Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)

Definitions

• Ovarian cancer in particular, epithelial ovarian cancer (EOC), is one of the deadliest gynecological cancers, ranking fifth in cancer death among women.
• EOC typically begins as small, borderline epithelial tumors on either the surface of the ovary, the fallopian tubes, or the mesothelium lining of the peritoneal cavity. These tumors grow and become well differentiated before metastasizing, primarily to the abdominal cavity but rarely to the lungs, liver, and brain.
• NCI National Cancer Institute
• SEER End Results
• Treatment options at this stage are limited based on the platinum-free interval (the length of time between platinum drug treatments) of the patient and the amenability of the subsequent disease to secondary debulking surgery, though the likelihood of survival is poor regardless.
• long-term maintenance therapy which consists of chemotherapeutics or biologies given after no residual disease is 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 significant increases in overall survival and these drugs are associated with significant adverse events.
• PARP poly ADP ribose polymerase
• the subject matter disclosed herein is based, in part, on the discovery that a fusion protein consisting of Avaren lectin and human IgGl Fc (AvFc) targets a unique biomarker of epithelial ovarian cancer (EOC).
• AvFc is selective for oligomannose glycans overrepresented on the surface of cancer cells.
• AvFc mediates anti-cancer activities including antibody-dependent cell-mediated cytotoxicity (ADCC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• the disclosure provides a method of killing an ovarian cancer (OVCA) cell, the method comprises contacting the OVCA cell with a polypeptide comprising an actinohivin or a variant thereof.
• the disclosure provides a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in an OVCA cell, the method comprises contacting the OVCA cell with a polypeptide comprising an actinohivin or a variant thereof.
• ADCC antibody-dependent cell-mediated cytotoxicity
• the disclosure provides a method of treating OVCA in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a polypeptide comprising an actinohivin or a variant thereof.
• the disclosure provides use of a polypeptide in manufacture of a medicament for treating OVCA in a subject in need thereof, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• the disclosure provides a polypeptide for use in treating OVCA in a subject in need thereof, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• the disclosure provides a method of reducing tumor size in a subject having OVCA, the method comprises administering to the subject an effective amount of a polypeptide comprising an actinohivin or a variant thereof, wherein the administering of the polypeptide reduces the tumor size.
• the disclosure provides use of a polypeptide in manufacture of a medicament for reducing tumor size in a subject having OVCA, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• the disclosure provides a polypeptide for use in reducing tumor size in a subject having OVCA, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• the disclosure provides a method of treating EOC in a subject in need thereof, the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 16.
• the disclosure provides use of a polypeptide in 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 wherein the method comprises administering to the subject an effective amount of the polypeptide.
• 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 wherein the method comprises administering to the subject an effective amount of the polypeptide.
• FIGs. 1A-1D show activity of Avaren-Fc (AvFc) against ovarian cancer (OVCA).
• FIGs. 1A-1B are immunohistochemistry of human epithelial ovarian cancer (EOC) tissue sections demonstrating oligomannose-dependent binding of AvFc to human OVCA tissue.
• EOC epithelial ovarian cancer
• FIG. 1A shows malignant and adjacent tissue stained with AvFc. Little to no staining is seen in the adjacent tissue while the malignant tissue is highly bound by AvFc. AvFc clearly delineates malignant from normal adjacent tissue as seen by the level of DAB staining.
• FIG. IB shows malignant and adjacent tissue stained with a non-sugar-binding mutant, AvFc lec . No binding is seen in either case, indicating that AvFc’s binding to human OVCA tissue is oligomannose- dependent.
• FIG. 1C shows binding of AvFc to OVCA cell lines A2780, SKOV3, ID8, and ID8- VEGF-DEFB29 as determined by single color flow cytometry.
• FIG. ID shows antibody- dependent cell-mediated cytotoxicity (ADCC) activity of AvFc against OVCA cell lines with a luciferase-based reporter cell line assay. AvFc potently induces ADCC against all 4 cancer cell lines with the highest degrees of activity seen against the ID8 and ID8-VEGF-DEFB29 cell lines.
• FIG. 2 shows binding of AvFc to murine and human OVCA cell lines determined by flow cytometry.
• AvFc was incubated with each cell line at concentrations ranging from 1.3 to 130 nM, followed by detection of AvFc with a goat antihuman Fc-FITC conjugate.
• a non- sugar-binding mutant, AvFc lec was used as a negative control.
• FIGs. 3A-3B are representative fluorescent micrographs of AvFc binding to the murine OVCA cell line ID8 (FIG. 3A) and the human cell line A2780 (FIG. 3B).
• FIG. 4 shows mouse ID8 OVCA challenge model.
• “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.
• polypeptide “peptide” or “protein” denotes a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g ., glycosylation or phosphorylation).
• a protein, peptide or polypeptide can comprise any suitable L-and/or D-amino acid, for example, common a-amino acids (e.g., alanine, glycine, valine), non-a-amino acids (e.g, b-alanine, 4-aminobutyric acid, 6- aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g, citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine).
• the amino, carboxyl and/or other functional groups on a peptide can be free ( e.g ., unmodified) or protected with a suitable protecting group.
• Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, “Protecting Groups in Organic Synthesis, ” John Wiley and Sons, 1991.
• the 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 a hapten) using methods known in the art.
• a protein, peptide or polypeptide can comprise one or more modifications (e.g, amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g, cyclizing modifications), A-methyl-a-amino group substitution), if desired.
• modifications e.g, amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g, cyclizing modifications), A-methyl-a-amino group substitution
• a protein, peptide or polypeptide can be an analog of a known and/or naturally-occurring peptide, for example, a peptide analog having conservative amino acid residue substitution(s).
• sequence identity refers to the extent to which two nucleotide sequences, or two amino acid sequences, have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage.
• sequence alignment and comparison typically one sequence is designated as a reference sequence, to which a test sequences are compared.
• sequence identity between reference and test sequences is expressed as the percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity.
• two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, 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.
• Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm.
• the alignment can include introduced gaps to provide for the maximal level of identity. 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.
• test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• a commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul etal ., 1990).
• Subject includes any human or nonhuman animal.
• Nonhuman animal includes all vertebrates, e.g. , mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
• the terms “subject” and “patient” are used interchangeably herein.
• Prevent means preventing that a disorder occurs in subject.
• “Responsive”, “responsiveness” or “likely to respond” refers to any kind of improvement or positive response, such as alleviation or amelioration of one or more symptoms, dimini shment of extent of disease, stabilized ( i.e ., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
• Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the disclosure is administered.
• vehicles may 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.
• histidine, sodium chloride, and sucrose may be used to formulate a polypeptide of the disclosure.
• These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g, filtration).
• the carrier may comprise sterile water and other excipients may be added to increase solubility or preservation.
• Injectable suspensions or solutions may also be prepared utilizing aqueous carriers along with appropriate additives.
• Suitable vehicles and formulations, inclusive of other human proteins, e.g, human serum albumin, are described, for example, in e.g, Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.
• the disclosure provides a method of killing an ovarian cancer (OVCA) cell, the method comprises contacting the OVCA cell with a polypeptide comprising an actinohivin or a variant thereof.
• OVCA ovarian cancer
• the disclosure provides a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in an OVCA cell, the method comprises contacting the OVCA cell with a polypeptide comprising an actinohivin or a variant thereof.
• ADCC antibody-dependent cell-mediated cytotoxicity
• contacting the OVCA cell with the polypeptide results in at least about 50% induction of ADCC compared to the baseline level (e.g. , pretreatment), for example, resulting in 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 induction of ADCC.
• contacting the OVCA cell with the polypeptide results in about
• I.5-20 fold induction of ADCC compared to the baseline level for example, 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, 5-16.5 fold, 5-16 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-
• the polypeptide comprises a wildtype actinohivin amino acid sequence (e.g, SEQ ID NO: 1) or a variant thereof.
• Actinohivin is a sugar-binding protein exhibiting an anti-human immunodeficiency virus activity, which was originally identified and isolated from the actinomycete K97-0003 strain (Chiba etal., Biochem Biophys Res Commun. 282:595-601 (2001)).
• the polypeptide comprises a wildtype actinohivin amino acid sequence (e.g, SEQ ID NO:l).
• the polypeptide comprises a variant of a wildtype actinohivin amino acid sequence (e.g ., SEQ ID NO: 1).
• the term “variant” refers to a polypeptide comprising an amino acid sequence that has at least about 70% sequence identity to a reference sequence, i.e ., a wild type actinohivin.
• 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:l-15.
• 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:l- 13.
• 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:l.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1.
• 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:2-15.
• 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:2- 13.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:2.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:3.
• 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 an amino acid sequence set forth in SEQ ID NO:4. [0053] 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 an amino acid sequence set forth in SEQ ID NO: 5.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:6.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:7.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 8.
• 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 an amino acid sequence set forth in SEQ ID NO:9.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 10.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 11.
• 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 an amino acid sequence set forth in SEQ ID NO: 12. [0061] 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 an amino acid sequence set forth in SEQ ID NO: 13.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 14.
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 15.
• the polypeptide is modified, e.g, with “GASDALIE” and/or “GnGn” modifications.
• GSDALIE modification refers to G236A/S239D/A330L/I332E mutations in the IgGl Fc domain (see, e.g, Ahmed, A. A., el al., Journal of structural biology 194(l):78-89 (2016)).
• GnGn modification refers to Fc glycan modifications to contain primarily terminal GlcNAc residues and lack plant-specific glycans (Strasser, R., el al., Plant Biotechnology Journal 6(4):392-402 (2008)).
• the polypeptide further comprises a fragment crystallizable domain of an antibody (Fc), a fragment antigen-binding domain of an antibody (Fab) or a single chain variable fragment of an antibody (scFv).
• the polypeptide further comprises an Fab.
• the polypeptide further comprises a scFv.
• the polypeptide further comprises an Fc.
• the polypeptide comprises the high-mannose glycan-binding (actinomycete-derived, oligomannose-binding) lectin Avaren and IgGl Fc (fragment crystallizable region (Fc) of human immunoglobulin Gl) (the “lectibody” Avaren-Fc (AvFc)).
• 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.
• the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16.
• the Fc region of the polypeptide is modified to optimize Pharmacokinetics (PK) and/or Pharmacodynamics (PD) properties, such as M428L and N434S mutations for extended plasma half-life (M.R. Gaudinski, et al ., PLoS Med. 15 (2016), el002493).
• PK Pharmacokinetics
• PD Pharmacodynamics
• the polypeptide is conjugated to a cytotoxic chemical, a DNA-damaging agent, a radioisotope or a combination thereof.
• the polypeptide is conjugated to a cytotoxic chemical.
• the cytotoxic chemical comprises a tubulin inhibitor (e.g ., a maytansinoid or an auristatin).
• the polypeptide is conjugated to a DNA-damaging agent.
• the DNA-damaging agent comprises a calicheamicin.
• the polypeptide is conjugated to a radioisotope.
• the radioisotope is lutetium-177.
• the polypeptide is plant-produced. In other embodiments, the polypeptide is produced in mammalian cells (e.g., CHO cells).
• 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 would interfere with diagnostic and/or therapeutic uses for the polypeptide, for example, enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• polypeptides that are isolated to a higher 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.
• the polypeptide is highly selective to (or specifically binds) malignant cells over noncancerous or normal healthy cells.
• the term “specifically binding” or “specifically binds” refers to preferential interaction, i.e ., significantly higher binding affinity, between the polypeptide and a malignant cell relative to a normal healthy cell.
• the binding affinity between the polypeptide and a malignant OVCA cell (e.g, an epithelial ovarian cancer (EOC) cell) relative to a normal healthy cell is at least about 2-fold higher, for example, 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.
• the binding affinity between the polypeptide and a malignant OVCA cell (e.g, an EOC cell) relative to a normal healthy cell is at least about 10-fold higher, for example, at least about: 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100-fold higher.
• the binding affinity between the polypeptide and a malignant OVCA cell (e.g, an EOC cell) relative to a normal healthy cell is about 2-10 times higher, for example 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.
• the binding affinity between the polypeptide and a malignant OVCA cell (e.g ., an EOC cell) relative to a normal healthy cell is about 10-100 times higher, for example, 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, 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.
• the EC50 of a polypeptide of the disclosure to an OVCA cell is about 1-10 nM, for example, about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-9, 2-9, 2-8, 3-8, 3-7, 4-7, 4-6 or 5-6 nM.
• the polypeptide specifically binds a high-mannose-type glycan epitope.
• Epitope refers to a portion of an antigen to which an antibody specifically binds.
• Epitopes typically 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.
• An epitope may be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come into close proximity in a three-dimensional space through the folding of the protein molecule.
• high-mannose-type glycan refers to asparagine-linked glycan (L -glycan) containing 5-9 terminal mannose residues attached to the chitobiose (GlcNAc2) core. High- mannose glycans are formed and attached to newly synthesized nascent polypeptides containing asparagine-X-serine/threonine sequences, where X can be any amino acid except for proline, in the endoplasmic reticulum of eukaryotic cells.
• glycans are then typically processed and matured into complex-type glycans containing fewer mannose residues as the nascent polypeptides undergo the secretory pathway through the Golgi apparatus.
• few high-mannose glycans remain attached to proteins that appear on the surface of healthy normal cells.
• unusually high-levels of high -mannose glycans are often found in cell-surface and secreted proteins produced by malignant cells.
• Non-limiting examples of high-mannose-type glycans include: Man9GlcNAc2 (Man 9), Man8GlcNAc2 (Man8), Man7GlcNAc2 (Man 7), Man6GlcNAc2 (Man6) and Man5GlcNAc2 (Man5).
• the high-mannose-type glycan epitope is OVCA-associated (e.g, EOC-associated).
• the high-mannose-type glycan epitope comprises one or more terminal al,2-linked mannose residues.
• the polypeptide specifically binds two or more high- mannose-type glycan epitopes, for example, 3, 4, 5 or more high-mannose-type glycan epitopes.
• the polypeptide specifically binds a highly glycosylated protein.
• the highly glycosylated protein comprises at least about 10 N-glycosylation sites, for example, at least about: 11, 12, 13, 14, 15, 16, 17 or 18 N- glycosylation sites. In particular embodiments, the highly glycosylated protein comprises about 13-16 N-glycosylation sites.
• the polypeptide specifically binds more than one highly glycosylated proteins, for example, 2, 3, 4, 5 or more highly glycosylated proteins.
• the OVCA cell is an EOC cell.
• the OVCA cell (e.g., EOC cell) is an in vitro cell. In certain embodiments, the OVCA cell (e.g., EOC cell) is an ex vivo cell.
• the OVCA cell is a cell of a subject described herein (e.g, a human patient).
• the OVCA cell e.g, EOC cell
• the OVCA cell is a mammalian cell, e.g, a cell from a dog, a cat, a mouse, a rat, a hamster, a guinea pig, a horse, a pig, a sheep, a cow, a chimpanzee, a macaque, a cynomolgus, or a human.
• the OVCA cell e.g, EOC cell
• the OVCA cell is a mouse cell.
• the OVCA cell is a primate cell.
• the OVCA cell e.g, EOC cell
• the OVCA cell (e.g, EOC cell) is a cell of an adult human patient, for example, 18-75 years of age, 18 years of age or older, or 40 years of age or older.
• the OVCA cell (e.g, EOC cell) is a cell of a juvenile human patient.
• the OVCA cell (e.g, EOC cell) is a cell of a human patient who is 18 years of age or younger, and/or 12 years of age or older.
• the OVCA cell (e.g, EOC cell) is a cell of a pediatric human patient.
• the OVCA cell (e.g ., EOC cell) is a cell of a subject newly diagnosed with OVCA (e.g., EOC).
• the OVCA cell (e.g, EOC cell) is a cell of a subject who has been diagnosed with OVCA (e.g, EOC) for at least about 1 month, at least about 1 year, at least about 5 years, or at least about 10 years.
• the OVCA cell (e.g, EOC cell) is a cell of an untreated subject (e.g, a human patient).
• the OVCA cell (e.g, EOC cell) is a cell of a subject who has received one or more prior anti-cancer therapies.
• the OVCA cell (e.g, EOC cell) is a cell of a subject who has relapsed and/or refractory ovarian cancer.
• the OVCA cell (e.g, EOC cell) is characterized by an abnormal surface accumulation of high-mannose glycans.
• cell surface high-mannose glycans on an OVCA cell (e.g, an EOC cell) of the disclosure are at least about 2 times higher than on a normal cell, for example, 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 times higher than on a normal cell.
• about 2-10 times higher than on a normal cell for example 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 than on a normal cell.
• the OVCA cell expresses a protein with an abnormal accumulation of high-mannose glycans on its cell surface.
• the OVCA cell e.g, EOC cell
• the OVCA cell is characterized by one or more tumor-associated glycobiomarkers.
• the OVCA cell is characterized by two or more tumor-associated glycobiomarkers.
• the disclosure provides a method of treating OVCA in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a polypeptide comprising an actinohivin or a variant thereof.
• the disclosure provides use of a polypeptide in manufacture of a medicament for treating OVCA in a subject in need thereof, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• the disclosure provides a polypeptide for use in treating OVCA in a subject in need thereof, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• the disclosure provides a method of reducing tumor size in a subject having OVCA, the method comprises administering to the subject an effective amount of a polypeptide comprising an actinohivin or a variant thereof, wherein the administering of the polypeptide reduces the tumor size.
• the disclosure provides use of a polypeptide in manufacture of a medicament for reducing tumor size in a subject having OVCA, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• the disclosure provides a polypeptide for use in reducing tumor size in a subject having OVCA, wherein the polypeptide comprises an actinohivin or a variant thereof, and wherein the treatment comprises administering to the subject an effective amount of the polypeptide.
• polypeptide comprising an actinohivin or a variant thereof may be any one of the polypeptides described herein.
• the disclosure provides a method of treating OVCA (e.g . , EOC) in a subject in need thereof, the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 16.
• the disclosure provides use of a polypeptide in 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 wherein the method comprises administering to the subject an effective amount of the polypeptide.
• OVCA e.g., EOC
• 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 wherein the method comprises administering to the subject an effective amount of the polypeptide.
• OVCA e.g, EOC
• the method comprises administering to the subject an effective amount of the polypeptide.
• a polypeptide described herein is provided in a composition, for example in a pharmaceutical composition.
• the composition (e.g, pharmaceutical composition) further comprises one or more pharmaceutically acceptable carriers, excipients, stabilizers, diluents or tonifiers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
• Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents ortonifiers 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 counter-ions (e.g, sodium), metal complexes (e.g, Zn-protein complexes); non-ionic surfactants (e.g, Tween), PLURONICSTM and polyethylene glycol (PEG).
• buffers e.g phosphate, citrate, histidine
• antioxidants e
• the composition (e.g, pharmaceutical composition) of the disclosure is formulated for a suitable administration schedule and route.
• administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous and topical, etc.
• the composition (e.g, pharmaceutical composition) of the disclosure is stored in the form of an aqueous solution or a dried formulation (e.g, lyophilized).
• the composition is formulated to be administered by infusion (e.g, intravenous infusion) or injection (e.g, intramuscular, subcutaneous, intraperitoneal or intratumoral injection).
• infusion e.g, intravenous infusion
• injection e.g, intramuscular, subcutaneous, intraperitoneal or intratumoral injection
• the composition is formulated to be administered by intravenous infusion.
• the composition is formulated to be administered by intramuscular injection.
• the composition is formulated to be administered by subcutaneous injection.
• the composition is formulated to be administered by intraperitoneal injection.
• the composition is formulated to be administered by intratumoral injection.
• the composition is formulated to be administered with one or more additional therapeutic agents as a combination therapy.
• additional therapeutic agents include a T cell expressing chimeric antigen receptor (CAR) (CAR-T cell), a natural killer cell expressing CAR (CAR-NK cell), a macrophage expressing CAR (CAR-M cell), a chemotherapeutic agent, an immune checkpoint inhibitor, a T- cell redirector, radiation therapy, surgery and a standard of care drug.
• the surgery comprises total abdominal hysterectomy, bilateral salpingo-oophorectomy, or omentectomy.
• the chemotherapeutic agent comprises cisplatin.
• the chemotherapy comprises a platinum-based drug (e.g, carboplatin) and ataxane (e.g, paclitaxel).
• a platinum-based drug e.g, carboplatin
• ataxane e.g, paclitaxel
• “in combination with” or the like encompass administration of the selected therapeutics or drugs to a single patient, and are intended to include treatment regimens in which the therapeutics or drugs are administered by the same or different route of administration or at the same or different time.
• composition referring to a product that results from combining a polypeptide that comprises an actinohivin or a variant thereof and one or more additional therapeutic agents includes both fixed and non-fixed combinations.
• “Fixed combination” refers to a single pharmaceutical composition comprising two or more compounds, for example, the polypeptide that comprises an actinohivin or a variant thereof and the one or more additional therapeutic agents are administered simultaneously in the form of a single entity or dosage.
• a pharmaceutical composition comprising the polypeptide that comprises an actinohivin or a variant thereof and the one or more additional therapeutic agents are provided as a fixed combination.
• Non-fixed combination refers to separate pharmaceutical compositions, wherein each comprises one or more compounds, for example, the polypeptide that comprises an actinohivin or a variant thereof and the one or more additional therapeutic agents are administered as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two or more compounds in the body of the subject.
• pharmaceutical composition comprising the polypeptide that comprises an actinohivin or a variant thereof and the one or more additional therapeutic agents are provided as a non-fixed combination.
• the polypeptide e.g ., AvFc
• the polypeptide is systemically administered to the subject at about 10-50 mg/kg, for example, at 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 at 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.
• the polypeptide e.g., AvFc
• polypeptide e.g, AvFc
• the polypeptide is systemically administered to the subject at about 25 mg/kg of every other day (Q2D) for 14 or 20 days (8 or
• the polypeptide e.g, AvFc
• the polypeptide is systemically administered to the subject at about 10-50 mg/kg of every 7 days (Q7D) for 1-2 months.
• a polypeptide or pharmaceutical composition of the disclosure e.g, AvFc
• the second therapeutic agent is an antibody (e.g, a monoclonal antibody (mAb)).
• the antibody can target an aspect of the cancer itself (such as a tumor-associated antigen) or a physiological/immunological process (such as checkpoint inhibitors, which prevent or delay T cell anergy and apoptosis), or targets endogenous VEGF and inhibits blood vessel formation in the tumor (e.g, bevacizumab).
• 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.
• a non limiting example of a chemotherapy combination comprises a platinum-based drug (e.g, carboplatin) and ataxane (e.g, paclitaxel).
• Non-limiting examples of immune checkpoint inhibitors include pembrolizumab (KEYTUDA ® , Merck), durvalumab (IMFINZI ® , Medimmune/AstraZeneca), dostarlimab (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), etc.
• a non limiting example of antibody-drug conjugates is mirvetuximab soravtansine, which targets the human folate receptor 1 (FOLR1).
• a non-limiting example of an adoptive immunotherapy is Chimeric Antigen Receptor T cells (CAR-T cells), e.g., targeting FOLR1 or Cytokine-Induced Killer cells (CIK cells).
• Cancer refers to an abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasize (spread) to other areas of a patient’s body.
• the OVCA is EOC.
• the OVCA (e.g, EOC) is characterized by one or more tumor- associated glycobiomarker. In certain embodiments, the OVCA (e.g, EOC) is characterized by two or more tumor-associated glycobiomarkers.
• the OVCA (e.g, EOC) is characterized by an abnormal cell- surface accumulation of high-mannose glycans.
• cell surface high- mannose glycans on an OVCA cell (e.g, an EOC cell) of the disclosure are about 2-10 times higher than on a normal cell.
• the OVCA (e.g, EOC) is characterized by cell-surface expression of a protein with an abnormal accumulation of high-mannose glycans.
• the OVCA e.g ., EOC
• the OVCA is chemo-resistant.
• the term “subject” refers to an animal (e.g., a mammal).
• the subject is a mammal.
• the subject is a mammal selected from the group consisting of a dog, a cat, a mouse, a rat, a hamster, a guinea pig, a horse, a pig, a sheep, a cow, a chimpanzee, a macaque, a cynomolgus, and a human.
• the subject is a primate.
• the subject is a human.
• subject in need thereof refers to a mammalian subject, preferably human, diagnosed with or suspected of having a disease (e.g, OVCA such as a EOC), whom will be or has been administered a polypeptide according to a method of the invention.
• a disease e.g, OVCA such as a EOC
• Subject in need thereof includes those subjects already with the undesired physiological change or disease as well as those subjects prone to have the physiological change or disease.
• Diagnosis may be performed by any method or technique known in the art.
• a subject to be treated according to the present disclosure may have been subjected to standard tests or may have been identified, without examination, as one at risk due to the presence of one or more risk factors associated with the disease or condition.
• the subject is an adult patient. In certain embodiments, the subject is a juvenile patient. In particular embodiments, the subject is a pediatric patient.
• the subject is 18-75 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 old.
• 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, 60 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.
• 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.
• the subject is 12 years of age or older.
• the subject is two years of age or older, for example, 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.
• 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.
• OVCA e.g ., EOC
• the subject is newly diagnosed with OVCA (e.g, EOC).
• 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 the OVCA.
• the subject is treatment naive.
• the subject has received one or more prior anti-cancer therapies.
• the one or more prior anti-cancer therapies comprises surgery, one or more chemotherapeutic agents, checkpoint inhibitors, targeted anti-cancer therapies or kinase inhibitors, or any combination thereof.
• the surgery comprises total abdominal hysterectomy, bilateral salpingo-oophorectomy, or omentectomy.
• the chemotherapy comprises a platinum-based drug (e.g, carboplatin) and a taxane (e.g, paclitaxel).
• a major issue in the current EOC management is the recurrence of chemo-resistant tumors.
• the subject has undergone primary therapy and achieved the no residual disease status.
• a polypeptide of the disclosure is administered complement to, or as a replacement for, existing maintenance therapies in the absence of disease recurrence.
• the polypeptide e.g, AvFc
• the polypeptide is formulated in a solution for intravenous (i.v.) infusion or intraperitoneal (i.p.) injection in an outpatient setting and is given to patients following completion of primary therapy.
• Administration of the drug in an outpatient clinic setting can also allow for close patient monitoring.
• a polypeptide of the disclosure e.g ., AvFc
• a polypeptide of the disclosure is administered as a complement to, or a replacement for, existing second-line therapies in the event of disease recurrence.
• the polypeptide e.g., AvFc
• the polypeptide is initially administered in an inpatient setting followed by administration in outpatient clinics for several weeks depending on patient condition, then followed by maintenance treatment in an outpatient setting as necessary.
• the subject is relapsed or resistant to treatment with one or more prior anti-cancer therapies.
• “Refractory” refers to a disease that does not respond to a treatment. A refractory disease can be resistant to a treatment before or at the beginning of the treatment, or a refractory disease can become resistant during a treatment. “Relapsed” refers to the return of a disease or the signs and symptoms of a disease after a period of improvement after prior treatment with a therapeutic.
• the subject has relapsed ovarian cancer.
• the subject has refractory ovarian cancer.
• the subject has relapsed and refractory ovarian cancer.
• Treat”, “treating” or “treatment” of a disease or disorder such as OVCA refers to accomplishing one or more of the following: reducing the severity and/or duration of the disorder, inhibiting worsening of symptoms characteristic of the disorder being treated, limiting or preventing recurrence of the disorder in subjects that have previously had the disorder, or limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the disorder.
• a therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g, treatment, healing, inhibition or amelioration of physiological response or condition, etc.).
• the full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
• a therapeutically effective amount may be administered in one or more administrations.
• a therapeutically effective amount may vary according to factors such as disease state, age, and weight of a mammal, mode of administration and the ability of a therapeutic, or combination of therapeutics, to elicit a desired response in an individual.
• the therapeutically effective amount of the polypeptide is sufficient to induce a cytotoxic effect.
• the cytotoxic effect comprises one or more Fc-mediated cytotoxic effects (e.g, ADCC).
• the cytotoxic effect e.g., ADCC
• the cytotoxic effect is induced (e.g., increased) by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.
• the cytotoxic effect (e.g, ADCC) is induced by about 1-90%, for example, 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%.
• the cytotoxic effect (e.g, ADCC) is induced by at least about 100%, for example, by at least about: 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,
• the cytotoxic effect (e.g, ADCC) is induced by about 1-15 folds, for example, by about: 1-14 folds, 1.5-14 folds, 1.5-13 folds, 2-13 folds, 2-12 folds, 2.5-12 folds,
• the therapeutically effective amount of the polypeptide is sufficient to inhibited (e.g, slowed and/or reduced) tumor growth.
• tumor growth is inhibited by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.
• tumor growth is inhibited by about 1-90%, for example, 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%.
• the therapeutically effective amount is sufficient to significantly decrease tumor burden, improve survival (e.g, extend survival and/or increase the likelihood of survival), or both.
• “Survival” refers to the patient remaining alive and can be estimated by the Kaplan- Meier method. “Survival” includes progression free survival (PFS) and overall survival (OS). [00147] “PFS” refers to the time from treatment to first disease progression or death. PFS can be assessed by, for example, Response Evaluation Criteria in Solid Tumors (RECIST). In some embodiments, the PFS is extended by at least about 1 month, for example, by 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. [00148] “OS” refers to the patient remaining alive for a defined period of time, such as about:
• the OS is extended by at least about 1 month, for example, by at least about: 1.5,
• the therapeutically effective amount of the polypeptide is sufficient to achieve no residual disease status (including any abdominal metastases), to prevent disease recurrence, or both.
• methods of treatment further comprise determining if a biological sample of the subject in need is characterized with high-mannose-type glycan epitopes.
• Diagnosing refers to methods to determine if a subject is suffering 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 prior diagnosed disease or condition, z.e., stratifying a patient population on likelihood to respond to treatment. Diagnosis is typically performed by a physician based on the general guidelines for the disease to be diagnosed or other criteria that indicate a subject is likely to respond to a particular treatment.
• the method further comprises: a) providing a biological sample from the subject; and b) determining presence or absence of an abnormal accumulation of the high- mannose glycan epitope in the biological sample.
• the biological sample comprises an ovarian biopsy (e.g. , an ovarian tumor biopsy).
• the biological sample comprises a blood sample.
• 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 actinohivin variant.
• a method of killing an ovarian cancer cell comprises contacting the ovarian cancer cell with a polypeptide comprising an actinohivin variant.
• polypeptide further comprises a fragment crystallizable domain of an antibody (Fc), and optionally, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16.
• Ovarian cancer is the deadliest gynecological cancer.
• Ovarian cancer in particular epithelial ovarian cancer (EOC)
• EOC epithelial ovarian cancer
• Treatment of this disease includes a combination of surgery and chemotherapy with platinum- based drugs and taxanes. While most patients respond to the first-line therapies, nearly all will experience fatal recurrent disease - most often chemoresistant.
• Recent FDA approval of PARP inhibitors and bevacizumab as maintenance therapies has increased the progression-free survival period after primary therapy but has had little effect on overall survival rates.
• High- mannose glycans occur early in the N-glycosylation pathway in the endoplasmic reticulum and are typically processed by mannosidases and glycosyltransferases prior to leaving the secretory pathway, and thus are not typically found on the surface of the cell under normal conditions.
• quantitative N-glycan analysis by mass spectrometry with formalin-fixed, paraffin- embedded tissues show that high-mannose glycans are overexpressed on the surface of OVCA tumors.
• high-mannose glycans were shown to be significantly elevated in the membrane glycoproteins of EOC cell lines compared to non-cancerous ovarian epithelial cells and may increase metastatic activity in SKOV3 cells.
• High-mannose glycans may be a useful EOC biomarker and a potentially druggable target.
• 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 IgGl that is highly expressed and can be efficiently produced in Nicotiana benthamiana plants. Data described herein indicate that AvFc has a particularly high selectivity for EOC tissue and can bind to and potently induce antibody-dependent cell-mediated cytotoxicity (ADCC) against several EOC cell lines (FIGs. 1A-1C).
• ADCC antibody-dependent cell-mediated cytotoxicity
• AvFc administration is well tolerated in multiple species including mice (both immunocompetent and deficient), rats, and rhesus macaques and is not cytotoxic or mitogenic to human peripheral blood mononuclear cells. It has been shown that repeated systemic administration of AvFc completely protected against hepatitis C virus challenge without causing any discernible toxicity in a human liver chimeric mouse model (hepatitis C virus also overexpresses high-mannose glycans on their surface). Based on these results concerning manufacturability, efficacy, and safety, AvFc may offer a powerful new option for EOC treatment by complementing or supplanting existing therapies for primary, secondary, or maintenance use. Such a therapy capable of improving overall survival in patients could potentially alter the paradigm of EOC management and introduce a new standard of care.
• second-line therapies include platins, paclitaxel, liposomal doxorubicin, or gemcitabine.
• Novel maintenance therapies like PARP inhibitors have increased progression-free survival but have yet to demonstrate benefit to overall survival. Additionally, second-line therapies are limited and generally ineffective. Furthermore, a recent analysis demonstrated that PARP inhibitors can be tremendously expensive and may not be cost-effective as maintenance therapies with incremental cost-effectiveness ratios of $235,000 and $287,000 per progression-free survival life-year. Therefore, any new intervention that improves progression-free survival and overall survival and would be welcomed.
• Avaren-Fc Avaren-Fc
• Avaren a novel antibody-like molecule called Avaren-Fc (AvFc)
• Avaren a novel antibody-like molecule called Avaren-Fc (AvFc)
• Avaren a novel antibody-like molecule fused to the Fc region of human IgGl
• High-mannose glycans are immature glycans that are found in unusually high proportions on the surface of ovarian cancer cells and thus may be a unique and druggable target.
• AvFc has shown high selectivity for ovarian cancer tissue and is capable of binding to and inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against a number of ovarian cancer cell lines (both human and mouse). Additionally, Avaren-Fc is not cytotoxic or mitogenic and displays no overt toxicity in animals.
• ADCC antibody-dependent cell-mediated cytotoxicity
• Example 1 demonstrates that AvFc binds to human ovarian cancer tissue in a high- mannose glycan-dependent manner.
• Immunohistochemistry was performed on a tissue array (US Biomax, Rockville, MD) (FIGs. 1 A-1B), which contained 3 Stage I HGSOC tissues from a 48-year-old (column a), 72-year-old (column b), and a 55-year-old patient (column c) and three adjacent normal ovary tissues (below).
• Immunohistochemistry of human ovarian epithelial cancer tissue sections shows that while malignant tissues were highly bound by AvFc, little to no staining was seen in the adjacent tissues (FIG. 1 A).
• an AvFc mutant deficient in high-mannose glycan-binding activity was incapable of binding to the tissue (FIG. IB), illustrating that the binding of AvFc is high-mannose glycan-mediated and that tissue from human patients is indeed covered 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 despite the fact that AvFc does not target a specific pro-tumorigenic molecule (like EGFR), it is selective enough for tumor tissues to be efficacious.
• Binding of AvFc to a number of OVCA cell lines was assessed by fluorescent staining (FIGs. 3A-3B) and flow cytometry (FIGs. 1C and 2). AvFc bound to these cell lines in a dose-dependent manner with saturation occurring at 13 nM (FIG. 2). The high level of binding suggests that AvFc would be a potent inducer of ADCC against these cancer cell lines.
• AvFc was capable of inducing ADCC with EC50 values in the low nanomolar range and with maximum fold induction values between 1.5-11.5- fold higher than the baseline (FIG. ID and FIG. 4). AvFc may have significant in vivo activity against OVCA and justifies the use the ID8 mouse OVCA model.
• Study 1-1 ID8 challenge experiments. 4xl0 6 ID8-luc cells in PBS will be implanted intraperitoneally on day 0. Intraperitoneal treatment with AvFc will begin on day 7 post implantation and continue Q2D for 28 days, with a dose level of 25 mg/kg or 10 mg/kg. This will be compared to the non-HMG-binding mutant version of AvFc, AvFc lec , as well as to 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 will be monitored through weekly measurements of abdomen circumference and body weight as well as twice weekly measurements of bioluminescence by injecting 150 mg/kg luciferin and using in vivo live animal imaging.
• Animals will be euthanized when reaching 35 g or when moribund. Following euthanasia, immune cells will be collected by peritoneal lavage, and immunophenotyping will be conducted by flow cytometry to assess if AvFc affects the composition of the immune cells in the tumor microenvironment.
• Results of the ID8 challenge model will be assessed.
• AvFc is expected to significantly increase survival and significantly decrease tumor burden as determined by abdominal circumference and bioluminescence.
• the model will be used to assess combination of AvFc treatment with cisplatin (which represents a 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.
• Study 2-1 Assessing impacts of AvFc Fc modifications on activity in vitro.
• the impacts of Fc modifications, in particular the GASDALIE and GnGn modifications, will be assessed on AvFc’s activity using an in vitro reporter-based ADCC assay targeting a number of human ovarian cancer cell lines.
• Modified forms of AvFc will be compared to wild type plant- produced AvFc as well as AvFc produced in CHO cells.
• Study 2-2 Assessing impacts of AvFc Fc modifications on activity in vivo. After assessing the impact of Fc modifications in vitro , the changes will be confirmed using the ID8 challenge model as described in Study 1-1.
• Study 2-3 Explore the use of AvFc as an ADC carrier. ADCs based on AvFc and various OVCA chemotherapeutics (such as paclitaxel) will be produced and their effects will be examined using in vitro cytotoxicity assays as well as primary cell ADCC assays.
• the target population for AvFc include patients diagnosed with any stage of EOC and who have completed the first-line standard of care, achieving no residual disease.
• In 2018 it was estimated that there were 22,240 new cases of OVCA diagnosed in the United States, with an average overall incidence of 11.8 per 100,000.
• EOC is by far the most common form of OVCA, making up around 90% of all new OVCA diagnoses.
• the vast majority of these patients will achieve no residual disease after first- line treatments, however nearly all of them will experience disease recurrence within a year. While the incidence and mortality rates of OVCA have decreased in the last few decades and are still trending downward, the trend is weak and OVCA is projected to be a major cause of gynecological cancer-related mortality for women for the foreseeable future.
• 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 include antibody-dependent cell-mediated cytotoxicity (ADCC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• SEQ ID NO: 1 is an amino acid sequence of a wild-type actinohivin polypeptide AS VTIRN AQTGRLLD SNYNGNVYTLP AN GGNY QRWT GPGDGT VRNAQTGRCLD SNYD GA V YTLP CN GGS Y QK WLF Y SN G YIQN VET GR VLD SN YN GN V YTLP AN GGN Y QK W YT G (SEQ ID NO: 1)
• SEQ ID NO:2 is an amino acid sequence of an actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant 1.
• SEQ ID NO: 3 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant ASGTIRN AET GRCLD SNYDGAV YTLPCNGGS Y QRWT GPGDGTVRNAET GRCLD SNYD GAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYDGAVYTLPCNGGSYQKWTG (SEQ ID NO:3)
• SEQ ID NO:4 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant
• SEQ ID NO: 5 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant
• SEQ ID NO: 6 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant
• SEQ ID NO: 7 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant 6
• SEQ ID NO: 8 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant 7.
• SEQ ID NO: 9 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant 8 or Avaren (actinohivin variant expressed in Nicotiana).
• SEQ ID NO: 10 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant
• SEQ ID NO: 11 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant
• SEQ ID NO: 12 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant 11 ASGTIRN AQTGRLLD SNYNGNVYTLP AN GGNY QRWT GPGDGT VRNAQTGRLLD SNYN GNVYTLPANGGNYQKWTGPGDGTIQNAQTGRVLDSNYNGNVYTLPANGGNYQKWTG (SEQ ID NO: 12)
• SEQ ID NO: 13 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant 12
• SEQ ID NO: 14 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant
• SEQ ID NO: 15 is an amino acid sequence of another actinohivin variant polypeptide made in accordance with the presently-disclosed subject matter and designated herein as variant
• SEQ ID NO: 16 is an amino acid sequence including the actinohivin variant polypeptide of SEQ ID NO:9 (Variant 8) fused, via a linker polypeptide, to an amino acid sequence comprising the fragment crystallizable (Fc) region of immunoglobulin (Ig) G, and referred to herein as AvFc.

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