancer cells are chemotherapy resistant.

[0166] 15. The method of any one of clauses 12-14, wherein the polypeptide induces killing of ovarian cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC).

[0167] 16. The method of any one of clauses 12-15, further comprising contacting the ovarian cancer cells with a second therapeutic agent.

[0168] 17. The method of any one of clauses 1-16, wherein the actinohibin variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-13, and optionally, the actinohibin variant comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0169] 18. The method of any one of clauses 1-17, wherein the polypeptide further comprises a fragment crystallizable domain (Fc) of an antibody, and optionally, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0170] 18. The method of any one of clauses 1-17, wherein the polypeptide further comprises a fragment antigen-binding domain of an antibody (Fab), or a single chain variable fragment of an antibody (scFv).

[0171] 20. The method of any one of clauses 1-19, wherein the polypeptide is modified or conjugated to a heterologous moiety, or a combination thereof. EXAMPLES

[0172] Ovarian cancer is the most lethal gynecologic cancer. Ovarian cancer (OVCA), particularly epithelial ovarian cancer (EOC), is the most lethal gynecologic cancer worldwide. Treatment of the disease includes a combination of surgery and chemotherapy with platinum-based drugs and taxanes. Most patients respond to first-line therapy, but nearly all will experience fatal recurrent disease (often chemotherapy resistant). Recent FDA approval of PARP inhibitors and bevacizumab as maintenance therapy has increased progression-free survival after first-line therapy but has provided little effect on overall survival.

[0173] The lack of novel or complementary second-line therapies, the lack of maintenance therapies that improve overall survival, and the growing evidence that high mannose glycans may be a unique druggable target for EOC provide a compelling argument for the proposed product. Aberrant glycosylation of cancer cell surfaces is a well-described phenomenon and is considered a hallmark of the disease. Recent advances in tumor glycobiology have demonstrated that various tumor types show increased levels of high mannose glycans on their surfaces and that these glycans may play a role in malignancy and metastasis. High mannose glycans arise early in the N-glycosylation pathway in the endoplasmic reticulum and are usually processed by mannosidases and glycosyltransferases before exiting the secretory pathway, and therefore are not usually found on the surface of cells under normal conditions. However, quantitative N-glycan analysis by mass spectrometry using formalin-fixed paraffin-embedded tissues shows that high mannose glycans are overexpressed on the surface of OVCA tumors. Furthermore, high mannose glycans have been shown to be significantly increased in membrane glycoproteins of EOC cell lines compared to noncancerous ovarian epithelial cells and may increase metastatic activity in SKOV3 cells. High mannose glycans may be a useful EOC biomarker and potentially a druggable target.

[0174] AvFc as a novel, first-in-class targeted therapy for EOC. Avaren-Fc is a potent antibody-like immunotherapeutic consisting of a high-mannose glycan-binding lectin fused to the Fc region of human IgG1, which is highly expressed and can be efficiently produced in Nicotiana benthamiana plants. The data described herein show that AvFc has particularly high selectivity for EOC tissues and can bind to and potently induce antibody-dependent cell-mediated cytotoxicity (ADCC) against several EOC cell lines (Figures 1A-1C). Furthermore, AvFc administration is well tolerated in multiple species, including mice (both immunocompetent and immunodeficient), rats, and rhesus monkeys, and is not cytotoxic or mitogenic to human peripheral blood mononuclear cells. Repeated systemic administration of AvFc has been shown to completely protect against hepatitis C viral load in a human liver chimeric mouse model without causing any discernible toxicity (hepatitis C virus also overexpresses high mannose glycans on its surface). Based on these results on manufacturability, efficacy, and safety, AvFc may offer a potential new option for EOC treatment by complementing or replacing existing therapies for primary, secondary, or maintenance use. Such a therapy, which can improve overall patient survival, could potentially change the paradigm of EOC management and introduce a new standard of care.

[0175] The majority of patients undergoing first-line therapy for EOC (surgery and chemotherapy), regardless of stage, will be free of residual disease and subsequently begin maintenance therapy. Currently, maintenance therapy for EOC consists of the PARP inhibitors rucaparib, olaparib, or niraparib and the angiogenesis inhibitor bevacizumab. Clinical trials with these drugs have demonstrated significant improvements in progression-free survival, but have not yet impacted overall survival. For example, the ARIEL3 trial of rucaparib showed that patients with BRCA-mutated EOC had a median progression-free survival of 16.6 months on rucaparib versus 5.4 months on the placebo control arm. Results for bevacizumab compared to chemotherapy are similar (median progression-free survival of 12.3 months versus 8.6 months). Neither study reported complete overall survival data, but an interim report of the OCEANS trial of bevacizumab showed no significant improvement (median overall survival 35.2 months with chemotherapy compared with 33.3 months with chemotherapy plus bevacizumab). In the event of failure of maintenance therapy and disease recurrence, the choice of second-line therapy depends on the platinum-free period and an assessment of the surgical resectability of the tumor mass for secondary cytoreduction surgery. In the case of platin-sensitive disease, second-line therapy includes platins, paclitaxel, liposomal doxorubicin, or gemcitabine. Unfortunately, recurrent EOC is in most cases chemoresistant, especially to platins. In this situation, several sequential single chemotherapy agents are used, including paclitaxel, liposomal doxorubicin, or gemcitabine, until the occurrence of subsequent disease progression or unacceptable toxicity, which is almost inevitable. Therefore, there is an urgent clinical need for new or complementary therapies to cure the disease, prolong progression-free survival and improve overall survival in patients.

[0176] Late-stage EOC patients face poor prognosis and limited treatment options. Novel maintenance therapies such as PARP inhibitors increase progression-free survival, but the benefit on overall survival has yet to be demonstrated. In addition, second-line therapies are limited and generally ineffective. Furthermore, a recent analysis showed that PARP inhibitors can be very expensive and may not be cost-effective as a maintenance therapy, with incremental cost-effectiveness ratios of $235,000 and $287,000 per year of progression-free survival. Therefore, any new therapeutic intervention that improves progression-free and overall survival would be welcomed.

[0177] As mentioned above, nearly all EOC patients will experience recurrence despite being free of residual disease after first-line therapy. Maintenance therapy can prolong the time to recurrence, but so far has little to no effect on survival, and second-line therapy is limited due to the prevalence of chemotherapy-resistant disease. Furthermore, there are no currently approved immunotherapies for EOC that target specific tumor biomarkers. Surgery, maintenance, and chemotherapy all demand great sacrifices from patients and healthcare providers alike, not only in terms of financial costs, but also physical and psychological costs. Therefore, novel therapies that can complement or replace current therapies to cure disease, extend progression-free survival, or improve overall survival in patients would be welcomed by the medical community.

[0178] The goal of this project is to develop novel treatments for ovarian cancer (OVCA).

[0179] Towards this end, a novel antibody-like molecule called Avaren-Fc (AvFc), consisting of the high mannose glycan-binding lectin Avaren fused to the Fc region of human IgG1, has been developed for the treatment of epithelial ovarian cancer. High mannose glycans are immature glycans found at an unusually high rate on the surface of ovarian cancer cells and therefore may be a unique druggable target. AvFc has shown high selectivity for ovarian cancer tissues and is able to bind to and induce antibody-dependent cell-mediated cytotoxicity (ADCC) against several ovarian cancer cell lines (both human and murine). Furthermore, Avaren-Fc is not cytotoxic or mitogenic and shows no obvious toxicity in animals.

[0180] Example 1. Activity of AvFc against OVCA Example 1 demonstrates that AvFc binds to human ovarian cancer tissue in a manner dependent on high mannose glycans. Immunohistochemistry (IHC) was performed on tissue arrays (US Biomax, Rockville, MD) containing three stage I HGSOC tissues from patients aged 48 (row a), 72 (row b), and 55 (row c) and three adjacent normal ovarian tissues (bottom) (Figures 1A-1B). Immunohistochemistry of human ovarian epithelial cancer tissue sections shows that malignant tissue is strongly bound by AvFc, while adjacent tissues show little to no staining (Figure 1A). Furthermore, the malignant and adjacent tissues were stained with the non-glycosylated mutant AvFc. lec- No binding was observed in either case when stained with β-lactam (Figure 1B). Thus, AvFc clearly distinguishes malignant tissue from normal adjacent tissue, and binding of AvFc to human ovarian cancer tissue is high-mannose glycan dependent.

[0181] Targeted immunotherapy has been quite successful against other types of cancer. Immunohistochemical analysis of AvFc binding to human high-grade serous ovarian cancer tissues showed that AvFc is highly selective for malignant tissue compared to adjacent non-malignant tissues (Figure 1A). Conversely, an AvFc mutant deficient in high mannose glycan binding activity (AvFc lec-) was unable to bind to the tissue (Figure 1B), indicating that AvFc binding is mediated by high-mannose glycans and that tissues from human patients are indeed coated with clusters of high-mannose glycans that can be distinguished by AvFc. This selectivity combined with the lack of toxicity seen in multiple animal models indicates that AvFc is sufficiently selective to be effective against tumor tissue, despite the fact that it does not target a specific tumorigenesis-promoting molecule (such as EGFR).

[0182] The binding of AvFc to several OVCA cell lines, including the human lines A2780 and SKOV3, and the mouse lines ID8 and ID8-VEGF-DEFB29, was assessed by fluorescent staining (Figures 3A-3B) and flow cytometry (Figures 1C and 2). AvFc bound to these cell lines in a dose-dependent manner, with saturation occurring at 13 nM (Figure 2). The high level of binding suggests that AvFc may be a potent inducer of ADCC against these cancer cell lines. Indeed, in a luciferase-based reporter cell ADCC assay, AvFc was able to induce ADCC and EC 50 The values ​​were in the low nanomolar range, and the maximum fold induction values ​​were 1.5-11.5-fold higher than baseline (Figure 1D and Figure 4). AvFc can have significant in vivo activity against OVCA, justifying the use of the ID8 murine OVCA model.

[0183] Example 2. Efficacy of AvFc in mice Female C57bl / 6 mice were challenged intraperitoneally (ip) with 2 million ID8 cells per mouse. Beginning 7 days after tumor challenge, mice were treated ip with either 25 mg / kg AvFc or vehicle control every 2 days for 28 days (days 7-35; 15 doses total) and survival was monitored after cessation of treatment until all animals died or reached euthanasia point (body weight >35 g). Survival curves (Figure 4) were determined to be significantly different by long rank test (P=0.0048; GraphPad Prism 8 software).

[0184] Additional in vivo data may be obtained to demonstrate the efficacy of AvFc in the ID8 mouse EOC model. The efficacy of AvFc against EOC may be evaluated using the mouse ID8 EOC challenge model in immunocompetent mice. This model is a standard model in the field of ovarian cancer research and has been used for the evaluation of therapeutic candidates. It may also be clinically relevant to test the efficacy of AvFc against human tumors, but currently available in vivo human tumor challenge models require the use of immunodeficient mice to successfully engraft and grow human-derived tumors in vivo. Here, the proposed primary mechanism of action of AvFc requires a functional immune system, so the proposed ID8 challenge study provides a suitable model.

[0185] Study 1-1: ID8 challenge experiment. 4x10 in PBS 6 ID8-luc cells can be implanted intraperitoneally on day 0. Intraperitoneal treatment with AvFc can begin on day 7 after implantation and continue for 28 days Q2D at dose levels of 25 mg / kg or 10 mg / kg. This is in combination with a non-HMG binding mutant version of AvFc, AvFc lec- , and cisplatin (5 mg / kg QW for 28 days), which has a history of efficacy in this model and can be used as a positive control. Disease progression can be monitored by injecting 150 mg / kg luciferin and measuring bioluminescence twice weekly using in vivo imaging of live animals, along with measuring abdominal circumference and body weight weekly. Animals can be euthanized when they reach 35 g or are moribund. After euthanasia, immune cells can be collected by peritoneal lavage and immunophenotyped by flow cytometry to assess whether AvFc influences immune cell composition in the tumor microenvironment.

[0186] The results of the ID8 loading model can be evaluated. AvFc is expected to significantly increase survival and significantly reduce tumor burden as determined by abdominal circumference and bioluminescence. This model can be used to evaluate the combination of AvFc treatment with cisplatin (which represents the standard of care chemotherapy for EOC and has been shown to provide efficacy in the proposed model) to test for any potential additive or synergistic effects.

[0187] Example 3. Optimization of AvFc Rationale: Changes to the Fc region of an antibody can alter its affinity for various Fc receptors (FcγR) and affect its PK / PD properties. The use of antibody-drug conjugates (ADCs) may further improve the therapeutic activity of AvFc.

[0188] Study 2-1: Evaluation of the impact of Fc modifications of AvFc on in vitro activity. The impact of Fc modifications, particularly GASDALIE and GnGn modifications, can be evaluated on the activity of AvFc using an in vitro reporter-based ADCC assay targeting several human ovarian cancer cell lines. The modified forms of AvFc can be compared to wild-type, plant-produced AvFc, and AvFc produced in CHO cells.

[0189] Study 2-2: Evaluation of the impact of Fc modifications of AvFc on in vivo activity. After evaluating the impact of Fc modifications in vitro, the changes can be confirmed using the ID8 loading model described in Study 1-1.

[0190] Study 2-3: Exploring the use of AvFc as an ADC carrier. ADCs based on AvFc and various OVCA chemotherapeutic drugs (such as paclitaxel) can be generated and their efficacy investigated using in vitro cytotoxicity assays and primary cell ADCC assays.

[0191] Milestone 3: Potential GLP toxicity studies and human studies may continue using the most efficacious form of AvFc.

[0192] Current chemotherapy drugs have yet to dramatically improve progression-free and overall survival in EOC patients, despite their significant costs. The use of novel first-in-class drugs targeting unique biomarkers such as AvFc has the potential to significantly improve patient outcomes. Complementing, synergizing with, or even completely replacing existing treatments could have a profound impact on the care of EOC patients.

[0193] The target population for AvFc includes patients diagnosed with any stage of EOC, who have completed first-line standard of care, and are free of residual disease. Currently, it is estimated that 67,000 people are living with OVCA in the United States, and globally, this number could reach 762,000. It was estimated that there were 22,240 new cases of OVCA diagnosed in the United States in 2018, with an overall average incidence rate of 11.8 per 100,000 people. EOC is by far the most common form of OVCA, constituting approximately 90% of all new OVCA diagnoses. Although the majority of these patients may be free of residual disease after first-line treatment, nearly all will experience disease recurrence within one year. The incidence and mortality of OVCA have been declining for the past several decades and are still declining, but this trend is weak and OVCA is projected to remain the leading cause of gynecologic cancer-related mortality in women for the foreseeable future.

[0194] AvFc is a first-in-class immunotherapeutic antibody-like molecule that is highly selective for OVCA-derived high mannose glycans and whose primary mechanism of action involves antibody-dependent cell-mediated cytotoxicity (ADCC). As a novel first-in-class agent that uses a unique biomarker (high mannose glycan), AvFc represents a new paradigm in OVCA immunotherapy. To date, there are no other high mannose glycan-binding agents in clinical or preclinical development for this indication, thus making AvFc stand out among the field of chemotherapeutics and biologics in development. Furthermore, the lack of specific tumor-associated antigens identified for OVCA means that AvFc is one of only a few immunotherapies in development that can directly bind and kill OVCA cells.

[0195] array SEQ ID NO:1 is the amino acid sequence of the wild-type actinohibin polypeptide. [ka]

[0196] SEQ ID NO:2 is the amino acid sequence of an actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 1. ASGTIRNAETGRLLDSNYDGAVYTLPANGGSYQRWTGPGDGTVRNAETGRLLDSNYDGAVYTLPANGGSYQKWTGPGDGTIQNAETGRLLDSNYDGAVYTLPANGGSYQKWTG (SEQ ID NO: 2)

[0197] SEQ ID NO:3 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant. ASGTIRNAETGRCLDSNYDGAVYTLPCNGGSYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYDGAVYTLPCNGGSYQKWTG (SEQ ID NO: 3)

[0198] SEQ ID NO:4 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 3. [ka]

[0199] SEQ ID NO:5 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 4. [ka]

[0200] SEQ ID NO:6 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 5. ASGTIRNAETGRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRVLDSNYNGNVYTLPANGGNYQKWTG (SEQ ID NO: 6)

[0201] SEQ ID NO:7 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 6. ASGTIRNAETGRCLDSNYDGNVYTLPCNGGSYQRWTGPGDGTVRNAETGRCLDSNYDGNVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYDGNVYTLPCNGGSYQKWTG (SEQ ID NO: 7)

[0202] SEQ ID NO:8 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 7. ASGTIRNAQTGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 8)

[0203] SEQ ID NO: 9 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 8 or Avaren (an actinohibin variant expressed in Nicotiana). ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 9)

[0204] SEQ ID NO:10 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 9. ASGTIRNAQTGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 10)

[0205] SEQ ID NO:11 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 10. ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 11)

[0206] SEQ ID NO:12 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 11. ASGTIRNAQTGRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAQTGRLLDSNYNGNVYTLPANGGNYQKWTGPGDGTIQNAQTGRVLDSNYNGNVYTLPANGGNYQKWTG (SEQ ID NO: 12)

[0207] SEQ ID NO:13 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 12. ASGTIRNAETGRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAETGRLLDSNYNGNVYTLPANGGNYQKWTGPGDGTIQNAETGRVLDSNYNGNVYTLPANGGNYQKWTG (SEQ ID NO: 13)

[0208] SEQ ID NO:14 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 13. ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYNGNVYTLPCNGGNYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 14)

[0209] SEQ ID NO:15 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter, designated herein as variant 14. ASGTIRNAETGRCLDSNYDGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGNVYTLPCNGGNYQKWTGPGDGTIQNAETGRCLDSNYDGNVYTLPCNGGNYQKWTG (SEQ ID NO: 15)

[0210] SEQ ID NO: 16 is an amino acid sequence comprising the actinohibin variant polypeptide (variant 8) of SEQ ID NO: 9 fused via a linker polypeptide to an amino acid sequence comprising the fragment crystallizable (Fc) region of immunoglobulin (Ig) G, referred to herein as AvFc. [ka]

[0211] References 1. Lengyel, E., Ovarian cancer development and metastasis.The American journal of pathology,2010.177(3):p.1053-1064. 2. Surveillance Research Program National Cancer Institute.SEER * Explorer:An interactive website for SEER cancer statistics[Internet].[cited 2020 April 2];Available from:seer.cancer.gov / explorer / . 3. Lheureux, S., et al., Epithelial ovarian cancer. The Lancet, 2019.393(10177):p.1240-1253. 4. Moore, K., et al., Maintenance olaparib in patients with newly diagnosed advanced ovarian cancer. New England Journal of Medicine, 2018.379(26):p.2495-2505. 5. Coleman,R.L.,et al.,Bevacizumab and paclitaxel-carboplatin chemotherapy and secondary cytoreduction in recurrent,platinum-sensitive ovarian cancer(NRG Oncology / Gynecologic Oncology Group study GOG-0213):a multicentre,open-label,randomised,phase 3 trial.The Lancet Oncology,2017.18(6):p.779-791. 6. Mirza,M.R.,et al.,Niraparib maintenance therapy in platinum-sensitive,recurrent ovarian cancer.New England Journal of Medicine,2016.375(22):p.2154-2164. 7. Aghajanian,C.,et al.,OCEANS:a randomized,double-blind,placebo-controlled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian,primary peritoneal,or fallopian tube cancer.Journal of clinical oncology,2012.30(17):p.2039. 8. Coleman,R.L.,et al.,Rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy(ARIEL3):a randomised,double-blind,placebo-controlled,phase 3 trial.The Lancet,2017.390(10106):p.1949-1961. 9. Hamanishi,J.,et al.,Safety and antitumor activity of anti-PD-1 antibody,nivolumab,in patients with platinum-resistant ovarian cancer.Journal of Clinical Oncology,2015.33(34):p.4015-4022. 10. Colombo,I.,et al.,Immunologic and genomic characterization of high grade serous ovarian cancer(HGSOC)in patients(pts)treated with pembrolizumab(Pembro)in the phase II INSPIRE trial.2017,American Society of Clinical Oncology. 11. Pearce,O.M.,Cancer glycan epitopes:biosynthesis,structure and function.Glycobiology,2018.28(9):p.670-696. 12. Oliveira-Ferrer,L.,K.Legler,and K.Milde-Langosch.Role of protein glycosylation in cancer metastasis.in Seminars in cancer biology.2017.Elsevier. 13. Loke,I.,et al.,Emerging roles of protein mannosylation in inflammation and infection.Molecular aspects of medicine,2016.51:p.31-55. 14. Everest-Dass,A.V.,et al.,N-glycan MALDI imaging mass spectrometry on formalin-fixed paraffin-embedded tissue enables the delineation of ovarian cancer tissues.Molecular & Cellular Proteomics,2016.15(9):p.3003-3016. 15. Zhang,X.,et al.,Discovery of specific metastasis-related N-glycan alterations in epithelial ovarian cancer based on quantitative glycomics.PLoS One,2014.9(2):p.e87978. 16. Anugraham,M.,et al.,Specific glycosylation of membrane proteins in epithelial ovarian cancer cell lines:glycan structures reflect gene expression and DNA methylation status.Molecular & Cellular Proteomics,2014.13(9):p.2213-2232. 17. Hamorsky,K.T.,et al.,Engineering of a Lectibody Targeting High-Mannose-Type Glycans of the HIV Envelope.Molecular Therapy,2019.27(11):p.2038-2052. 18. Dent,M.,et al.,Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model.Cellular and molecular gastroenterology and hepatology,2021.11(1):p.185-198. 19. Zhong,L.,et al.,Cost-Effectiveness of Niraparib and Olaparib as Maintenance Therapy for Patients with Platinum-Sensitive Recurrent Ovarian Cancer.J Manag Care Spec Pharm,2018.24(12):p.1219-1228. 20. Bray,F.,et al.,Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA:A Cancer Journal for Clinicians,2018.68(6):p.394-424. 21. Torre,L.A.,et al.,Ovarian cancer statistics,2018.CA:A Cancer Journal for Clinicians,2018.68(4):p.284-296. 22. Ghisoni,E.,et al.,Ovarian Cancer Immunotherapy:Turning up the Heat.International Journal of Molecular Sciences,2019.20(12):p.2927. 23. Richardson,D.L.,S.M.Seward,and K.N.Moore,Antibody Drug Conjugates in the Treatment of Epithelial Ovarian Cancer.Hematology / Oncology Clinics of North America,2018.32(6):p.1057-1071. 24. ImmunoGen Inc.ImmunoGen Announces Top-Line Results from Phase 3 FORWARD I Study of Mirvetuximab Soravtansine in Ovarian Cancer.2019 [cited 2020 April 13];Available from:http: / / investor.immunogen.com / news-releases / news-release-details / immunogen-announces-top-line-results-phase-3-forward-i-study. 25. Sertkaya,A.,et al.,Key cost drivers of pharmaceutical clinical trials in the United States.Clin Trials,2016.13(2):p.117-26. 26. Wei,H.,et al.,Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin.PloS one,2013.8(12):p.e84927-e84927. 27. Guo,Z.,et al.,PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer.PloS one,2014.9(2). 28. van Brummelen,E.M.,et al.,Antidrug antibody formation in oncology:clinical relevance and challenges.The oncologist,2016.21(10):p.1260. 29. Choi,Y.,et al.,Studying cancer immunotherapy using patient-derived xenografts(PDXs)in humanized mice.Experimental & Molecular Medicine,2018.50(8):p.99. 30. BCC Publishing.Therapies and Diagnostics for Ovarian Cancer:Global Markets to 2022.2018;Available from:www.bccresearch.com / market-research / healthcare / therapies-and-diagnostics-for-ovarian-cancer-report.html. 31. Chiba H.et al.,Actinohivin,a novel anti-HIV protein from an actinomycete that inhibits syncytium formation:isolation,characterization,and biological activities.Biochem Biophys Res Commun.2001 Mar 30;282(2):595-601. 32. Ahmed AA et al.,Structural characterization of GASDALIE Fc bound to the activating Fc receptor FcγRIIIa.J Struct Biol.2016 Apr;194(1):78-89. 33. Strasser R et al., Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J.2008 May;6(4):392-402. 34. Gaudinski et al.,VRC 606 Study Team.Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS:A Phase 1 open-label clinical trial in healthy adults.PLoS Med.2018 Jan 24;15(1):e1002493.

[0212] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition for use in a method of treating ovarian cancer in a subject in need thereof, said pharmaceutical composition comprising a polypeptide comprising actinohibin or a variant thereof, said method comprising administering a therapeutically effective amount of said pharmaceutical composition to the subject.

2. The pharmaceutical composition of claim 1 , wherein the ovarian cancer is epithelial ovarian cancer.

3. The pharmaceutical composition of claim 1, wherein the subject has undergone first-line therapy and achieved a state of no residual disease.

4. The pharmaceutical composition of claim 1 , wherein the subject has recurrent or refractory ovarian cancer.

5. The pharmaceutical composition of claim 1 , wherein the ovarian cancer is chemotherapy resistant.

6. 2. The pharmaceutical composition of claim 1, wherein the therapeutically effective amount is an amount sufficient to reduce tumor burden, improve progression-free survival, improve overall survival, or a combination thereof.

7. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for intravenous (i.v.) administration.

8. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

9. The pharmaceutical composition of claim 1 , wherein the subject is a human.

10. The pharmaceutical composition of claim 9 , wherein the pharmaceutical composition is administered in an outpatient clinical setting.

11. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is administered in a clinical inpatient setting.

12. A pharmaceutical composition for use in a method of killing ovarian cancer cells, the pharmaceutical composition comprising a polypeptide comprising actinohibin or a variant thereof, the method comprising contacting the ovarian cancer cells with the pharmaceutical composition.

13. The pharmaceutical composition of claim 12, wherein the ovarian cancer cells are epithelial ovarian cancer cells.

14. The pharmaceutical composition of claim 12, wherein the ovarian cancer cells are chemotherapy resistant.

15. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition induces the killing of the ovarian cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC).

16. The pharmaceutical composition of claim 12, wherein the method further comprises contacting the ovarian cancer cells with a second therapeutic agent.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 15, and optionally the actinohibin variant comprises the amino acid sequence set forth in SEQ ID NO:

9.

18. 18. The pharmaceutical composition of claim 17, wherein the polypeptide further comprises a fragment crystallizable domain (Fc) of an antibody, and optionally, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

16.

19. 18. The pharmaceutical composition of claim 17, wherein the polypeptide further comprises a fragment antigen-binding domain of an antibody (Fab), or a single chain variable fragment of an antibody (scFv).

20. 18. The pharmaceutical composition of claim 17, wherein the polypeptide is modified or conjugated to a heterologous moiety, or a combination thereof.

Images