Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5434—IL-12
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
  • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2800/00—Nucleic acids vectors
  • C12N2800/90—Vectors containing a transposable element

Definitions

• fusion polypeptides and preparations comprising such, specifically including, for example, IL-12- fusion polypeptides (e.g., as described in published International Patent Application WO2020/263399).
• provided technologies achieve efficient and effective production of preparations of a phosphorylated form of such fusion polypeptides.
• the present disclosure identifies the source of a problem with certain manufacturing technologies utilized to prepare relevant fusion polypeptides (e.g., fusion polypeptides in which a metal binding polypeptide is conjugated with an immunomodulatory domain, such as an IL-12 immunomodulatory domain, and in particular phosphorylated form(s) of such fusion polypeptide(s)).
• relevant fusion polypeptides e.g., fusion polypeptides in which a metal binding polypeptide is conjugated with an immunomodulatory domain, such as an IL-12 immunomodulatory domain, and in particular phosphorylated form(s) of such fusion polypeptide(s)
• an immunomodulatory domain such as an IL-12 immunomodulatory domain
• such provided methods may comprise a step of co-expressing in a host cell the fusion polypeptide with a kinase that phosphorylates it so that the fusion polypeptide and kinase are expressed in a ratio within a range of about 4:1 to about 10:1.
• a phosphorylation degree e.g., an average number of phosphate molecules per polypeptide
• methods according to the present disclosure provide (e.g., achieve production of preparation(s) of) a phosphorylated form of a fusion polypeptide having enhanced metal hydroxide (e.g., alum) conjugation.
• the present disclosure provides a surprising discovery that improved (e.g., relative to a method using an affinity chromatography capture step and/or using a host cell wherein in the expression ratio of the fusion polypeptide and kinase are not within the range of about 4:1 to about 10:1) methods of manufacturing a phosphorylated form of a fusion polypeptides can be developed.
• improved e.g., relative to a method using an affinity chromatography capture step and/or using a host cell wherein in the expression ratio of the fusion polypeptide and kinase are not within the range of about 4:1 to about 10:1
• the present disclosure demonstrates that typical methods of manufacturing phosphorylated form(s) of a fusion polypeptide via affinity chromatography do not achieve degree(s) of phosphorylation as described herein.
• affinity chromatography may be desirable, or even necessary, to effectively isolate phosphorylated form(s) of relevant fusion polypeptide(s)
• present disclosure surprisingly demonstrates that provided methods lacking any affinity chromatography step can achieved superior results.
• methods including affinity chromatography steps may have various disadvantages such as, for example, lengthy and/or expensive purification processes, typically involving multiple steps, which together can result significant losses in product recovery.
• the present disclosure demonstrates that provided methodologies can achieve improved product quality (e.g., reduced level of one or more unwanted species, e.g., product-related species), and/or reduced impurity loads (e.g., host cell protein, DNA) as compared with certain conventional methods, including methods that include one or more affinity chromatography steps.
• Page 2 of 144 11666457v1 Attorney Docket: 2013762-0017 [0008]
• the present disclosure provides particularly useful methods that achieve producing of high purity preparations of phosphorylated fusion polypeptide as described herein.
• Provided technologies provide improvements over and/or solve problems associated with, various conventional manufacturing technologies.
• provided manufacturing methods do not include an affinity chromatography capture step; alternatively or additionally, in some embodiments, provided manufacturing methods (e.g., specifically provided purification methods) include one or more purification steps selected from an anion chromatography capture step; and a hydrophobic interaction chromatography polishing step.
• provided technologies involve an anion chromatography step followed (e.g., following immediately) by a hydrophobic interaction step. In some embodiments, a hydrophobic interaction step is followed by a second anion chromatography step.
• such provided technologies are applied to cell extracts, for example as may be prepared from engineered mammalian cells as provided by the present disclosure (e.g., engineered mammalian cells that express a fusion polypeptide as described herein and a kinase that phosphorylates such fusion polypeptide, such as engineered mammalian cells that express the fusion polypeptide and kinase within a range of about 4:1 to about 10:1, e.g., at a ratio of about 8:1.
• Figure 1 provides an exemplary schematic of fusion polypeptide metal- hydroxide complexes of the present disclosure.
• FIG. 2 provides a diagram of an exemplary fusion polypeptide of the present disclosure comprising a first (p40) and second (p35) IL12 immune agonist moieties and a metal-hydroxide binding polypeptide with a plurality of phosphorylation sites.
• ABP alum-binding peptides.
• Figures 3A-C show viability (A) and viable cell density (VCD) (B) during 1L fed-batch run of stable pool.
• FIG. 4A-B show viability (A) and viable cell density (B) during 1L fed- batch run of stable pools representing different ratios of IL12 fusion protein to FAM20C.
• FIG. 5 shows productivity (titer) during 1L fed-batch run of stable pools representing different ratios of IL12 fusion protein to FAM20C assessed by the bio-layer interferometry (BLI) method.
• Figure 6 shows viability of stable pool 6880 P1 (8:1) after transfection and corresponding recovery.
• Figures 7A-C show (A) viability, (B) viable cell density, and (C) titer during 1L fed-batch culture of stable pool 6880 P1 (8:1).
• Figure 8 shows entire chromatogram and zoom of elution peak during first chromatography of stable pool 6880 P1 clarified harvest by Anion Exchange Chromatography (GigaCap Q).
• Figure 9 shows qualitative analysis of Anion Exchange Chromatography elution fractions on non-reducing SDS-PAGE. Fractions 13-25 were pooled. Volume was 18 mL and concentration 6.57 mg/mL.
• Figure 10 shows entire chromatogram and zoom of elution peak during first chromatography of stable pool 6880 P1 clarified harvest by Hydroxyapatite Chromatography as a second chromatography step.
• Figure 11 shows qualitative analysis of hydroxyapatite chromatography fractions of 6880 P1 on non-reducing SDS-PAGE.3 ug per lane.
• FT1 (lane 14) was taken inline halfway through the load.
• FT2 (lane 15) was taken inline at the end of load, maximum absorbance.
• Pool A Fractions 6-9 form each run.10 mL at 2.63 mg/mL.
• Pool B Fractions 10-13 from each run.10 mL at 1.30 mg/mL.
• Figure 12 shows qualitative analysis of hydrophobic interactions chromatography (Phenyl) fractions of 6880 P1 on non-reducing SDS-PAGE as a second chromatography step.
• Figures 13A-C show chromatography (A) and qualitative analysis of Phenyl chromatography fractions of 6880 P1 on non-reducing SDS-PAGE. Purity of eluted fraction Page 4 of 144 11666457v1 Attorney Docket: 2013762-0017 is determined by analytical size-exclusion chromatography (SEC-HPLC) (B). Protein eluted with 0.5 M AmSulfate is shown in dotted line or with 0.75 M followed by 0.5 M AmSulfate is shown in solid line (C).
• SEC-HPLC analytical size-exclusion chromatography
• Figure 15 shows qualitative analysis of Q HP anion exchange chromatography fractions of 6880 P1 on non-reducing SDS-PAGE when utilized as third chromatography step.
• Figures 16A-B show purity analysis of Heparin chromatography fractions (A upper panel), Phenyl chromatography fractions (A lower panel), and Q HP anion exchange chromatography fractions of 6880 P1 on SEC-HPLC (B) when utilized as first, second and third chromatography steps respectively
• Figure 17 shows chromatogram of ANK101 (IL-12 fusion polypeptide) polished after TOYOPEARL GigaCap Q-650S Chromatogram during multi cycling. Blue line is A280nm traces for cycles 9, orange line is A280nm traces for cycles 10, Green line is A280nm traces for cycles 11, and purple line is A280nm traces for cycles 12.
• Figure 18 shows full view chromatograms of reduced RP HPLC Profiles for IL-12 fusion polypeptide Reference Standard (top panel) and IL-12 fusion polypeptide GMP Drug Substance Batch (bottom panel).
• Figure 19 shows full view chromatograms of reducing CE-SDS Electropherograms for IL-12 fusion polypeptides Reference Standard (top panel) and GMP Drug Substance Batch (bottom panel).
• Figure 20 shows full view of SE HPLC Chromatograms for IL-12 fusion polypeptide Reference Standard (top panel) and GMP Drug Substance Batch (bottom panel).
• Figure 21 shows full view of AEX HPLC Chromatograms for IL-12 fusion polypeptide Reference Standard (top panel) and GMP Drug Substance Batch (bottom panel).
• Figure 22 shows expanded view of Deglycosylated ESI-MS Processed Spectra Profiles of IL-12 fusion polypeptide Drug Substance In-Process Samples Prior to Excipient (Polysorbate 20) addition for Reference Standard (top panel) and GMP Drug Substance Batch (bottom panel).
• Figure 23 shows Total Ion Current Chromatogram - Deglycosylated Samples for Drug Product GMP batch (top panel), Reference Standard (middle panel), and Assay Control (NEC) (bottom panel).
• the peak at RT ⁇ 16.5 min corresponded to a mass of approximately 34,778 Da and was assigned to PNGaseF. Only the main peak was considered for data collection.
• Figure 24 shows deconvoluted MS Spectra – Reduced and Deglycosylated Samples for Reference Standard (lower graph) and Drug Product GMP batch (upper graph).
• Figure 25 shows full view of Neutral N-Linked Oligosaccharide Profiles of IL-12 fusion polypeptide Reference Standard (upper panel/P4130826ARS) and GMP Drug Substance Batch (lower panel/1205114).
• Administration typically refers to application of a composition to a subject or system.
• routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
• administration may be systemic; in some embodiments, administration may be local.
• administration may be enteral; in some embodiments, administration may be parenteral.
• administration may be by injection (e.g., intramuscular, intratumoral, intravenous, or subcutaneous injection).
• injection may involve bolus injection, drip, perfusion, or infusion.
• administration in accordance with the present disclosure is by intratumoral injection.
• Affinity As is known in the art, “affinity” is a measure of the tightness with which two or more binding partners associate with one another. Those skilled in the art are aware of a variety of assays that can be used to assess affinity, and will furthermore be aware Page 6 of 144 11666457v1 Attorney Docket: 2013762-0017 of appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay.
• affinity is assessed over a plurality of concentrations (e.g., of one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitor entities (e.g., that might be present in a relevant – e.g., physiological – setting). In some embodiments, affinity is assessed relative to a reference (e.g., that has a known affinity above a particular threshold [a “positive control” reference] or that has a known affinity below a particular threshold [ a “negative control” reference”]. In some embodiments, affinity may be assessed relative to a contemporaneous reference; in some embodiments, affinity may be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.
• agent in general, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc, or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc).
• entity e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc, or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof
• phenomenon e.g., heat, electric current or field, magnetic force or field, etc.
• the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof.
• the term may be used to refer to a natural product in that it is found in and/or is obtained from nature.
• the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
• an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form.
• potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them.
• agonist may be used to refer to an agent, condition, or event whose presence, level, degree, type, or form correlates with increased level or activity of another agent (i.e., the agonized agent or the target agent).
• an agonist may be or include an agent of any chemical class such as, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant activating activity.
• an Page 7 of 144 11666457v1 Attorney Docket: 2013762-0017 agonist may be direct (in which case it exerts its influence directly upon its target, for example by physically binding to such target); in some embodiments, an agonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level and/or activity of the target is altered).
• Amino acid in its broadest sense, as used herein, the term “amino acid” refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
• an amino acid has the general structure H2N–C(H)(R)–COOH.
• an amino acid is a naturally-occurring amino acid.
• an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid.
• Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
• Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
• an amino acid including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above.
• an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure.
• such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
• such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
• amino acid may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
• Animal refers to a member of the animal kingdom. In some embodiments, “animal” refers to humans, of either sex and at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development.
• the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a horse, a sheep, cattle, a primate, and/or a pig).
• animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
• an animal may be a transgenic animal, genetically engineered animal, and/or a clone.
• Binding typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts – including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently, electrostatically, or otherwise associated with a carrier entity and/or in a biological system or cell). Binding between two entities may be considered “specific” if, under the conditions assessed, the relevant entities are more likely to associate with one another than with other available binding partners.
• Buffer typically describes a solution that based on the amount of acid or alkali in it will cause changes in pH. ⁇ buffered solution resists changes in pH by the presence of its acid-base components. Typical buffers that are utilized in biological manufacturing processes maintain the pH of the solution within a physiological range. Conventional buffer components include, but are not limited to, organic and inorganic salts, acids and bases. Commonly used buffers include but are not limited to TRICINE and BICINE, MES, PIPES, HEPES, MOPS, PBS.
• a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
• precancerous e.g., benign
• malignant pre-metastatic
• metastatic metastatic
• non-metastatic e.g., metastatic
• present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant.
• a relevant cancer may be characterized by a solid tumor.
• a relevant cancer may be characterized by a hematologic tumor.
• examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; Page 9 of 144 11666457v1 Attorney Docket: 2013762-0017 sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.
• Characteristic sequence element refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, presence of a characteristic sequence element correlates with presence or level of a particular activity or property of the polymer. In some embodiments, presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides).
• a characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., contiguously linked monomers).
• a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share the sequence element.
• Chemotherapeutic agent has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example specifically including agents utilized and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation.
• chemotherapeutic agents are useful in the treatment of cancer.
• a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g.
• microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhihitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or Page 10 of 144 11666457v1 Attorney Docket: 2013762-0017 more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity).
• HDACs histone deacetylase inhibitors
• topoisomerase inhibitors e.g., inhibitors of topoisomerase I and/or topoisome
• a chemotherapeutic agent may be or comprise one or more of Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and/or analogs thereof (e.g.
• DM1 Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof.
• a chemotherapeutic agent may be utilized in the context of an antibody- drug conjugate.
• a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLL1-doxorubicin, hRS7-SN- 38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2- P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P- Dox, hLL1-Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin
• a chemotherapeutic agent may be one described as utilized in an antibody-drug conjugate as described or discussed in one or more of Govindan et al, TheScientificWorldJOURNAL 10:2070, 2010, –2089).
• a chemotherapeutic agent may be or comprise one or more of farnesyl-thiosalicylic acid (FTS), 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), ⁇ -tocatrienol, salinomycin, or curcumin [0045]
• FTS farnesyl-thiosalicylic acid
• CSH 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide
• E2 estradiol
• TMS tetramethoxystilbene
• ⁇ -tocatrienol salinomycin
• curcumin Curcumin
• the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to Page 11 of 144 11666457v1 Attorney Docket: 2013762-0017 administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
• “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
• combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).
• Dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
• a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
• a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.
• a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
• Epitope refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component.
• an epitope is comprised of a plurality of chemical atoms or groups on an antigen.
• such chemical atoms or groups are surface- exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when Page 12 of 144 11666457v1 Attorney Docket: 2013762-0017 the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).
• Excipient refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
• Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• expression As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence.
• a gene product can be a transcript.
• a gene product can be a polypeptide.
• expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
• Functional As used herein, the term “functional” is used to refer to a form or fragment of an entity that exhibits a particular property and/or activity.
• Fragment A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole.
• a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polymer.
• monomeric units e.g., residues
• a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or Page 13 of 144 11666457v1 Attorney Docket: 2013762-0017 more of the monomeric units (e.g., residues) found in the whole polymer.
• the whole material or entity may in some embodiments be referred to as the “parent” of the fragment.
• Gene refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and/or a polypeptide product).
• a gene includes coding sequence (i.e., sequence that encodes a particular product); in some embodiments, a gene includes non-coding sequence.
• a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences.
• a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.).
• Gene product or expression product As used herein, the term “gene product” or “expression product” generally refers to an RNA transcribed from the gene (pre- and/or post-processing) or a polypeptide (pre- and/or post-modification) encoded by an RNA transcribed from the gene.
• Genome As used herein, the term “genome” refers to the total genetic information carried by an individual organism or cell, represented by the complete DNA sequences of its chromosomes.
• HMWS High Molecular Weight Species
• Host cell refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
• host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence).
• exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, Page 14 of 144 11666457v1 Attorney Docket: 2013762-0017 S. pombe, P. pastoris, P.
• the cell is a human, monkey, ape, hamster, rat, or mouse cell.
• the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO Kl, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3 A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell.
• CHO e.g., CHO Kl, DXB-11 CHO, Veggie-CHO
• COS e.g
• the cell comprises one or more viral genes.
• Host Cell Protein(s) or 'dzCP(s): As used herein, refers to proteins that may be present in a cell extract or preparation, for example because they were produced by or otherwise contained in or on a host cell in which a fusion polypeptide (e.g., a phosphorylated or unphosphorylated fusion polypeptide) as described herein is produced, and that are not the fusion polypeptide.
• provided technologies e.g., provided manufacturing methods, such as provided purification methods
• exclude or reduce HCPs from preparation(s) of the fusion polypeptide e.g., from preparations of phosphorylated fusion polypeptide as described herein).
• reduced HCP preparation describes a preparation that contains reduced HCPs relative, for example, to that amount present before application of a relevant purification step (e.g., as provided herein) and/or relative to that achieved through a different purification technology.
• a relevant purification step e.g., as provided herein
• provided technologies achieve production of fusion polypeptide preparations (e.g., preparations of phosphorylated fusion polypeptide) in which HCP are undetectable, for example, using e.g., an ELISA method.
• removal of HCP may be monitored or assessed, for example, during or after purification of a fusion polypeptide (e.g., a phosphorylated form thereof) as described herein, for example from a host cell which may, in some embodiments, be an engineered mammalian cell as described herein (e.g., that expresses the fusion polypeptide and a kinase that phosphorylates it at a ratio within a range of about 4:1 to 10:1, for example at a ratio of about 8:1.
• a fusion polypeptide e.g., a phosphorylated form thereof
• a host cell which may, in some embodiments, be an engineered mammalian cell as described herein (e.g., that expresses the fusion polypeptide and a kinase that phosphorylates it at a ratio within a range of about 4:1 to 10:1, for example at a ratio of about 8:1.
• an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc).
• comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance).
• in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
• in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
• Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
• isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
• a substance is "pure” if it is substantially free of other Page 16 of 144 11666457v1 Attorney Docket: 2013762-0017 components.
• a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
• carriers or excipients e.g., buffer, solvent, water, etc.
• a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature.
• a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated” polypeptide.
• a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated” polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
• Linker as used herein, is used to refer to that portion of a multi-element agent that connects different elements to one another.
• a polypeptide whose structure includes two or more functional or organizational moieties or domains often includes a stretch of amino acids between such moieties or domains that links them to one another.
• a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two moieties or domains associated with one another by the linker.
• a polyptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.
• a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.
• Modulator is used to refer to an entity whose presence or level in a system in which an activity of interest is observed correlates with a change in level and/or nature of that activity as compared with that observed under otherwise comparable conditions when the modulator is absent.
• a modulator is an activator, in that activity is increased in its presence as compared with that observed under otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator is an antagonist or inhibitor, in that activity is reduced in its presence as compared with otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator interacts directly with a target entity whose activity is of interest. In some embodiments, a modulator interacts indirectly (i.e., directly with an intermediate agent that interacts with the target entity) with a target entity whose activity is of interest.
• a modulator affects level of a target entity of interest; alternatively or additionally, in some embodiments, a modulator affects activity of a target entity of interest without affecting level of the target entity. In some embodiments, a modulator affects both level and activity of a target entity of interest, so that an observed difference in activity is not entirely explained by or commensurate with an observed difference in level [0064]
• Moiety is a defined chemical group or entity with a particular structure and/or or activity, as described herein. Typically, a “moiety” is part of, less than the entirety of, a molecule or entity.
• mutant refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a mutant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “mutant” of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A mutant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements.
• a small molecule may have a characteristic core structural element (e.g., a Page 18 of 144 11666457v1 Attorney Docket: 2013762-0017 macrocycle core) and/or one or more characteristic pendent moieties so that a mutant of the small molecule is one that shares the core structural element and the characteristic pendent moieties but differs in other pendent moieties and/or in types of bonds present (single vs double, E vs Z, etc.) within the core, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function, a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three- dimensional space.
• a characteristic core structural element e.g., a Page 18 of 144 11666457v1 Attorney Docket: 2013762-00
• a mutant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone.
• a mutant polypeptide shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
• a mutant polypeptide does not share at least one characteristic sequence element with a reference polypeptide.
• the reference polypeptide has one or more biological activities.
• a mutant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a mutant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a mutant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide.
• Operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element.
• control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest.
• patient refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., Page 19 of 144 11666457v1 Attorney Docket: 2013762-0017 mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human.
• a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.
• pharmaceutical composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
• active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
• pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for a particular route of administration, e.g., as described herein.
• Pharmaceutically acceptable As used herein, the phrase “pharmaceutically acceptable” is used to refer to an agent or entity that, within the scope of sound medical judgment, is suitable for use in contact with tissues of human beings and/or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• compositions or vehicles such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; Page 20 of 144 11666457v1 Attorney Docket: 2013762-0017 gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alg
• Polypeptide As used herein refers to a polymeric chain of amino acids.
• a polypeptide has an amino acid sequence that occurs in nature.
• a polypeptide has an amino acid sequence that does not occur in nature.
• a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
• a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
• a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids.
• a polypeptide may comprise D-amino acids, L-amino acids, or both.
• a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion.
• a polypeptide is not cyclic and/or does not comprise any cyclic portion.
• a polypeptide is linear.
• a polypeptide may be or comprise a stapled polypeptide.
• the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
• exemplary polypeptides within the class whose amino acid Page 21 of 144 11666457v1 Attorney Docket: 2013762-0017 sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
• a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
• a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
• a conserved region that may in some embodiments be or comprise a characteristic sequence element
• Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
• a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
• a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
• Predetermined By predetermined is meant deliberately selected, for example as opposed to randomly occurring or achieved.
• Pure As used herein, an agent or entity is “pure” if it is substantially free of other components.
• an agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
• Recombinant as used herein, is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/
• one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc).
• Reference standard As used herein describes a standard or control relative to which a comparison is performed.
• an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
• a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
• a reference or control is a historical reference or control, optionally embodied in a tangible medium.
• a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
• Specific binding refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur.
• a binding agent that interacts with one particular target when other potential Page 23 of 144 11666457v1 Attorney Docket: 2013762-0017 targets are present is said to "bind specifically" to the target with which it interacts.
• specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity.
• specific binding is assessed by performing such detections or determinations across a range of concentrations.
• the term “specific”, when used herein with reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities or states.
• an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets.
• specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute.
• specificity may be evaluated relative to that of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding agent. In some embodiments specificity is evaluated relative to that of a reference non-specific binding agent. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its target entity. In some embodiments, binding agent binds with higher on-rate, lower off-rate, increased affinity, decreased dissociation, and/or increased stability to its target entity as compared with the competing alternative target(s).
• Specificity As is known in the art, “specificity” is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.
• Subject As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, Page 24 of 144 11666457v1 Attorney Docket: 2013762-0017 disorder or condition.
• a subject does not display any symptom or characteristic of a disease, disorder, or condition.
• a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
• a subject is a patient.
• a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
• Therapeutic agent refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
• a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• Therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
• a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
• the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
• the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
• a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
• treatment refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
• such treatment may be of a subject who does not exhibit signs of the relevant Page 25 of 144 11666457v1 Attorney Docket: 2013762-0017 disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
• such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
• treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition.
• treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
• treatment may be prophylactic; in some embodiments, treatment may be therapeutic.
• a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
• a tumor is associated with, or is a manifestation of, a cancer.
• a tumor may be a disperse tumor or a liquid tumor.
• a tumor may be a solid tumor.
• Variant As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements.
• a variant by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
• a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three- dimensional space and/or contributing to a particular structural motif and/or biological function;
• a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three- dimensional space.
• a variant polypeptide or nucleic acid may differ Page 26 of 144 11666457v1 Attorney Docket: 2013762-0017 from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
• moieties e.g., carbohydrates, lipids, phosphate groups
• a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
• a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
• a reference polypeptide or nucleic acid has one or more biological activities.
• a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
• a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions.
• a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference.
• a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference.
• a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference.
• a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions Page 27 of 144 11666457v1 Attorney Docket: 2013762-0017 or deletions as compared to the reference.
• a reference polypeptide or nucleic acid is one found in nature.
• a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.
• Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• vector refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
• viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• certain vectors are capable of directing the expression of genes to which they are operatively linked.
• Viral inactivation or removal describes inactivation or removal of a virus that may be contained in a sample such as, for example, a cell extract or a fusion polypeptide preparation.
• a virus present in a sample may have originated from a source material (e.g. a host cell); alternatively or additionally, in some embodiments, a virus present in a sample may have been introduced, e.g., during processing of such source material.
• pH viral inactivation involves comprises exposing a virus (e.g., a sample containing a virus) to a pH that inactivates (e.g., has been established to inactivate) the virus.
• Wild-type has its art-understood meaning and refers to a form of an entity (e.g., a polypeptide or nucleic acid) that has a structure and/or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered) state or context.
• entity e.g., a polypeptide or nucleic acid
• more than one “wild type” form of a particular polypeptide or nucleic acid may exist in nature, for example as “alleles” of a particular gene or normal variants of a particular polypeptide..
• that form (or those Page 28 of 144 11666457v1 Attorney Docket: 2013762-0017 forms) of a particular polypeptide or nucleic acid that is most commonly observed in a population (e.g., in a human population) is the “wild type” form.
• the present disclosure provides particularly methods of manufacturing, including purifying, phosphorylated forms of a fusion polypeptide (e.g., as described in published International Patent Application WO2020/263399).
• the present disclosure relates to manufacturing of phosphorylated forms of fusion polypeptides comprising (a) an immunomodulatory polypeptide that comprises an immune agonist moiety; and (b) a metal-hydroxide binding polypeptide whose amino acid sequence includes a plurality of phosphorylation sites, so that it can adopt phosphorylated and unphosphorylated forms.
• Fusion Polypeptides [0089] In some embodiments, fusion polypeptides according to the present disclosure can adopt phosphorylated and unphosphorylated forms.
• Immunomodulatory polypeptide [0090] In some embodiments, fusion polypeptides of the present disclosure comprise at least one immunomodulatory polypeptide.
• a fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immune agonist moieties). In some such embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides that are the same; in some such embodiments, all immunomodulatory polypeptides in a fusion polypeptide in accordance with the present disclosure are the same. In some such embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides that at are different from one another. [0092] In some embodiments, an immunomodulatory polypeptide is or comprises at least one immune agonist moiety.
• an immune agonist moiety is or comprises a functional fragment of a parent (e.g., a wild type) polypeptide; for example, in some embodiments, an immunomodulatory polypeptide is or comprises a functional fragment Page 29 of 144 11666457v1 Attorney Docket: 2013762-0017 that is a signaling competent fragment. In some embodiments, an immunomodulatory polypeptide comprises one, two, three, four, five, or six immune agonist moieties. [0093] Thus, in some embodiments, an immunomodulatory polypeptide may include more than one immune agonist moiety which, in various embodiments, may be the same or different.
• a fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immune agonist moieties) that include at least two subtypes of immunomodulatory polypeptides (e.g., immune agonist moieties) – for example so that the fusion polypeptide includes at least two of a first subtype and at least two of a second subtype.
• an immunomodulatory polypeptide e.g., an immune agonist moiety
• activates or inhibits activity of a cell of the immune system e.g., is signaling competent.
• signal competency is characterized in that, when assessed for binding to a particular binding partner, an immune agonist moiety or moieties or functional fragments thereof displays binding comparable to that of a reference standard (e.g., a wild-type polypeptide).
• signal competency is characterized in that, when assessed for a biological effect, e.g., in vitro or in vivo, an immune agonist moiety or moieties or functional fragments thereof displays said biological effect comparable to that of a reference standard (e.g., a wild-type polypeptide).
• an immunomodulatory polypeptide e.g., an immune agonist moiety
• a response stimulatory moiety is, for example, but without limitation, a cytokine, a chemokine, an agonistic antibody, an immune checkpoint inhibitor, or a combination thereof.
• an immunomodulatory polypeptide promotes differentiation and proliferation of immune cells (e.g., T cells). In some embodiments, an immunomodulatory polypeptide enhances production of interferon gamma.
• an immunomodulatory polypeptide comprises an interleukin-12 (IL-12) immunomodulatory polypeptide (e.g., an IL-12 immune agonist moiety).
• IL-12 is a pro-inflammatory cytokine that plays an important role in innate and adaptive immunity.
• Wild type IL-12 is a heterodimeric protein comprising two subunits, p35 (IL-12A; GenBenk GeneID: 3592) and p40 (IL-12B; GenBank GeneID: 3593), connected by disulfide bonds. Binding of IL-12 to the IL-12 receptor complex (IL-12R ⁇ 1 / IL-12R ⁇ 2) on T cells and Natural Killer (NK) cells leads to signaling via signal transducer and activator of transcription 4 (STAT4) and subsequent interferon ⁇ (IFN- ⁇ ) production and secretion. [0101] IL-12 subunits, IL-12A and IL-12B, can also form heterodimers with other IL- 12 family members.
• IL-12A may also dimerize with Epstein-Barr virus induced gene 3 (EBI3) to form IL-12 family member
• EBI3 Epstein-Barr virus induced gene 3
• IL-35 and IL-12B may dimerize with a p19 monomer, to form IL-12 family member, IL23.
• IL-12 plays important roles in the innate and adaptive immune response, and dysregulation of IL-12 has been implicated in a number of disease states.
• an immunomodulatory polypeptide disclosed herein is or comprises an IL-12 immune agonist moiety.
• an immunomodulatory polypeptide disclosed herein comprises a plurality of IL-12 immune agonist moieties. In some embodiments, an immunomodulatory polypeptide disclosed herein comprises exactly two IL-12 immune agonist moieties. In some embodiments, two or more IL-12 immune agonist moieties of a plurality of (e.g., two) IL-12 immune agonist moieties are the same moiety. In some such embodiments, a plurality (e.g., two) IL-12 immune agonist moieties are Page 31 of 144 11666457v1 Attorney Docket: 2013762-0017 different moieties. In some such embodiments, an IL-12 immune agonist moiety comprises an IL-12A polypeptide or functional fragment thereof.
• an IL-12 immune agonist moiety comprises an IL-12B polypeptide or functional fragment thereof.
• immunomodulatory polypeptide disclosed herein comprises an IL-12 immune agonist moiety that comprises an IL-12A polypeptide or functional fragment thereof and an IL-12 immune agonist moiety that comprises an IL-12B polypeptide or functional fragment thereof.
• an IL-12B immune agonist moiety is located N- terminal to an IL-12A immune agonist moiety in an immunomodulatory polypeptide.
• an IL-12A immune agonist moiety is located N-terminal to an IL-12B immune agonist moiety in an immunomodulatory polypeptide.
• an immunomodulatory polypeptide comprising a plurality (e.g., two) IL-12 immune agonist moieties are linked directly.
• an immunomodulatory polypeptide comprising a plurality (e.g., two) IL-12 moieties are linked via a first linker.
• linkers are discussed elsewhere herein.
• an immunomodulatory polypeptide disclosed herein comprises an IL-12A and/or IL-12B immune agonist moiety comprising a variant.
• an IL12 variant comprises a mutation.
• an immunomodulatory polypeptide disclosed herein comprises an IL-12A and/or IL-12B immune agonist moiety that is a functional fragment thereof (e.g., a signaling competent fragment).
• an immunomodulatory polypeptide comprises a functional IL-12A fragment.
• an immunomodulatory polypeptide comprises a functional IL-12B fragment.
• an immunomodulatory polypeptide comprises a full-length IL-12A and a functional IL-12B fragment.
• an immunomodulatory polypeptide comprises a full-length IL-12B and a functional IL-12A fragment.
• an IL-12A or IL-12B fragment comprises or consists of at least 5%, 10 %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the monomeric units (e.g., residues) as found in wild- type IL-12A or IL-12B.
• an immunomodulatory polypeptide disclosed herein comprises an IL-12A and/or IL-12B immune agonist moiety that is a human IL-12A and/or IL-12B immune agonist moiety.
• an immunomodulatory polypeptide disclosed herein comprises an IL-12B immune agonist moiety having at least 80 % sequence identity to SEQ ID NO: 3, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO: 3.
• an immunomodulatory polypeptide disclosed herein comprises an IL-12A immune agonist moiety having at least 80 % sequence identity to SEQ ID NO: 4, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO: 4.
• an immunomodulatory polypeptide disclosed herein comprises an IL-12B immune agonist moiety having at least 80 % sequence identity to SEQ ID NO: 3 and an IL- 12A immune agonist moiety having at least 80 % sequence identity to SEQ ID NO: 4.
• an immunomodulatory polypeptide disclosed herein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO: 5.
• a hydroxyl replacement e.g., with phosphate groups
• a metal hydroxide e.g., aluminum hydroxide
• immunomodulatory polypeptides according to the present invention can adopt phosphorylated and unphosphorylated forms.
• an immunomodulatory polypeptide comprises at least one amino acid that can be phosphorylated. In some embodiments, an immunomodulatory polypeptide comprises at least one kinase targets motif. In some embodiments, an immunomodulatory polypeptide does not comprise a kinase targets motif. In those embodiments, the immunomodulatory polypeptide may still comprise an amino acid that can be phosphorylated. In some embodiments, an immunomodulatory polypeptide comprising one or more phosphorylated amino acids contributes to metal strong binding to metal hydroxide, e.g., aluminum hydroxide.
• an immunomodulatory polypeptide comprises at least one phosphorylated serine. In some embodiments, an immunomodulatory polypeptide comprises at least two phosphorylated serine residues. In some embodiments, an immunomodulatory polypeptide comprises at least three phosphorylated serine residues. In some embodiments, an immunomodulatory polypeptide comprises at least four phosphorylated serine residues.
• an immunomodulatory polypeptide comprises at least five phosphorylated serine residues, such as six serine residues, such as seven serine residues, such as eight serine residues, such as nine serine residues, such as ten serine residues.
• Metal-hydroxide binding peptide [0115]
• fusion polypeptides of the present disclosure comprise at least one metal-binding polypeptide.
• a fusion polypeptide comprises two or more metal- binding polypeptides (e.g., two or more alum-binding polypeptides).
• a fusion polypeptide comprises two or more metal-binding polypeptides that are the same; in some such embodiments, all metal-binding polypeptides in a fusion polypeptide in accordance with the present disclosure are the same. In some such embodiments, a fusion polypeptide comprises two or more metal-binding polypeptides that at are different from one another. [0118] As discussed above, a metal-binding peptide can be fused to an immunomodulatory polypeptide allowing for strong binding to a metal hydroxide, such as aluminum hydroxide. Various immunomodulatory polypeptides can be fused to metal- binding peptides.
• metal-binding polypeptides adsorbed to alum in serum can be used to retain proteins and peptides in tumors.
• a metal-hydroxide binding polypeptide comprises an amino acid sequence that includes a plurality of phosphorylation sites, so that it can adopt phosphorylated and unphosphorylated forms.
• a metal-hydroxide binding polypeptide comprises at least one kinase target motif.
• a target kinase motif comprises an amino acid that is phosphorylated by a kinase.
• Amino acids that are typically phosphorylated include a hydroxyl, such as serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y) residues.
• a kinase motif refers to the amino acid sequence immediately N- and/or C- terminal to the amino acid residue capable of being phosphorylated.
• many kinases comprise structural features that confer specificity such that the kinase phosphorylates a particular amino acid (e.g., serine, threonine, or tyrosine) of a particular kinase target motif.
• Kinase target motifs recognized are highly diverse depending on the particular type of kinase.
• the present disclosure provides metal-hydroxide binding polypeptides comprising one or more kinase target motifs of a secretory pathway kinase.
• the secretory pathway which is the pathway by which a cell secretes proteins and/or other biomolecules into the extracellular space, refers to the endoplasmic reticulum (ER), Golgi apparatus (Golgi), cell membrane, and lysosomal storage compartments as well as the vesicles that travel between them.
• Secretory pathway kinases are localized throughout the secretory pathway (e.g., at the ER, Golgi, etc.) and function to phosphorylate proteins Page 35 of 144 11666457v1 Attorney Docket: 2013762-0017 destined for secretion (Sreelatha et al. Biochimica et biophysica acta vol.1854,10 Pt B (2015): 1687-93).
• a relevant kinase is a naturally occurring secretory pathway kinase (e.g., is endogenously targeted to the secretory pathway to function).
• a secretory pathway kinase comprises a signal sequence that targets the kinase to the secretory pathway.
• Naturally occurring human secretory pathway kinases include, for example, four-jointed box kinase 1, Fam20A, Fam20B, Fam20C, vertebrate lonesome kinase (VLK), SGK196, and Fam69A, Fam69B, and Fam69C.
• a relevant kinase is a non-naturally occurring secretory pathway kinase.
• a non-naturally occurring kinase is produced by linking a secretory signal peptide to a kinase endogenously localized to a non-secretory pathway cellular compartment.
• a kinase target motif is a target kinase motif of a secretory pathway kinase.
• a secretory pathway kinase target kinase motif comprises an S-X-E motif.
• Fam20C phosphorylates serine and has been shown to phosphorylate kinase targets motif comprising the amino acid sequence Ser-X-Glu (e.g., S-X-E), Ser-X-pSer (e.g., S-X-pS), and Ser-X-Gln-X-X-Asp-Glu-Glu (S-X-Q-X-X-D- E-E) wherein X is any amino acid, and pS is phosphorylated serine (Mercier, et al (1981) Biochimie, 63: 1-17; Mercier et al (1971) Eur J.
• Ser-X-Glu e.g., S-X-E
• Ser-X-pSer e.g., S-X-pS
• Ser-X-Gln-X-X-Asp-Glu-Glu S-X-Q-X-X-D- E-E
• a target kinase motif comprises the amino acid sequence SEEE. In some embodiments, a target kinase motif comprises the amino acid sequence SEEA. In some embodiments, a target kinase motif comprises the amino acid sequence SEEQ. In some embodiments, a target kinase motif comprises the amino acid sequence SEE.
• a metal-hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, a metal-hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, a metal-hydroxide binding polypeptide comprises more than four S-X-E motifs.
• a metal-hydroxide binding polypeptide comprises eight S-X-E Page 36 of 144 11666457v1 Attorney Docket: 2013762-0017 motifsIn some embodiments, the number of target kinase motifs (e.g., S-X-E motifs) contributes to the number of phosphorylated residues on a metal-hydroxide binding polypeptide. In some embodiments, a metal-hydroxide binding polypeptide comprises eight S-E-E motifs. [0125] In some embodiments, a metal-hydroxide binding polypeptide is a metal- hydroxide binding polypeptide whose amino acid sequence includes a plurality of phosphorylation sites.
• a plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, a plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, a plurality of phosphorylation sites comprises more than four S-X-E motifs. In some embodiments, a plurality of phosphorylation sites comprises more than eight S-X-E motifs.
• the number of target kinase motifs contributes to the number of phosphorylated residues on a metal-hydroxide binding polypeptide.
• the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs are directly adjacent (e.g., linked) to the next target kinase (e.g., S-X-E motif).
• the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs are separated (e.g., linked) to the next target kinase motif (e.g., S-X-E motif) by a spacer.
• the spacer comprises at least one glycine residue.
• the spacer comprises a plurality of glycine residues.
• the spacer comprises three glycine residues.
• the spacer comprises at least four glycine residues.
• the spacer has a sequence that comprises four glycine residues.
• the spacer has an amino acid sequence comprising GGGSGGGG. In some embodiments, the spacer has an amino acid sequence comprising GGGEGGGG. In some embodiments, the spacer has an amino acid sequence comprising GGGGG. In some embodiments, the spacer has an amino acid sequence comprising GGGG. [0127] In some embodiments, a metal-hydroxide binding polypeptide comprises four S-X-E motifs and three spacers comprising four glycine residues. In some embodiments, a Page 37 of 144 11666457v1 Attorney Docket: 2013762-0017 metal-hydroxide binding polypeptide comprises six S-X-E motifs and five spacers comprising four glycine residues.
• a metal-hydroxide binding polypeptide comprises eight S-X-E motifs and seven spacers comprising four glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises eight S-X- E motifs and eight spacers comprising four glycine residues. In some embodiments, a metal- hydroxide binding polypeptide comprises eight motifs with the amino acid sequence, SEE, and eight spacers comprising four glycine residues. [0128] In some embodiments, a metal-hydroxide binding polypeptide comprise an ending sequence (e.g., an amino acid sequence) at the c-terminus of the fusion polypeptide.
• ending sequence e.g., an amino acid sequence
• an ending sequence comprises a plurality of amino acid residues.
• a plurality of amino acid residues comprises GGGG.
• an ending sequence comprises the amino acid sequence GGGGS.
• a desired (e.g., optimal) number of kinase target motifs and/or spacing of kinase motifs may be determined based on one or more of, for example, desired phosphate content to achieve strong metal-hydroxide retention and/or avoidance of one or more manufacturing challenges (e.g., which the present disclosure appreciates may be associated with highly phosphorylated elements).
• a desired (e.g., optimal) number and/or spacing of kinase motifs results in an exposure of the polypeptide to the kinase to achieve a desired level of fusion polypeptide phosphorylation.
• improved fusion polypeptides as described herein results in one or more of improved reproducibility, consistency, and/or production of a homogenously-phosphorylated fusion polypeptide.
• provided technologies achieve reproducible manufacturing of comparable preparations (e.g., preparations that are consistently within established parameters) of fusion polypeptides (e.g., phosphorylated fusion polypeptides) and/or complexes as described herein.
• provided technologies achieve reduced immunogenicity compared to an appropriate reference standard.
• degree of phosphorylation e.g., the average number of phosphate molecules per polypeptide
• degree of phosphorylation is 0.5-7, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 0.5-6, 0.5-5, 0.5-4, 1-6, 2-6, 3-6, 4-6, 5-6, 7-8, 8-9, 9-10, 10-11, 11-12 or 13-14.
• degree of phosphorylation is 3, 3.5
• Page 38 of 144 11666457v1 Attorney Docket: 2013762-0017 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7.7.8, 7.9, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, or 12.
• fusion polypeptides as described herein may include one or more linkers and/or spacers.
• fusion polypeptides comprise an immunomodulatory polypeptide comprising a first and a second immune agonist moiety.
• a first and a second immune agonist moiety are linked via a first linker.
• fusion polypeptides of the present disclosure comprise an immunomodulatory polypeptide and a metal-hydroxide binding polypeptide.
• an immunomodulatory polypeptide and a metal-hydroxide binding polypeptide are linked via a second linker.
• a first linker and/or a second linker is a polypeptide linker.
• a polypeptide linker is synthetic.
• a synthetic polypeptide linker may comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides.
• polypeptide linkers of the present disclosure are at least one amino acid in length and can be any suitable number of amino acids.
• a polypeptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.
• a first linker comprises a polypeptide linker.
• a first linker comprises or consists of a Glycine-Serine (Gly-Ser or G-S linker).
• Gly-Ser linker is a polypeptide linker that consists of glycine and serine residues.
• a Gly-Ser linker comprises an amino acid sequence of (Gly4Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
• a Gly-Ser linker is (Gly4Ser)1.
• a Gly-Ser linker is (Gly4Ser)2.
• a Gly-Ser linker is (Gly4Ser)3.
• a Gly-Ser linker is (Gly4Ser)4. In some embodiments, a Gly-Ser linker is (Gly4Ser)5. In some embodiments, a Gly-Ser linker is Page 39 of 144 11666457v1 Attorney Docket: 2013762-0017 (Gly4Ser)6. In some embodiments, a Gly-Ser linker is (Gly4Ser)7. In some embodiments, a Gly-Ser linker is (Gly 4 Ser) 8. In some embodiments, a Gly-Ser linker is (Gly 4 Ser) 9. In some embodiments, a Gly-Ser linker is (Gly4Ser)10.
• a second linker comprises a polypeptide linker. In some embodiments, a second linker comprises a plurality of glycine residues. In some embodiments, a second linker comprises a polypeptide linker with the amino acid sequence, GGGGSGGGG. In some embodiments, a second linker comprises a polypeptide linker with the amino acid sequence, GGGGEGGGG.
• an immunomodulatory polypeptide or a metal- hydroxide-binding polypeptide utilized in accordance with the present disclosure is a variant of a relevant reference polypeptide (e.g., a wild type polypeptide or functional portion thereof).
• a variant shows at least 70% identity to its reference polypeptide. In some such embodiments, a variant shows at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to its references polypeptide [0140] In some embodiments, a variant comprises one or more conservative or otherwise non-disruptive modifications (e.g., substitutions, deletions or additions) relative to its reference. In some embodiments, a variant is free of any disruptive modifications (e.g., substitutions, deletions or additions) so that an immunomodulatory polypeptide maintains one or more functional characteristics of the reference.
• conservative or otherwise non-disruptive modifications e.g., substitutions, deletions or additions
• maintains means an immunomodulatory polypeptide display comparable activity (e.g., signaling competency or binding) compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide).
• an immunomodulatory polypeptide maintains at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher activity compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide).
• the present disclosure provides phosphorylated fusion polypeptides comprising an immunomodulatory polypeptide and a metal-hydroxide binding polypeptides, wherein the phosphorylated fusion polypeptide, when exposed to a metal- hydroxide forms a complex therewith.
• a complex is formed via adsorption of the phosphorylated fusion polypeptide to a metal-hydroxide. Without wishing to be bound by any one theory, it is hypothesized adsorption of a phosphorylated fusion polypeptide to a metal hydroxide occurs by ligand exchange.
• Ligand exchange for example, is a substitution or exchange of a surface hydroxyl by another ligand.
• substitution or exchange of a surface hydroxyl occurs by a hydroxyl-replacement group (e.g., a phosphate group).
• a metal-hydroxide is a substance that includes at least one hydroxyl group bound to a metal.
• a metal-hydroxide can adsorb fusion polypeptides comprising a hydroxyl- replacement moiety.
• a hydroxyl-replacement moiety is a phosphate group.
• a metal-hydroxide is selected based on its inherent qualities or characteristics. In some embodiments, a metal-hydroxide is selected due to its biocompatibility for use in a subject (e.g., a mammal, e.g., a human). In some embodiments, a metal-hydroxide is aluminum hydroxide (e.g., alum). In some embodiments, a metal- hydroxide is iron-hydroxide. A skilled artisan will recognize any number of metal-hydroxide may be successfully utilized in accordance with the present disclosure.
• Phosphorylated Fusion Polypeptide Preparations [0144] Among other things, the present disclosure provides preparations of fusion polypeptides, and particularly of phosphorylated fusion polypeptides, with particularly desirable characteristics (e.g., level of purity of such phosphorylated fusion polypeptide and/or degree of phosphorylation, such as average degree of phosphorylation and/or distribution of particular phosphoforms of the fusion polypeptide, within achieved preparations).
• particularly desirable characteristics e.g., level of purity of such phosphorylated fusion polypeptide and/or degree of phosphorylation, such as average degree of phosphorylation and/or distribution of particular phosphoforms of the fusion polypeptide, within achieved preparations.
• the present disclosure identifies the source of a problem with certain methods of manufacturing of relevant fusion polypeptides, including, for Page 41 of 144 11666457v1 Attorney Docket: 2013762-0017 example, in their ability to generate preparations having a suitable degree and/or consistency (e.g., homogeneity) of phosphorylation, and/or in their ability to generate preparations of sufficient purity.
• the present disclosure provides particularly high purity preparations of phosphorylated fusion polypeptides, including high purity preparations characterized by a particular degree and/or consistency of phosphorylation.
• the present disclosure notes that either too high or too low degree of phosphorylation can negatively impact activity of a fusion polypeptide preparation.
• the present disclosure provides particular useful methods of manufacturing a phosphorylated form of a fusion polypeptide.
• the present disclosure provides methods of manufacturing a phosphorylated form of fusion polypeptides disclosed herein by contacting the fusion polypeptide with a kinase.
• the present methods of manufacturing as described herein provide certain fusion polypeptide preparations that are characterized by particularly desirable degree of phosphorylation degree.
• provided technologies achieve manufacturing of desirable preparations as described herein (e.g., with level and/or consistency of phosphorylation and/or degree of purity as described herein) with a particularly high yield.
• the present invention provides methods of manufacturing a high purity preparation of a phosphorylated form of a fusion polypeptide.
• a high purity phosphorylated fusion polypeptide preparation comprises more phosphorylated fusion polypeptides relative to unphosphorylated fusion polypeptide.
• a high purity phosphorylated fusion polypeptide preparation comprises predominantly phosphorylated fusion polypeptides compared.
• a high purity phosphorylated fusion polypeptide preparation comprises at least 90% phosphorylated fusion polypeptides, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% phosphorylated fusion polypeptides.
• a fusion polypeptide preparation comprises an unphosphorylated form of a fusion polypeptide.
• a fusion polypeptide preparation comprises a phosphorylated form of the fusion polypeptide.
• a fusion polypeptide preparation comprises a mixture of both unphosphorylated and phosphorylated forms of a fusion polypeptide.
• a fusion polypeptide preparation is a preparation comprising fusion polypeptide wherein one or more characterization attributes are assessed and determined to meet a release and/or acceptance criteria (e.g., as described herein). Examples of such product quality attributes include, but are not limited to, degree of phosphorylation and/or heterogeneity of phosphorylation.
• fusion polypeptides having phosphorylated and unphosphorylated forms described herein are made in host cells using techniques for exogenous expression.
• fusion polypeptide preparations comprise phosphorylated fusion polypeptides.
• a fusion polypeptide preparation is a high purity preparation of a phosphorylated form of a fusion polypeptide.
• a fusion polypeptide preparation comprises a mixture of both unphosphorylated and phosphorylated forms of a fusion polypeptide.
• a fusion polypeptide preparation comprises more fusion polypeptide that are phosphorylated than fusion polypeptide that are unphosphorylated.
• a phosphorylated fusion polypeptide preparation comprises fusion polypeptides with varying degrees of phosphorylation as described herein above.
• fusion polypeptide preparations are prepared in a Tris buffer of about pH 7 to about pH 8.
• a fusion polypeptide preparation comprises a salt (e.g., NaCl).
• Manufacturing Phosphorylated Fusion Polypeptides Expression in Host Cells [0153]
• fusion polypeptides are manufactured by production in a host cell, such as a mammalian cell. Typically, such host cell (e.g., such mammalian cell) will have been engineered to express the fusion polypeptide.
• a polynucleotide e.g., DNA or RNA
• a polynucleotide encoding a fusion polypeptide of the present disclosure may be prepared, e.g., for introduction into a host cell.
• sequences encoding fusion polypeptides may be excised from DNA using restriction enzymes, may be amplified from plasmids or genomic polynucleotide sequences using, for example, polymerase chain reaction, or may be synthesized using chemical synthesis techniques. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide encoding fusion polypeptides of the present disclosure. [0155] Recombinant polynucleotides encoding fusion polypeptides of the present disclosure may be cloned into a vector capable of expressing a fusion polypeptide.
• a vector is a viral vector.
• a vector is a non-viral vector.
• a vector is a plasmid.
• a vector is a transposon.
• a vector capable of expression comprises a recombinant polynucleotide that encodes a fusion polypeptide of the present disclosure is operatively linked to a sequence or sequences that regulates expression of the polynucleotides (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.).
• a regulatory sequence or sequences that control expression are selected to achieve a desired level of expression.
• more than one sequence that controls expression are utilized.
• more than one sequence that controls expression are utilized to achieve a desired level of expression of a plurality of recombinant polynucleotides that encode a plurality polypeptides.
• a plurality of recombinant polypeptides are expressed from the same vector (e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic.).
• a plurality of recombinant polypeptides are expressed, each of which is expressed from a separate vector.
• a vector capable of expression comprising a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure is used to express a fusion polypeptide in a host cell.
• Page 44 of 144 11666457v1 Attorney Docket: 2013762-0017
• a host cell may be selected from a variety of the available and known host cells (e.g., Human Embryonic Kidney (HEK) cells, suspension HEK293 cells, Chinese Hamster Ovary cells) suitable expressing fusion polypeptides disclosed herein.
• HEK Human Embryonic Kidney
• HEK293 cells suspension HEK293 cells
• Chinese Hamster Ovary cells suitable expressing fusion polypeptides disclosed herein.
• a variety of methods are available to introduce a vector into host cells.
• a vector may be introduced into host cells using transfection.
• transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection.
• a vector may be introduced into a host cell using transduction.
• host cells e.g., producer cells
• host cells are used to manufacture a phosphorylated form of a fusion polypeptide.
• host cells expressing a fusion polypeptide and/or kinase are cultured in a single use bioreactor (e.g., 50L to 4000L) or a stainless steel bioreactor (e.g., 50L to 4000L).
• host cell are cultured at a temperature ranging from 30 ⁇ C to 40 ⁇ C. In some embodiments, the temperature is lower during the production phase (e.g., 33 ⁇ C). In some embodiments, the cell extract is harvested by a 2 or 3 stage filters followed by terminal sterile 0.22 ⁇ m filtration.
• a nucleic acid encoding a fusion polypeptide is introduced into a host cell, such that the fusion polypeptide is expressed by the host cell.
• a nucleic acid encoding a kinase that phosphorylates a fusion polypeptide is introduced into a host cell so that the host cell expresses the kinase.
• both a nucleic acid encoding a fusion polypeptide and a nucleic acid encoding a kinase that phosphorylates it are introduced into the same host cell; in some such embodiments, a single nucleic acid molecule may encode both.
• a nucleic acid molecule introduced into a cell is an RNA (e.g., an mRNA); in some such embodiments, encoded polypeptide(s) (e.g., a fusion polypeptide and/or a kinase) is/are expressed from such RNA.
• a nucleic acid molecule introduced into a cell is a DNA (e.g., a single stranded DNA or a double stranded DNA).
• a nucleic acid is introduced into a cell so that coding sequences integrate into the host cell (e.g., into its Page 45 of 144 11666457v1 Attorney Docket: 2013762-0017 genome); in some such embodiments encoded polypeptide(s) (e.g., a fusion polypeptide and/or a kinase) is/are expressed therefrom, [0164]
• a nucleic acid molecule introduced into a cell includes one or more expression elements, e.g., that may regulate expression of such encoded polypeptide(s).
• a nucleic acid molecule introduced into a cell may be designed or intended to become associated (e.g., by integration) with one or more regulatory elements in a host cell.
• a vector e.g., a transposon
• sequences encoding a fusion polypeptide and/or a kinase as described herein is used to express a fusion polypeptide and/or a kinase in a host cell.
• a host cell may be selected from a variety of the available and known host cells (e.g., Human Embryonic Kidney (HEK) cells, suspension HEK293 cells, Chinese Hamster Ovary cells) suitable expressing fusion polypeptides disclosed herein.
• a host cell is a mammalian cell.
• a variety of methods are available to introduce a nucleic acid (e.g., a vector, such as an expression vector) into host cells.
• a nucleic acid may be introduced into host cells using transfection.
• transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection.
• a nucleic acid may be introduced into a host cell using transduction. In some embodiments, a nucleic acid may be introduced into a host cell using electroporation. In some embodiments, a nucleic acid may be introduced into a host cell using particle delivery, e.g., polymer particle delivery, lipid particle delivery, gold particle delivery, etc. Phosphorylation [0168] In some embodiments, the present disclosure provides a method of manufacturing a phosphorylated form of fusion polypeptides disclosed herein by contacting the fusion polypeptide with a kinase.
• a nucleic acid such as a nucleic acid encoding a fusion polypeptide and/or a kinase is introduced into a host cell.
• a fusion polypeptide is contacted with a kinase by co-expressing a fusion polypeptide in a host cell with a kinase.
• co-expression is achieved by introducing two vectors, one comprising a recombinant polynucleotide encoding a fusion polypeptide and one comprising a recombinant polynucleotide encoding a kinase, into a host cell.
• co-expression is achieved by introducing a single, multi-cistronic (e.g., bi-cistronic) vector that comprises a plurality of recombinant polynucleotides (e.g., such as a transposon).
• a recombinant polynucleotide encodes a fusion polypeptide and a recombinant polynucleotide encodes a kinase.
• a transformed host cell is cultured following introduction of a vector (e.g., a transposon) into a host cell.
• a vector e.g., a transposon
• a kinase upon co-expression of a fusion polypeptide and a kinase in a host cell, a kinase can contact a fusion polypeptide and phosphorylate it.
• co-expression is achieved by introducing two vectors, one comprising a recombinant polynucleotide encoding a fusion polypeptide and one comprising a recombinant polynucleotide encoding a kinase, into a host cell.
• two vectors are introduced at a ratio of vector encoding fusion polypeptide to vector encoding a kinase introduced into a host cell optimized to achieve a desired, relative level of expression of fusion polypeptide to kinase.
• a ratio of vector encoding fusion polypeptide to vector encoding a kinase is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30: 1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
• co-expression is achieved by introducing one vector comprising both a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase (e.g., a bi-cistronic vector) into a host cell.
• co-expression is achieved by introducing one transposon comprising both a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase into a host cell.
• a transposon is a DNA transposon.
• the transposon or part thereof is integrated in the host cell genome by an integration enzyme (i.e., by an integrase enzyme, such as a DDE/D integrase enzyme).
• an integration enzyme is delivered to the host cell as mRNA.
• an integration enzyme is a Page 47 of 144 11666457v1 Attorney Docket: 2013762-0017 PiggyBac enzyme.
• an integration enzyme is a Leap-In Transposase.
• the transposon or part thereof is not integrated into the genome by random integration.
• a single copy of a polynucleotide encoding a fusion polypeptide of the present disclosure is integrated into specific multiple host cell genomic loci.
• an integration of a polynucleotide encoding a fusion polypeptide of the present disclosure is irreversible. Irreversible integration of the fusion polypeptide into the host cell genome may ensure stable integration. Hereby allowing production of a very stable cell line.
• a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polynucleotide encoding a kinase are operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.).
• a sequence or sequences that control expression are selected to achieve a desired level of expression.
• multiple regulatory nucleotide sequences that control expression e.g., promoters
• a ratio is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30: 1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
• a regulatory nucleotide sequence is a promoter.
• a specific ratio e.g., 2:1, 4:1, 8:1 or 15:1 is achieved using a single transposon with two promoters to express the fusion polypeptide and the kinase.
• a single separate transposon comprises promoters of differing strength to produce the desired ratio (e.g., 8:1).
• the promoter is a CMV or EF1a promoter.
• a fusion polypeptide is under the control of a CMV promoter or EF1a promoter.
• the promoter is a SV40 or Ubc promoter.
• a kinase is under the control of a SV40 promoter or Ubc promoter.
• a ratio of fusion polypeptide to kinase is 8:1.
• transformed host cells i.e., host cells into which a nucleic acid, such as a nucleic acid encoding a fusion polypeptide and/or a kinase
• a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer.
• a transformed host cells are cultured for at least 1 day, 2 days, 3 days, 4 days, 5 Page 48 of 144 11666457v1 Attorney Docket: 2013762-0017 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or longer.
• Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art.
• host cells secret a phosphorylated form of a fusion polypeptide to the cell extract.
• a host cell may show increased secretion of phosphorylated fusion polypeptides to the cell extract compared to an unhosphorylated fusion polypeptides. In some embodiments, a host cell secrets comparable levels of phosphorylated fusion polypeptide and unphosphorylated fusion polypeptides to the cell extract. In some embodiments, a cell extract comprises an unphosphorylated form of a fusion polypeptide. In some embodiments, a cell extract comprises a phosphorylated form of the fusion polypeptide. In some embodiments, a cell extract comprises a mixture of both unphosphorylated and phosphorylated forms of a fusion polypeptide.
• a cell extract comprises more phosphorylated forms of a fusion polypeptide than unphosphorylated forms of a fusion polypeptide.
• a host cell secretes a phosphorylated form of a fusion polypeptide to the cell extract, but does not secrete a kinase. In some embodiments, a host cell does not secrete a kinase or only secretes a small amount of a kinase.
• a host cell extract comprises a phosphorylated form of a fusion polypeptide. In some embodiments, a host cell extract comprises host cell proteins and/or host cell nucleotides.
• a phosphorylated form of a fusion polypeptide is harvested from transformed host cells and clarified by centrifugation.
• a transformed host cell is characterized in that a culture thereof produces the fusion protein with a titer of at least 200 mg/L, such as at least 250 mg/L, such as at least 300 mg/L, such as at least 350 mg/L, such as at least 400 mg/L, such as at least 450 mg/L, such as at least 500 mg/L, such as at least 550 mg/L, such as at least 600 mg/L, such as at least 650 mg/L, such as at least 700 mg/L, such as at least 750 mg/L, such as at least 800 mg/L, such as at least 850 mg/L, such as at least 900 mg/L, such as at least 950 mg/L, such as at least 1 g/L or more.
• one or more serine residues at position 43, 281, 306, 311, 316, 365 or 481 of SEQ ID NO: 2 are phosphorylated. In some embodiments, one or more serine residues at position 43, 154, 281, 306, 311, 316, 365 or 481 of SEQ ID NO: 2 are phosphorylated. In some embodiments, one or more serine residues at position 43, 154, 168, 281, 306, 311, 316, 365 or 481 of SEQ ID NO: 2 are phosphorylated.
• one or more serine residues at position 43, 154, 168, 281, 306, 311, 316, 365, 406 or 481 of SEQ ID NO: 2 are phosphorylated. In some embodiments, at least serine residue at position 481 of SEQ ID NO: 2 is phosphorylated.
• the present disclosure provides technologies (e.g., manufacturing technologies) that are or comprise methods of purification – e.g., methods that comprise one or more purification steps.
• a phosphorylated form of a fusion protein is purified from an extract of cell described herein.
• a purification step may include removal of commonly aberrant products (e.g., residual protein, host cell contaminants (e.g., host DNA and/or protein, etc.)) from a cell extract.
• a manufacturing method comprising one or more purification steps results in a high purity preparation of a phosphorylated form of a fusion polypeptide (See, for example, Examples 10-11).
• a high purity preparation of a phosphorylated form of a fusion polypeptide comprises reduced host cell proteins comparable to a cell extract described herein above. In some embodiments, such high purity preparation does not comprise any host cell proteins.
• a high purity preparation comprises less than 100 ng/mg host cell proteins, such as less than 50 ng /mg, such as less than 40 ng/mg, such as less than 30 ng/mg, such as less than 20 ng/mg, such as less than 10 ng/mg, such as less than 9 ng/mg, such as less than 8.5 ng/mg host cell proteins.
• a high purity preparation of a phosphorylated form of a fusion polypeptide comprises reduced host cell DNA comparable to a cell extract described herein above. In some embodiments, such high purity preparation does not comprise any host cell DNA.
• a high purity preparation comprises less than 10 pg/mg host cell DNA, such as less than 9 pg/mg, such as less than 8 pg/mg, such as less than 7 pg/mg, such as less than 6 pg/mg, such as less than 5 pg/mg, such as less than 4 pg/mg, such as less Page 50 of 144 11666457v1 Attorney Docket: 2013762-0017 than 3 pg/mg, such as less than 2 pg/mg, such as less than 1.5 pg/mg, such as less than 1 pg/mg, such as less than 0.9 pg/mg, such as less than 0.8 pg/mg, such as less than 0.7 pg/mg host cell DNA.
• a high purity preparation comprises a low degree of in- process compounds.
• in-process compounds may be Tropolone, Pluronic, PDMS, Octamethylcyclo tetrasiloxane D4, TDAO, and/or Fam20C).
• a high purity preparation comprises less than 1 mg/mL TDAO, such as less than 0.9 mg/mL TDAO such as less than 0.8 mg/mL TDAO such as less than 0.7 mg/mL TDAO, such as less than 0.6 mg/mL TDAO, such as less than 0.5 mg/mL TDAO, such as less than 0.4 mg/mL TDAO, such as less than 0.3 mg/mL TDAO.
• a high purity preparation comprises less than 5000 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 4000 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 3000 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 2500 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 2000 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 1800 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 1000 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 750 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 500 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 300 ng Fam20C/mg IL-12 fusion polypeptide, such as less than 200 ng Fam20C
• a phosphorylated form of a fusion polypeptide can be purified by a including one or more chromatography purification steps. In some embodiments, one or more conventional chromatography steps is used. In some embodiments, a conventional chromatography step utilizes anion or cation exchange, hydrophobic interactions, hydroxyapatite chromatography. [0182] In some embodiments, a phosphorylated form of a fusion polypeptide can be purified by including one or more purification steps selected from an ion chromatography step (e.g., an anion chromatography step) and a hydrophobic interaction chromatography step. Accordingly, resulting in separation of the phosphorylated fusion polypeptide from impurities.
• an ion chromatography step e.g., an anion chromatography step
• a substrate utilized for purification e.g., for chromatography
• properties such that, in accordance with the present disclosure, permits a different retention time for a fusion polypeptide relative to any other undesirable components in the preparation of fusion polypeptide.
• a phosphorylated form of a fusion polypeptide is not purified by an affinity-based purification method.
• provided purification technologies do not utilize affinity chromatography.
• a phosphorylated form of a fusion polypeptide may be eluted from a solid substrate.
• elution may be carried out using specific elution.
• specific elution is completed by challenging a polypeptide-substrate complex by an agent or agents that will complete for complexation with either a substrate or a polypeptide, releasing a polypeptide into solution.
• elution may be carried out using non-specific elution.
• non-specific elution is completed by manipulating solvent or buffer conditions (e.g., increasing concentration of a buffer, e.g., an imidazole buffer) to reduce the associate rate constant, resulting in dissociation of the polypeptide from the substrate.
• First chromatography step [0186]
• methods according to the present disclosure comprise at least one ion chromatography step (e.g., an anion chromatography step). In some embodiments, methods according to the present disclosure comprise at least one anion chromatography step.
• a first chromatography step is a capturing step (e.g., an anion chromatography capturing step).
• a capturing step e.g., an anion chromatography capturing step.
• polypeptide phosphorylation confers variability in a polypeptide’s charge, permitting separation of differentially phosphorylated polypeptides using ion-exchange chromatography (e.g., anion-exchange chromatography).
• Anion-exchange chromatography is a form of ion exchange where a negatively charged biomolecule (e.g., a phosphorylated form of a fusion Page 52 of 144 11666457v1 Attorney Docket: 2013762-0017 polypeptide disclosed herein) binds to a positively charged solid substrate (e.g., resin).
• the positively charged solid substrate hereby captures the negatively charged fusion polypeptide from the cell extract and at the same time remove positively charged impurities from the cell extract as well as fusion polypeptide aggregates, as aggregates seem to have a less negatively charge.
• anion-exchange chromatography can be used to resolve polypeptides with different numbers of phosphorylated amino acid residues (e.g., differentially phosphorylated polypeptides).
• Anion exchange chromatography can enrich a fusion polypeptide preparation for highly phosphorylated species (e.g., fusion polypeptides having more than 6 phosphorylation sites). Accordingly, in some embodiments, a preparation having a high concentration of phosphorylated fusion polypeptide and a low concentration of positively charged impurities is generated through use of an anion-exchange chromatography step (e.g., as a first step).
• a step of purifying a phosphorylated form of a fusion polypeptide from a cell extract comprises an anion chromatography capture step.
• an anion chromatography capture step is a first capture step.
• anion exchange chromatography utilizes an ion exchange resin with covalently bound positively charged groups, such as quaternary amino groups.
• commercially available anion exchange resins include Q Sepharose, DEAE Sepharose, ⁇ , GigaCap Q 650M and 650S.
• Binding a negatively charged biomolecule (e.g., a phosphorylated form of a fusion polypeptide) to an anion exchange material comprises, in some embodiments, exposing the negatively charged biomolecule to the resin under appropriate conditions (e.g., pH/conductivity) hereby immobilizing the biomolecule to the anion exchange resin through ionic interactions between the negatively charged biomolecule and a charged group or charged groups of the ion exchange material.
• a wash step may entail passing an appropriate buffer through the chromatography resin to wash out unwanted material such as host cell proteins or host cell nucleotides.
• a wash buffer may include varying conditions such as pH, conductivity, with the goal of dissociating impurities that are non-specifically bound with the chromatography resin.
• a wash step utilizes a mixture of an equilibration and an elution buffer.
• Page 53 of 144 11666457v1 Attorney Docket: 2013762-0017 [0192] Phosphorylated forms of fusion polypeptides can be eluted from a solid substrate (e.g, a positively charged resin) using an elution.
• a negatively charged agent e.g., a phosphorylated form of a fusion polypeptide
• a buffer that decreases interaction between the anion exchange resin and the negatively charged agent e.g., a phosphorylated fusion polypeptide
• such an elution buffer may have higher concentration of salt and/or different pH so that dissociation of the negatively charged agent from the chromatography resin is promoted.
• a gradient elution buffer e.g., a buffer with increasing salt concentrations
• a buffer is, for example, a Tris buffer.
• a linear gradient of Tris buffer is utilized.
• a linear gradient of Tris buffer comprises over a linear gradient from 20 mM Tris, pH 7.1 to 20 mM Tris, 1 M NaCl, pH 7.1 over a pre-defined period of time.
• a linear gradient is conducted over a period of 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes, or longer.
• a first anion chromatography capture step uses a Tris buffer.
• a first anion chromatography capture step is performed at a pH of about 6 to about 9, such as about 7 to about 8. [0195]
• a first anion chromatography capture step is performed using capturing beads.
• a first step capture bead has a diameter of at least 50 ⁇ m, such as at least 55 ⁇ m, such as at least 60 ⁇ m, such as 65 at least ⁇ m, such as at least 70 ⁇ m, such as at least 75 ⁇ m.
• a first capture bead is a GigaCap Q 650M.
• a first anion chromatography capture step comprises an immobilizing step (e.g., binding a phosphorylated fusion polypeptide to a chromatography column), a pre-elution wash step, and an elution step (e.g., eluting a phosphorylated fusion polypeptide).
• a resin bead of about 50 to about 100 micrometer particle size (mean), such as 75 micrometer particle size (mean) is utilized for immobilizing a Page 54 of 144 11666457v1 Attorney Docket: 2013762-0017 phosphorylated fusion polypeptide.
• an immobilization composition having a low salt concentration is used during immobilization of a phosphorylated fusion polypeptides to a chromatography column (e.g., 0 M sodium chloride is used).
• a pre-elution composition having an intermediate salt concentration, comparable to the salt concentration of the immobilization composition and the elution composition is utilized during a pre-elution wash step.
• an elution composition having a high salt concentration is used during the elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 350 mM sodium chloride is used).
• a first anion chromatography capture step is performed at a pH within the range of about 7 to about 8 (e.g., 7.4).
• a flow rate of 200-400 cm/h is used (e.g., 300 cm/h).
• Second chromatography step [0197]
• methods according to the present disclosure comprise at least one hydrophobic interaction chromatography step.
• a hydrophobic interaction step is performed after a first anion chromatography step.
• a phosphorylated form of a fusion protein according to the present disclosure has low hydrophobicity (e.g., due to its phosphorylation degree and hence its charge) which can be used to separate it from hydrophobic impurities.
• hydrophobic host cell impurities e.g., host cell proteins
• fusion polypeptide aggregates are separated from a preparation of a phosphorylated form of a fusion polypeptide.
• a preparation e.g., a high purity preparation
• a hydrophobic interaction step may, in some embodiments, separate product or process related impurities, e.g., hos cell proteins or aggregated product (e.g., fusion polypeptide aggregates) from a phosphorylated form of a fusion polypeptide based on differences in hydrophobic interactions of the phosphorylated fusion polypeptide and the impurities with a hydrophobic material.
• a step may in some embodiments be referred to as a polishing step.
• hydrophobic interaction resins include, but are not limited to, hydrophobic ligands such as alkyl groups ranging from 2 to 8 carbon atoms, or aryl groups such as phenyl.
• Binding a negatively charged agent (e.g., a phosphorylated form of a fusion polypeptide) to a hydrophobic interactions resin comprises, in some embodiments, exposing the biomolecule to the resin under appropriate conditions (pH/conductivity) hereby immobilizing the biomolecule to the hydrophobic resin through hydrophobic interactions between the biomolecule and a non-polar group of the hydrophobic interactions material.
• Hydrophobic interaction bindings typically happen at a high concentration salt (e.g., 1 to 1.8 M Ammonium Sulfate).
• a phosphorylated form of a fusion polypeptide is immobilized at a high salt concentration (e.g., 1.4 M sodium sulfate).
• a phosphorylated form of a fusion polypeptide is eluted with a linear gradient ranging from 1.4 M ammonium sulfate to 0 M ammonium sulfate.
• a wash step may entail passing an appropriate buffer through the chromatography resin to wash out unwanted material such as less hydrophobic host cell proteins.
• a wash buffer may have different pH, so that dissociating of the low hydrophobicity agent from impurities that are non-specifically bound with the chromatography resin is promoted.
• a wash step utilizes a mixture of an equilibration and an elution buffer.
• a low hydrophobicity agent e.g., a phosphorylated form of a fusion polypeptide
• a buffer that decreases interaction between the hydrophobic interactions resin and the negatively charged agent e.g., a phosphorylated fusion polypeptide
• such an elution buffer may have lower concentrations of salts or changes in pH that promote dissociation of the bio molecule from the chromatography resin.
• a phosphorylated form of a fusion polypeptide is eluted at a low salt concentration (e.g., 750 mM sodium sulfate).
• an immobilization composition having a high salt concentration is used during immobilization of a phosphorylated fusion polypeptides to a chromatography column (e.g., 1.4 M ammonium sulfate is used).
• an elution composition having a low salt concentration is used during the elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 740 mM ammonium sulfate is used).
• a hydrophobic interaction chromatography step is performed at a pH within the range of about 7 to about 8 (e.g., 7.4).
• methods according to the present disclosure comprise a first anion chromatography step and a second anion chromatography step. In some embodiments, methods according to the present disclosure comprise a first anion chromatography step and a second anion chromatography step, wherein the first anion chromatography step is a capture step and the second anion chromatography step is a polishing step. In some embodiments, a second anion chromatography step is performed after a hydrophobic interaction chromatography step.
• methods according to the present disclosure comprise the following steps: i) a first anion chromatography step; ii) a hydrophobic interaction chromatography step; and iii) a second anion chromatography step.
• a second anion chromatography step is performed using capturing beads.
• a second step capture bead has a diameter of at the most 50 ⁇ m, the most 45 ⁇ m, the most 40 ⁇ m, the most 35 ⁇ m.
• a first capture bead is a GigaCap Q 650S.
• a second anion chromatography capture step comprises an immobilizing step (e.g., binding a phosphorylated fusion polypeptide to a chromatography column), a pre-elution wash step, and an elution step (e.g., eluting a phosphorylated fusion Page 57 of 144 11666457v1 Attorney Docket: 2013762-0017 polypeptide).
• a resin bead of about 10 to about 50 micrometer particle size (mean), such as 35 micrometer particle size (mean) is utilized for immobilizing a phosphorylated fusion polypeptide.
• an immobilization composition having a low salt concentration is used during immobilization of a phosphorylated fusion polypeptides to a chromatography column (e.g., 0 M sodium chloride is used).
• a pre-elution composition having an intermediate salt concentration, comparable to the salt concentration of the immobilization composition and the elution composition e.g., 274 mM sodium chloride is used
• an elution composition having a high salt concentration is used during the elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 355 mM sodium chloride is used).
• a first anion chromatography capture step is performed at a pH within the range of about 7 to about 8 (e.g., 7.3). In some embodiments, a flow rate of 200-400 cm/h is used (e.g., 300 cm/h).
• methods according to the present disclosure comprise a viral inactivation step or viral removal step. In some embodiments, a viral inactivation step or viral removal step is performed before or after any of the chromatography steps described herein above. In one embodiment, a viral inactivation step or viral removal step is performed prior to the first chromatography step (e.g., a first anion chromatography step).
• a viral inactivation step comprises pH inactivation or chemical inactivation (e.g., through use of a chemical agent such as a surfactant).
• a viral inactivation step comprises utilizes a detergent as, without being bound a particular theory, IL12-ABP is sensitive and might aggregate at low pH.
• a viral inactivation step comprises utilizes a detergent selected from Myristyldimethylamine N- oxide, TDAO, Triton X-100 or Polysorbates.
• a viral removal step comprises a filtration step.
• the present disclosure provides technologies for characterizing fusion polypeptides (e.g., phosphorylated or Page 58 of 144 11666457v1 Attorney Docket: 2013762-0017 unphosphorylated) or preparations thereof and/or of complexes comprising such fusion polypeptides and metal hydroxides, or preparations of such complexes.
• one or more characterizations may be performed during and/or following one or more steps of a manufacturing process (e.g., a purification process) as described herein.
• a particular preparation process may be modified or terminated in light of a characterization (e.g., if a particular preparation fails to meet one or more specifications).
• characterization may involve assessment of one or more of metal-hydroxide retention, degree of phosphorylation, heterogeneity of phosphorylation, signaling activity, and/or efficacy. See exemplary suitable characterizations assays under “Data Analysis” in Example 3.
• the amount of each phosphorylated fusion polypeptide is measured.
• the amount of each phosphorylated fusion polypeptide is measured according to one or more of a variety of methods available in the art.
• phosphorylated fusion polypeptides are measured using a malachite green assay, analytical ion exchange, spectrophotometer, colorimetric assays, intact mass analysis by mass spectroscopy, and/or western blot. See exemplary suitable characterizations assays under “Data Analysis” in Example 3.
• Exemplary characterization of phosphate content [0211]
• degree of phosphorylation e.g., of fusion polypeptides of the present disclosure is characterized.
• degree of phosphorylation can be determined by a colorimetric method.
• a colorimetric method is or comprises a malachite green assay.
• a malachite green assay is based on quantification of a green complex formed between Malachite green, molybdate, and free orthophosphate which can be measured (e.g., using a spectrophotometer or plate reader).
• degree of phosphorylation e.g., the average number of phosphate molecules per polypeptide
• degree of phosphorylation is about 4-12, about 5-11, about 6-10, about 7-9, or about 7.5-8.5 (See, for example, exemplary degree of phosphorylation in Example 10).
• degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7.7.8, 7.9, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, or 11.
• degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7.7.8, 7.9, 7.10, 8.0, or 8.1.
• heterogeneity of phosphorylation of fusion polypeptides of the present disclosure and/or preparations thereof are characterized.
• heterogeneity of phosphorylation is a measurement of the degree of phosphorylation within a given preparation of fusion polypeptide.
• heterogeneity of phosphorylation is a measurement of the degree of phosphorylation across a plurality of preparations of fusion polypeptide.
• heterogeneity of phosphorylation is a measurement of location of particular phosphate groups on a polypeptide within a given preparation of fusion polypeptide. In some embodiments, heterogeneity of phosphorylation is a measurement of location of particular phosphate groups on a polypeptide across a plurality of preparations of fusion polypeptide.
• a variety of technologies is available for measurement of heterogeneity of phosphorylation. For example, in some embodiments, degree of phosphorylation can be determined by a chromatography method, as described herein above.
• differentially phosphorylated polypeptides are dephosphorylated.
• dephosphorylation comprises use of a phosphatase (e.g., a lambda phosphatase).
• a fusion polypeptide is incubated with a phosphatase for a period of time and at a temperature that permits activity of said phosphatase and dephosphorylation of said fusion polypeptide.
• dephosphorylation occurs at an incubation temperature of approximately 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or higher.
• dephosphorylation occurs for an incubation time of 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, or longer.
• dephosphorylation occurs for an incubation time of 25-65 minutes, 30-60 minutes, 35-55 minutes, 40-50 minutes, 30-65 minutes, 35-65 minutes, 40-65 minutes, 45-65 minutes, 50-65 minutes, or 55-65 minutes.
• differentially phosphorylated polypeptides are dephosphorylated prior to separation.
• differentially phosphorylated polypeptides of the present disclosure are assessed relative to an appropriate reference standard (e.g., a dephosphorylated and/or non-phosphorylated form of a fusion polypeptide).
• an appropriate reference standard e.g., a dephosphorylated and/or non-phosphorylated form of a fusion polypeptide.
• the amount of each differentially phosphorylated polypeptide is measured. In some embodiments, the amount of each differentially phosphorylated polypeptide is measured according to a variety of methods available in the art.
• differentially phosphorylated polypeptides are measured using a malachite green assay, analytical ion exchange, spectrophotometer, colorimetric assays, and/or western blot.
• a metal-hydroxide e.g., aluminum hydroxide
• fusion polypeptides of the present disclosure when exposed to a metal-hydroxide (e.g., aluminum hydroxide) forms a complex therewith.
• retention of a fusion polypeptide of the present disclosure on a metal- hydroxide is characterized. A variety of methods are available to measure metal-hydroxide retention.
• metal-hydroxide retention can be measured by ellipsometry, surface plasmon resonance, optical waveguide lightmode spectroscopy, attenuated total internal reflectance-infrared spectroscopy, circular dichroism spectroscopy (CD), total internal reflectance-infrared spectroscopy (TIRF), and other high resolution microscopy techniques.
• metal-hydroxide retention is characterized using an in vitro assay. For example, fusion polypeptides at a known concentration are mixed with an excess of metal-hydroxide. The concentration of free, non-complexed fusion polypeptides is quantified and compared to a standard curve to determine metal-hydroxide retention.
• the concentration of free, non-complexed fusion polypeptide can be assessed according to a variety of method known to those of skill in the art. For example, and without limitation, in Page 61 of 144 11666457v1 Attorney Docket: 2013762-0017 some embodiments, free, non-complexed, fusion polypeptides are quantified by enzyme- linked immunosorbent assay (ELISA), western blot, bicinchoninic acid assay, or Bradford assay.
• ELISA enzyme- linked immunosorbent assay
• fusion polypeptides as described herein are characterized for activity (e.g., signaling activity).
• activity is characterized by assessing signaling activity (e.g., signaling competency) compared to an appropriate reference standard.
• An appropriate reference standard can be, for example, a wild-type polypeptide and/or a fusion polypeptide lacking a metal-hydroxide binding polypeptide.
• a variety of methods are available to assess signaling competency. In some embodiments, for example, signaling competency is assessed using an in vitro- or in vivo- based activity assay. [0223] In some embodiments, signaling activity is assessed with an in vitro activity assay. In some embodiments, an in vitro activity assay comprises measuring activation or inhibition of downstream signaling of a fusion polypeptide. In some embodiments, measuring activation or inhibition of downstream activity comprises use of a reporter (e.g., a reporter assay).
• a reporter e.g., a reporter assay
• a reporter assay measures activity using a detectable molecule (e.g., a reporter) that correlates with fusion polypeptide activity.
• a reporter comprises a fluorescent, bioluminescent, and/or other detectable probe known to those of skill in the art.
• a reporter comprises use of a gene reporter.
• a gene reporter for example, can be activated upon signaling elicited from a polypeptide. For example, upon activation of gene reporter transcription, a detectable product or enzyme that can be activated upon addition of substrate, generating a detectable product and/or by-product, can be utilized.
• an enzyme useful in accordance with a reporter assay is, for example, luciferase or an alkaline Page 62 of 144 11666457v1 Attorney Docket: 2013762-0017 phosphatase (e.g., secreted alkaline phosphatase, SEAP).
• a HEK- blue-IL12 reporter assay is utilized.
• signaling activity is assessed with an in vivo activity assay.
• a fusion polypeptide is administered to a subject (e.g., a mouse, non-human primate, human, etc.) and activity is assessed.
• activity is assessed, for example, by measuring activation or inhibition of downstream signaling of a fusion polypeptide as compared to an appropriate reference standard (e.g., activity of a wild- type polypeptide).
• an appropriate reference standard e.g., activity of a wild- type polypeptide.
• a variety of methods are available to measure activation or inhibition of downstream signaling of a fusion polypeptide. For example, and without limitation, differential gene expression, protein expression, and/or alterations in post-translational modifications induced by a fusion polypeptide can be measured.
• Exemplary efficacy characterization [0226] In some embodiments, efficacy can be characterized according to one or more of a variety of methods that are available.
• a fusion polypeptide (or complex thereof) as described herein is administered (e.g., by intratumoral or peritumoral injection) to a subject (e.g., mouse, non-human primate, human, etc.) and efficacy is determined in comparison to an appropriate reference standard.
• a subject e.g., mouse, non-human primate, human, etc.
• efficacy is determined in comparison to an appropriate reference standard.
• An appropriate reference standard can be, for example, a wild-type polypeptide and/or a polypeptide lacking a metal-hydroxide binding polypeptide, or having a metal-hydroxide binding polypeptide in a non-binding (e.g., non-phosphorylated) state.
• efficacy is determined pre-clinically in an animal model (e.g., in mice, rats, non-human primates, etc.).
• a fusion polypeptide is administered (e.g., by intratumoral or peritumoral injection) to an animal model.
• an animal model is an animal model with a tumor (e.g., an animal model of cancer).
• a cancer animal model is generated by inoculating said animal model with tumor cells.
• an animal model is inoculated with tumor cells at the flank region.
• an animal model is inoculated with tumor cells in a clinically relevant region (e.g., a mammary fat pad).
• an animal model of cancer is administered a fusion polypeptide (e.g., a preparation thereof such as a preparation of a phosphorylated fusion polypeptide) of the present disclosure.
• an animal model of cancer is administered a reference standard (e.g., a wild-type polypeptide and/or a polypeptide lacking a metal-hydroxide binding polypeptide).
• one or more of a variety of available, pre-determined measurements for efficacy known in the art is/are assessed over time relative to an appropriate reference standard (e.g., a wild-type polypeptide and/or a polypeptide lacking a metal- hydroxide binding polypeptide).
• an appropriate reference standard e.g., a wild-type polypeptide and/or a polypeptide lacking a metal- hydroxide binding polypeptide.
• efficacy of a fusion polypeptide e.g., a preparation thereof such as a preparation of a phosphorylated fusion polypeptide is determined clinically.
• a fusion polypeptide is administered (e.g., by intratumoral, peritumoral injection, or into a tumor-draining lymph node) to a subject with a tumor.
• a variety of available, pre-determined measurements for efficacy known in the art such as, for example, tumor volume and/or percent survival are assessed over time relative to a subject with a tumor administered reference standard (e.g., a treatment in the art of known efficacy and/or placebo).
• Fusion polypeptide-metal hydroxide complex formation and preparation [0230]
• a phosphorylated form of a fusion polypeptide described herein when exposed to a metal-hydroxide (e.g., aluminum hydroxide) forms a complex therewith.
• fusion polypeptides comprise hydroxyl replacement groups (e.g., phosphate groups) for adsorption via ligand exchange with a metal hydroxide.
• a fusion polypeptide can, via electrostatic interactions, form a complex with a metal hydroxide.
• a phosphorylated form of a fusion polypeptide comprising an IL-12 immunomodulatory domain as described herein, when exposed to an aluminum hydroxide forms a complex therewith. Accordingly forming an IL-12 complex according to the present disclosure.
• a fusion polypeptide metal-hydroxide complex e.g., an IL-12 complex
• mixing Page 64 of 144 11666457v1 Attorney Docket: 2013762-0017 occurs in a buffer (e.g., a tris-buffered saline buffer). In some such embodiments, a buffer does not contain phosphate.
• a buffer does not contain a substance or substances that solubilize a metal-hydroxide (e.g., citric acid, malic acid, or lactic acid).
• a metal-hydroxide e.g., citric acid, malic acid, or lactic acid.
• buffers that contain phosphate compete with, and can hinder, complex formation.
• mixing occurs for a duration of time at a particular temperature. In some such embodiments, a duration of time is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 45 minutes.
• a particular temperature is approximately 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.
• the present disclosure provides a fusion polypeptide-metal hydroxide complex preparation comprising a fusion polypeptide- metal hydroxide complex according to the present disclosure.
• a preparation comprising a fusion polypeptide-metal hydroxide complex comprises any of a variety of suitable metal-hydroxide known in the art.
• the present disclosure provides a pharmaceutical composition comprising a fusion polypeptide disclosed herein.
• the pharmaceutical composition is formulated as a fusion polypeptide-metal hydroxide complex.
• a pharmaceutical composition comprises a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant.
• Table 1 Exemplary Amino Acid Sequences SEQ ID Pol e tide Se ence D L T E S SI M C E D E Page 65 of 144 11666457v1 Attorney Docket: 2013762-0017 2 Mature IL-12 IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSG fusion KTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP l tid KNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAAT T P K N Q I E G P T T P D SS T G P T T P K N Q I S Q S P P S Y S W Y R S N S S S R K M S 11666457v1 Attorney Docket: 2013762-0017 STTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQII LDKGMLVA
• a transposon system used in the present Example is designed to efficiently and consistently deliver IL-12 fusion polypeptides and hFAM20Cs to a host cell (e.g., CHO cells).
• the transposon system comprises a transposon construct (DNA) and a cognate transposon enzyme (mRNA) (also termed leap in enzyme or transposase).
• the transposon construct was generated by synthetizing nucleotide sequences coding for IL-12 fusion polypeptide and hFAM20C and inserted them in a dual open reading frame (ORF) expression construct.
• the transposon construct was also designed to express and deliver a glutamine synthase (GS).
• GS glutamine synthase
• the transposase belongs to the DDE/D integrase family and catalyze an efficient cut-and-paste (excision followed by integration) process to integrate their cognate transposons into the target genome.
• the enzymatic mechanism of action results in single copy integrants in multiple genomic loci.
• This homologous integration mechanism is several orders of magnitude more efficient than non-homologous recombination. Moreover, it eliminates the formation of concatemers as well as deleted and rearranged transgenes.
• Leap- Page 70 of 144 11666457v1 Attorney Docket: 2013762-0017 In mediated transposition enriches the integration sites in transcriptionally active genomic regions.
• One of the notable features of the Leap-In Transposase mediated stable integration mechanism is the strong comparability between the Leap-In mediated stable pools and the monoclonal cell lines derived from those pools. The comparability applies to both productivity and basic global physicochemical attributes.
• the transposase enzyme is delivered as mRNA.
• transposase a gene that is excreted from human cells.
• the transposase mRNA manufacturing process utilizes recombinant protein components but is free of animal-derived raw materials.
• CHO cell line A glutamine synthase (GS) null Chinese hamster ovary (CHO) cell line (GS- CHO cell line) was develop and used as parental cell line (production cell line). The glutamine synthetase (GS) gene is knocked out so that the resulting cells cannot synthesize their own glutamine and are completely dependent on glutamine supplementation for growth. GS is present on the transposon construct used for transfection so that successful transfectants can be selected in glutamine-free medium.
• IL-12 fusion protein:Fam20C ratio studies [0242] Experiments were performed to establish the ratio between IL-12 fusion protein:Fam20C for optimal cell growth and high productivity of the fusion polypeptide.
• GS-CHO cells and Leap-In transposon systems as described in Example 1 are used in Example 2.
• the DNA sequences of IL-12 fusion polypeptide and hFAM20C in the transposon construct were confirmed by Sanger sequencing.
• the DNA sequence of the entire stable expression plasmid was confirmed by Oxford nanopore sequencing.
• Transfection [0253] Non-transfected GS-CHO cells were maintained in shake flasks with growth medium and kept in shaker incubators at 5% CO2, 37°C, and 70-80% relative humidity.
• transfections of the transposon system were performed using a Thermo Fisher Neon electroporation device. Post- transfection recovery was performed for 24 to 48 hours in growth medium in T25 flasks under static conditions. After the recovery period, the transfectant cultures were introduced to metabolic selection by media exchanging the cells into a glutamine-free formulation.
• Fed-batch production and fed-batch run [0255] Fed-batch production runs were performed for pool ranking in TubeSpin bioreactors. Working volume was 10 mL, and shaker speed 240 rpm.
• SDA Page method For reduced SDS-PAGE gels, clarified supernatant was prepared in NuPAGETM LDS Sample Buffer (4X) (Thermo Fisher catalog number NP0008) and reduced using NuPAGETM Sample Reducing Agent (10X) (Catalog number NP0009) as per vendor protocol. The reduced samples were run on NuPAGE Novex 4-12 Bis-Tris Protein Gels (Catalog numbers NP0329BOX, WG1403BX10) and stained using InstantBlueTM Protein Stain (Novus Biologicals catalog number ISB1L-1L) as per vendor protocol.
• NuPAGETM LDS Sample Buffer (4X) Thermo Fisher catalog number NP0008
• NuPAGETM Sample Reducing Agent (10X) Catalog number NP0009
• the reduced samples were run on NuPAGE Novex 4-12 Bis-Tris Protein Gels (Catalog numbers NP0329BOX, WG1403BX10) and stained using InstantBlueTM Protein Stain (Novus Biologicals catalog number IS
• GS-CHO cells were transfected with an expression construct expressing an IL- 12 fusion polypeptide and a Fam20C kinase to generate a transfected cell pool (6880 P1) that produces IL-12 fusion polypeptide and hFAM20C that phosphorylates the IL-12 fusion polypeptide.
• a 8:1 ratio of IL-12 fusion polypeptide:Fam20C was used.
• the cells were seeded into selection medium at 0.3x106 cells/mL. This selective pressure was maintained throughout the selection period. Viable cell density and percent viability were determined 2-3 times weekly during selection. Viability during selection is graphed in Figure 6.
• Figure 6 shows a common decline in cell viability after transfection, but importantly the cell line shows good recovery and long term high Page 75 of 144 11666457v1 Attorney Docket: 2013762-0017 viability (e.g., day about 18 to day 43 of culturing).
• a healthy and stable of cells transfected with a construct having a 8:1 ratio of IL-12 fusion polypeptide:Fam20C appeared was generated.
• Cell productivity and fusion polypeptide quality [0260] A fed-batch production run of 10 mL culture was initiated in TubeSpins for cell pool 6880 P1 to estimate productivity and protein quality. Productivity was measured over the course of the fed-batch by a bio-layer interferometry (BLI) method.
• BBI bio-layer interferometry
• IL12 fusion polypeptide concentration was calculated in ForteBio Data Analysis HT 11.1 by measuring the observed binding rate, compared to a standard curve using purified IL-12 (protein version ID 67074.1.a) between 1.56 to 100 ⁇ g/ml using a Four parameter logistic (4PL) curve. Clarified supernatant was diluted into 1X Kinetics Buffer (ForteBio) so that the measured concentration was within the range of the standard curve. Additional qualitative analysis was done by SDS-PAGE. The performance parameters of pool 6880 P1 during the fed-batch production evaluation are shown in Figures 7A-C.
• Transfected cells show viability above 90% during the 14-days fed-batch run (Figure 7A) and they were capable of producing between 100 mg/L (at day 7) and 150 mg/L (at day 14) IL-12 fusion polypeptide ( Figure 7C).
• Figure 7A Malachite Green Testing of purified IL-12 fusion polypeptide from the Day 14 harvest of Pool 11370.
• IL-12 fusion polypeptides were purified using a first anion chromatography step and a hydrophobic interaction chromatography step chromatography step. This purification step did not include a second anion exchange polishing step.
• IL-12 fusion polypeptides demonstrated about 5 phosphates per fusion polypeptide.
• Example 3 Bioreactor production of IL-12 fusion polypeptide
• Transfected GS-CHO cells from Example 2 was further cultured and tested.
• Only GS-CHO cells expressing detectable level of IL-12 fusion polypeptide were sorted at 1 cell per well into a 96-well plate using a fluorescence-activated cell sorter. In total, ⁇ 5700 wells were targeted to be inoculated with a single cell.
• a total of 644 wells were identified as containing growing cells. From these wells, 539 cell lines were screened for product expression; 122 colonies expressed detectable levels of product.
• a total of 80 high ranked cell lines were transferred to suspension culture.
• an assessment of the growth and productivity of 24 high ranked cell lines was undertaken in a miniature bioreactor system (ambr® 250 system; TAP Biosystems / Sartorius Stedim Biotech). The selection of the 24 cell lines was based on ranking by IL-12 fusion polypeptide product concentration evaluated by static productivity assessment and acceptable growth of cell lines (viable cell concentration (VCC) on day of subculture consistently above 1.0 x 106 cells/mL and culture viability above 90 %). Twenty-three cell lines were successfully evaluated. The concentration of the IL-12 fusion polypeptide at harvest ranged from 189.1 to 742.8 mg/L, as determined by Octet analysis.
• the system uses glass fiber biosensors coated with a special optical layer and the capturing molecule attached to the tip.
• the tip is dipped into the samples containing a target molecule.
• the target molecule binds to the capture molecule, and the two form a molecular layer.
• white light is directed into the biosensor, two beams are reflected to the back end: 1. from the tip as a reference, 2. from the molecular layer.
• the interference of the two beams causes a wavelength shift, which is a function of the molecular layer Page 78 of 144 11666457v1 Attorney Docket: 2013762-0017 thickness and corresponds to the number of molecules on the tip surface.
• the wavelength shift, expressed in nm, is plotted in real time to generate binding curves.
• Concentration dependent binding curves were acquired for standards, controls and samples, which represented the rate of increase in molecular layer thickness as product bound to the capture molecules on the biosensor tip surface under constant conditions. Binding rates were determined from the binding curves using Octet® Data Analysis software. The binding rates of standards of known concentrations were plotted as a standard curve, which was used to estimate the product concentrations in the samples and to rank them accordingly.
• the UPLC-MS high throughput N-glycan analysis platform comprises of high throughput glycan preparation using a GlykoPrep Rapid 2-AB kit with the AssayMAP Bravo Liquid Handler followed by UPLC and mass spectrometry analysis.
• the sample preparation workflow consisted of an automated purification and normalization step followed by the N- glycans release by digestion with the enzyme peptide-N glycosidase F, separation from the glycoproteins, fluorescent labelling with the fluorophore 2-aminobenzamide (2-AB) and clean-up for analysis.
• the labelled glycans were analyzed by hydrophilic interaction UPLC coupled with electrospray time of flight mass spectrometry.
• the monomeric component was identified by its characteristic retention time and position relative to calibration markers. Aggregate analysis was performed using a TSKgel® G3000SWXL column (Hichrom Ltd). Product components were detected by A280 nm measurements and peak chromatograms were analyzed using EmpowerTM 2 software (Waters Corporation). The proportion of sample components was determined by calculation of the peak areas of each component relative to the total integrated peak area used to measure the product concentration in the samples. Peptide mapping mass spectrometry (PMAP-MS) Analysis PMAP-MS analysis was performed using the Waters Acquity UPLC and Waters XevoG2 QTO. Samples underwent trypsin digestion. MS was calibrated for mass assignment, resolution and sensitivity prior to analysis.
• PMAP-MS protein mapping mass spectrometry
• Glu-fibrinogen lockspray was acquired as a reference scan every 30 seconds. Data analysis was performed using MassLynx and BiopharmaLynx software. The focus of this PMAP-MS analysis was comparison of peaks within the Total ion chromatograms (TIC). MS1 and MS / MS data was acquired for all samples. Only MS1 data was used for analysis, with the MS / MS dataset providing additional information where needed. The following were not covered in this analysis: Page 80 of 144 11666457v1 Attorney Docket: 2013762-0017 estimation of sequence coverage, clipping, pyroglutamation, deamidation, glycation and oxidation.
• Electrospray Ionisation mass spectrometry (ESI-MS) Analysis was performed using the Waters Acquity UPLC and Waters XevoG2 QTOF. Samples were deglycosylated and reduced as per UKSL-1795 without buffer exchange due to sample limitation (clone buffer components were compatible with this method).
• LCMS analysis was performed using the XevoG2 QTOF mass spectrometer operated in positive ion mode over a range of 600m/z to 4000m/z. Data analysis was performed using Protein Metrics software (Version 3.9), utilising the parsimonious deconvolution algorithm. Appropriate processing parameters were selected for the product to be analysed and assigned masses were reported from this processing.
• Thermo ScientificTM Phosphoprotein Phosphate Estimation Assay Kit utilizes the alkaline hydrolysis of phosphate groups from seryl and threonyl residues in phosphoproteins. The liberated phosphate forms a complex with the addition of the ammonium molybdate. The malachite green reagent then forms a secondary complex with the phosphomolybdate to produce a colour change reaction. The absorbance of the sample is measured using a microplate reader and is estimated against a 5-point standard curve of known phosphate concentrations.
• Sialic Acid Determination Sialic acid determination in the test samples were performed by RP-HPLC.
• the sample preparation workflow consisted of dilution of the test sample followed by hydrolysis using TFA solution. The hydrolysed test sample was then labelled with DMB solution to form fluorescent reaction products.
• RP-HPLC with fluorescent detection, quantitation of Nacetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) was performed versus external standards.
• the samples were analysed by reverse phase (RP)- HPLC using Agilent 1100/1200 Series HPLC systems.
• RP-UPLC with Fluorescence detection FLD is used to quantify the relative percent of total purity of IL-12 fusion polypeptide product.
• Reversed phase chromatography separates sample components on the basis of differences in the hydrophobicity. Samples are injected onto the column (stationary phase) and the interaction with the hydrophobic surface removes them from the flowing mobile phase. Sample components bind to the column depending on the strength and nature of hydrophobic and polar interactions. The product is eluted by increasing the organic strength of the mobile phase. Less hydrophobic components elute earlier and more hydrophobic components elute later.
• Table 7 PMAP-MS Analysis of Phosphorylation in AKTA purified ANK-101 from the FMB1 cultures of the 8 lead candidate cell lines. 11666457v1 Attorney Docket: 2013762-0017 [0280] Finally, differences were observed in the intact protein characterization data for 2 of the cell lines which were indicative of product fragmentation in these cell lines. [0281] Characterization data for lead cell lines Table 8: Summary of growth and productivity data of the 8 selected cell lines expressing IL- 12 fusion polypeptide in FMB1. Table 9: Summary of Malachite green analysis of AKTA purified IL-12 fusion polypeptide from the FMB1 cultures of the 8 lead candidate cell lines.
• Example 4 Exemplary purification overview
• the present Examples demonstrates exemplary methods of purifying IL-12 fusion polypeptide from transfected cells.
• An exemplary purification method includes: x Depth filtration or centrifugation for harvesting the bioreactors (typically a 2-week, fed batch run as e.g., as in Examples 2 or 3) to remove cells and cell debris. x Viral inactivation by low pH or detergent for CHO based processes.
• a first Chromatography Step such as a capture step (e.g., an Anion Exchange Capture Chromatography Step) Page 84 of 144 11666457v1 Attorney Docket: 2013762-0017 x
• a second Chromatography Step such as a polishing step (e.g., Hydrophobic Interactions Chromatography) x
• a third Chromatography Step such as a final polishing step (e.g., an Anion Exchange Capture Chromatography Step) x UF/DF for formulating the bulk drug in the right buffer and excipients
• Example 6 Conventional Capture Chromatography [0285] Equilibrating the resin/column with an appropriate buffer that conditions the column to the right pH (e.g., 6.8-7.4) and conductivity (Salt, such as NaCl).
• Example 7 Purification - 1
• a first anion exchange chromatography (AEX) step followed by a second hydrophobic interaction chromatography step can be used to separate a phosphorylated form of a fusion polypeptides from lower and higher molecular weight impurities (e.g., aggregated IL-12 fusion polypeptides) as well as host cell impurities and that is possible to obtain a high purity preparation of a phosphorylated form of a fusion polypeptide.
• a first anion chromatography (AEX) step is used to capture IL-12 fusion polypeptide from a cell extract.
• a hydroxyapatite Chromatography Capture step or 2) a Hydrophobic interaction polishing chromatography step is used as a subsequent polishing step.
• Culture harvest and clarification [0288] Fed-Batch harvest was concluded at Day 14 and clarified by centrifugation. Stored frozen in 200 mL aliquots. Aliquots were thawed in room temperature bath and Page 85 of 144 11666457v1 Attorney Docket: 2013762-0017 clarification was performed by secondary depth filtration. Feasibility tests were performed after flushing filters with DI H2O and equilibrating with TBS (20 mM Tris pH 7.4, 150 mM NaCl).
• Anion chromatography capture step [0289] An exemplary method of anion exchange capture chromatography is shown below.
• Equilibration buffer (A): 20 mM Tris pH 7.4
• Elution buffer (B): 20 mM Tris pH 7.4 + 1M NaCl
• Cleaning buffer 0.5N NaOH Load prep: Clarified harvest was diluted 1:1 (v:v) with DI H2O. Eluted fractions were stored at 4°C overnight.
• Figure 8 shows entire chromatogram and zoom of elution peak during first chromatography of stable pool 6880 P1 clarified harvest by Anion Exchange Chromatography (GigaCap Q).
• the SDS-PAGE gel demonstrates that IL-12 fusion polypeptide (band ⁇ 75,000 Dalton) is captured by the resin, and anion exchange capture chromatography can be used to separate IL-12 fusion polypeptides from lower and higher molecular weight impurities (Figure 9).
• IL-12 fusion polypeptide was first captured by AEX Gigacap Q (5 mL) and hereafter purified (polished) by a polishing hydroxyapatite capture chromatography step.
• An exemplary method of hydroxyapatite capture chromatography is included below.
• IL-12 fusion polypeptide was first captured by AEX Gigacap Q (5 mL) and hereafter purified (polished) by either Heparin HP or Phenyl HP chromatography.
• An exemplary method of Phenyl HP chromatography is included below.
• Example 8 Purification - 2 [0298] This Example demonstrates that Phenyl Chromatography reduces impurities and Q Chromatography is capable of separate product from aggregated product and isolate particular IL-12 fusion polypeptide phosphorylation levels. [0299] Cells were harvested and clarified as in Example 7. Affinity chromatography capture step [0300] An exemplary method of affinity capture chromatography is shown below.
• Phenyl HP chromatography An exemplary method of Phenyl HP chromatography is included below.
• Load prep Adjust Heparin elution pool to 1.5M Ammonium Sulfate with 4.0M Ammonium Sulfate stock. Allow 30 min, filter load with 0.45 micron filter (vacuumflask).
• FIG. 16 shows a non-reduced stain free 4-10 % SDS-PAGE gel loaded with heparin or Phenyl factions (3 ⁇ g sample per lane). Elution fraction (lane 7) demonstrated significant purification when compared to lane 9 (load of Phenyl chromatography). In addition, SEC-HPLC analysis ( Figure 16A) showed higher purity of the Phenyl pool over its load (Heparin Sepharose eluate). Anion chromatography capture step [0304] An exemplary method of anion exchange capture chromatography is shown below.
• Example 9 Purification - 3 [0307] This Example demonstrates that a purification process using three chromatography steps provides a highly purified IL-12 fusion polypeptide product (>98% SEC-HPLC, and >82 % and >55 %, for non-reduced and reduced RP HPLC respectively) having reduced levels of host cell proteins (HCP), DNA, low molecular species (LMWS), high molecular species (HMWS) to acceptably low levels and detergent levels.
• HCP host cell proteins
• LMWS low molecular species
• HMWS high molecular species
• IL-12 fusion polypeptide having desired phosphorylated levels, exhibiting about 7 - 8 phosphorous molecules per molecule of protein by malachite green assay.
• LSRT Lab Scale Run Through
• An exemplary purification process for ANK-101 includes three chromatography steps; x TOYOPEARL GigaCap Q-650M anion exchange chromatography (equivalent to Q chromatography as used in Example 7 and 8), x Capto Phenyl HS hydrophobic interaction chromatography (equivalent to the Phenyl chromatography as used in Example 7 and 8), and x TOYOPEARL GigaCap Q-650S anion exchange chromatography (equivalent to the Q chromatography as used in Example 7 and 8).
• the process also included a viral inactivation step prior to loading onto the first chromatography step.
• the second chromatography step was followed by an intermediate ultrafiltration / diafiltration (UF/DF).
• the third step was followed by a virus reduction filtration sizing and a final dilution.
• Detailed purification workflow x TDAO viral inactivation x TOYOPEARL GigaCap Q-650M anion exchange chromatography (AEX) x Capto Phenyl HS hydrophobic interaction chromatography (HIC) x Intermediate Ultrafiltration/diafiltration (UF/DF) x TOYOPEARL GigaCap Q-650S anion exchange chromatography (AEX) x Planova BioEX Viral Reduction Filtration (VRF) x UF/DF and Excipient Addition [0312]
• the TOYOPEARL GigaCap Q-650M chromatography step was performed at a linear flow rate of 300 cm/h using a loading capacity of 21.4 g/L resin determined during this stage.
• the average step recovery was 100%, determined based on octet data and the average step recovery was 85.7%, determined based on RP HPLC data.
• the Capto Phenyl HS chromatography step was performed at a loading capacity of 15.0 g/L and a linear flow rate of 275 cm/h using 20 mM tris, 100 mM sodium Page 94 of 144 11666457v1 Attorney Docket: 2013762-0017 chloride, 1.4 M ammonium sulphate pH 7.4 as equilibration buffer.
• IL-12 fusion polypeptide was eluted with 20 mM tris, 100 mM sodium chloride, 740 mM ammonium sulphate pH 7.4.
• the average step recovery was 65.4%.
• Further polishing experiments were evaluated on Ceramic Hydroxyapatite (CHT XT) and TOYOPEARL GigaCap Q-650S. Data obtained for TOYOPEARL GigaCap Q-650S showed desired product quality, low impurity levels and high efficiency to separate phosphorylated species, using an isocratic elution step. A three-chromatography step process was designed for the purification of IL-12 fusion polypeptide. The average step recovery was 51.3%.
• the Planova BioEX filter sizing results suggested 1.0 m2 membrane for GMP scale to process material in 5 h. In some methods, the maximum volumetric throughput within a 5 h processing time is 262.6 L/m2.
• the final product was diluted into 20 mM tris, 50 mM sodium chloride pH 7.3 to a protein concentration of 2.040 g/L. This purification step successfully recovered 1326.17 mg (83.8%). Excipients were added to the diafiltered product to achieve a final composition of 20 mM tris, 50 mM sodium chloride, 10 mM methionine, 150mM sucrose, 0.02% polysorbate pH 7.3 at a protein concentration of 2.006 g/L. The estimated cumulative process yield was 19.7%. The estimated cumulative process yield was 19.7 %.
• Intact LC ESI-MS showed 6X, 7X, 8X, 9X, 10X, 11X and 12X phosphorylation sites present in the bulk purified IL-12 fusion polypeptide product. Intact LC ESI-MS also showed + 9 Phosphates as the most predominant species on the bulk purified product.
• Page 95 of 144 11666457v1 Attorney Docket: 2013762-0017 TOYOPEARL GigaCap Q-650M anion exchange chromatography as a first step [0318]
• anion exchange chromatography e.g., TOYOPEARL GigaCap Q-650M
• TOYOPEARL GigaCap Q-650M can be used as a first capturing step for IL-12 fusion polypeptide.
• anion exchange chromatography As a first capture step achieves significant reduction in Host Cell Proteins (HCP) and higher purity. Anion exchange chromatography also enrich the preparation for highly phosphorylated species.
• Hydrophobic interaction chromatography as a second chomatography step for IL-12 fusion polypeptide demonstrated high purity and Host Cell Proteins (HCP) clearance and further demonstrated superior performance both in terms of product recovery ( ⁇ 55%), and purity (98.74% monomer, 89.9% NR RP-HPLC, 67% Red RP-HPLC, 92 ppm HCP).
• HCP Host Cell Proteins
• An exemplary method of hydrophobic interaction chromatography, using a linear gradient elution approach, is described in the table below.
• Table 24 HIC chromatography Gradient Evaluating Operating Conditions.
• An exemplary method of hydrophobic interaction chromatography, using a step or isocratic condition approach is described in the table below.
• Page 99 of 144 11666457v1 Attorney Docket: 2013762-0017 Table 25: HIC chromatography Isocratic Elution Operating Conditions.
• Page 100 of 144 11666457v1 Attorney Docket: 2013762-0017
• Table 26 summary of HIC isocratic evaluation conditions T over multiple column runs (cycling the column 10 times). Conditions resulted in superior performance both in terms of product recovery ( ⁇ 55%), and purity (98.74% monomer, 89.9% NR RP-HPLC, 67% Red RP-HPLC, 92 ppm HCP).
• Page 101 of 144 11666457v1 Attorney Docket: 2013762-0017 [0328] An exemplary method of hydrophobic interaction chromatography is described in the table below.
• Table 28 HIC chromatography Operating Conditions. as a step [0329]
• anion exchange chromatography e.g., TOYOPEARL GigaCap Q-650S
• the concentration of sodium chloride in the post-load wash and the elution buffer are optimized in order to select optimally phosphorylated IL12 fusion polypeptide and purify the product from aggregated species and other impurities (Host cell protein and host cell DNA).
• the cycle was performed using a 25 CV gradient of sodium chloride for the elution step and a loading capacity of 10.0 g/L of resin.
• the elution peak was fractionated, and fractions were analyzed by SEC-HPLC, RP HPLC and malachite green assay. Different fractions were combined, and mock pools were also submitted for additional impurity analysis (DNA and HCP). Analytical results are summarized on Table 29.
• Table 29 Summary of TOYOPEARL GigaCap Q-650S Gradient Elution – Cycle 1 individual fractions analysis. k pool analysis.
• Table 33 Summary of TOYOPEARL GigaCap Q-650S for Isocratic Elution, using 27.8% and 33.2% B (buffer B – 20 mM Tris, 1.0 Sodium Chloride pH 7.3) for PLW2 and Elution respectively (Cycle 3 – Individual Fraction Analysis).
• Table 34 Summary of TOYOPEARL GigaCap Q-650S for Isocratic Elution, using 27.8% and 33.2% Buffer B (containing 1M Sodium Chloride) for PLW2 and Elution respectively (Cycle 3 – Mock Pool Analysis).
• Page 106 of 144 11666457v1 Attorney Docket: 2013762-0017 [0333]
• Figure 17 shows multiple cycles of the GigaCap 650S column performed under the optimized conditions.
• Table 37 Summary of TOYOPEARL GigaCap Q-650S Multi-cycling.
• Table 38 Summary of Impurity Analysis.
• Page 107 of 144 11666457v1 Attorney Docket: 2013762-0017
• Table 39 GP HPLC Analysis lts are shown below.
• Table 40 Summary of Phosphorylation analysis [ ] n-process sampes or eac cromatograpy step were anayze or O (exemplary viral inactivation buffer) detergent detection assay at Eurofins. The results are shown below.
• Table 41 Summary of TDAO quantification Page 108 of 144 11666457v1 Attorney Docket: 2013762-0017 Summary [0336]
• the purification process may in some of the manufacturing methods includes three chromatography steps (e.g., TOYOPEARL GigaCap Q-650M anion exchange chromatography; Capto Phenyl HS hydrophobic interaction chromatography followed by TOYOPEARL GigaCap Q-650S anion exchange chromatography).
• a retrovirus inactivation step in the form of detergent treatment of the cell culture supernatant can be included before TOYOPEARL GigaCap Q-650M chromatography.
• a Planova BioEx virus reduction filtration can also be included after the final polishing chromatography step.
• an intermediate UF/DF step can be introduced after Capto Phenyl HS to reduce the conductivity and exchange the buffer for the next chromatography step.
• a cleaning procedure can be implemented that includes sodium hydroxide, WFI and guanidine hydrochloride as this might improve resin regeneration.
• an intermediate ultrafiltration / diafiltration can be introduced after Capto Phenyl HS chromatography to condition the material prior to the final polishing step.
• the [0340] IL-12 fusion polypeptide can be slightly concentrated and buffer exchanged into 20 mM tris pH 7.3.
• the final conditions of the step were determined as loading at 20 g/L, using 274 mM sodium chloride in the PLW2 buffer and 355 mM sodium chloride in the elution buffer.
• the eluate collection was defined to start when A280nm ⁇ 20 mAU on the upslope and end when A280nm ⁇ 500 mAU on the downslope. It should be noted that specification conductivities for both PLW2 and elution buffer were tighten and are critical to run the final polishing step.
• a filtering step is included in the manufacturing process.
• the TOYOPEARL GigaCap Q-650S eluate comprising an IL-12 fusion polypeptide is diluted to 1-3 g/L (e.g., 2.040 g/L) into an appropriate buffer (e.g., 20 mM tris, 355 mM sodium chloride pH 7.3) and named Bulk Purified Product (BBP).
• BBP Bulk Purified Product
• GP HPLC analysis demonstrated that the IL-12 fusion polypeptide product was purified to greater than 98% in the Bulk Purified Product during this purification process.
• the non-reduced and reduced RP HPLC also show high purity to greater than 82 % and 55 %, respectively.
• the process also successfully reduced levels of HCP, DNA, LMWS and HMWS to acceptably low levels. Detergent clearance through the purification process to acceptable levels was observed.
• the Bulk Purified Product shows desired phosphorylated levels, exhibiting about 7 phosphorous molecules per molecule of protein by malachite green assay.5X, 6X, 7X, 8X, 9X, 10X, 11X and 13X phosphorylated species were observed in the final bulk product by Intact ESI-MS in comparison to reference standard batch (C-150921- 0079) which showed 3X, 4X, 5X, 6X, 7X, 8X and 9X phosphorylated mass species. Additionally, fragmentation in 5X, 7X, 9X, 11X and 13X phosphorylated species was also observed in final bulk product.
• Example 10 Exemplary 500 L scale and 1000 L scale Production [0346] This Example manufacturing of two batches of an IL-12 fusion polypeptide drug substance batch and 1000 L current Good Manufacturing Practice (GMO)). Both batches produce comparable high-quality IL-12 fusion polypeptide drug substance.
• the IL-12 fusion polypeptide drug substance showed high purity (e.g., low fragmentation and/or low aggregation), high heterogeneity, suitable phosphorylation, and/or high activity.
• IL-12 fusion polypeptide drug substance was manufactured at a: x 500 L scale bioreactor batch (analytical reference standard batch) (P4130826ARS); and x 1000 L scale bioreactor batch (Good Manufacturing Practice (GMP) batch) (1205114).
• the 500 L scale bioreactor batch (P4130826ARS) was manufactured using multiple rounds of bind and elute chromatography (comprising a first anionic chromatography step, a hydrophobic chromatography step; and a second anionic chromatography step) and two diafiltrations with no additional purification or processing prior to dispensing and storage.
• the 1000 L scale GMP batch (1205114) was manufactured using multiple rounds of bind and elute chromatography (comprising a first anionic chromatography step, a hydrophobic chromatography step; and a second anionic chromatography step) and two diafiltrations.
• both the 500 and 1000 L scale involve the following steps: [0350] a) Inoculum Expansion where enough viable cells, through serial subculture, serve as inoculum for the production bioreactor. The contents of each vial of cell bank are thawed and cells sedimented by centrifugation. After resuspension in warmed growth medium, the cells are transferred into shake flasks.
• the cultures are expanded by sub- cultivation in shake flasks and then roller bottles using growth medium under an atmosphere of 5% CO 2 in air until sufficient cells are generated to inoculate a single use inoculum bioreactor.
• the inoculum is further expanded in subsequent inoculum bioreactors until sufficient cells are generated to inoculate the single-use production bioreactor; Page 111 of 144 11666457v1 Attorney Docket: 2013762-0017 [0351]
• b) Cell Culture in Production Bioreactor either 500 L or 1000 L where the production bioreactor step is to propagate cell growth under controlled conditions to express the recombinant protein of desired quality that can be subsequently purified from the harvest filtrate during subsequent downstream processing steps.
• the required amount of growth medium Prior to cell culture, the required amount of growth medium is added to the production bioreactor and is allowed to equilibrate with respect to pH, temperature, and dissolved oxygen tension then the cells are transferred aseptically from the inoculum bioreactor into the production bioreactor. Throughout the cell culture process, various additives and feeds are added aseptically to the bioreactor according to feed addition criteria; [0352] c) Harvest remove cells and cell debris and to provide a suitable product stream for the purification process. The production bioreactor is chilled prior to harvest, then the cells and cell debris removed by filtering the culture through depth filters that range in nominal pore size from 9.0 to ⁇ 0.1 ⁇ m.
• the clarified supernatant is then aseptically filtered into sterile bioprocess containers through a 0.22 ⁇ m filter; [0353] d) TDAO treatment and Anion Exchange Chromatography (Toyopearl GigaCap Q 650M) where N,N-Dimethyltetradecylamine N-oxide (TDAO) is added to the clarified harvest.
• TDAO N,N-Dimethyltetradecylamine N-oxide
• the processed harvest supernatant is loaded onto an anion exchange column. After loading, the column is washed allowing impurities to be removed and then the bound protein is eluted as a single fraction using a high salt buffer; [0354] e) Hydrophobic Interaction Chromatography (HIC, Capto Phenyl, High Sub) where the resin binds the protein, allowing impurities to flow through the column. In-process product is diluted and loaded onto a HIC column.
• HIC Hydrophobic Interaction Chromatography
• the percentage main peak, area of group A and B peaks for the IL-12 fusion polypeptide GMP drug substance batch and IL-12 fusion polypeptide reference standard is shown in Table 43.
• the chromatograms for the GMP batch and reference standard are presented in Figure 18.
• Table 43 Reduced RP HPLC Results for IL-12 fusion polypeptide (ANK-101) Reference Standard (P4130826ARS) and GMP Drug Substance Batch (1205114). . of peaks A and group of peaks B were detected for both the samples.
• the RP HPLC profiles of GMP drug substance batch were comparable to reference standard. All results met the GMP specification current at the time of testing, of ⁇ 60.0% area of Main Peak and report % Area of Group A and B peaks.
• SE HPLC Purity Size Heterogeneity (aggregation) - SE HPLC
• SE HPLC was performed on IL-12 fusion polypeptide reference standard and GMP drug substance batch to assess the levels of main peak, high molecular weight species Page 116 of 144 11666457v1 Attorney Docket: 2013762-0017 (HMW) and low molecular weight species (LMW) as part of batch release testing.
• HMW high molecular weight species
• LMW low molecular weight species
• Table 44 SE HPLC Testing Results for IL-12 fusion polypeptide (ANK-101) Reference Standard (P4130826ARS) and GMP Drug Substance Batch (1205114).
• HMW 1 polypeptide GMP drug substance batch
• Slightly higher levels of HMW peaks was found in GMP Drug Substance batch when compared to Reference Standard. This was consistent with the results by Sedimentation Velocity Analytical Ultra- Centrifugation (SV-AUC), where HMW species were more consistently detected at higher levels for GMP Drug Substance batch. LMW species were not detected for both samples.
• SV-AUC Sedimentation Velocity Analytical Ultra- Centrifugation
• AEX HPLC Aggregation measured by Anion exchange (AEX) HPLC
• AEX HPLC was performed on IL-12 fusion polypeptide reference standard and GMP drug substance batch to assess the levels of main peak 1 and 2, acidic and basic peaks as part of batch release characterization. The results are shown in Table 45 and chromatogram is presented in Figure 21. Page 117 of 144 11666457v1 Attorney Docket: 2013762-0017 Table 45: AEX HPLC Testing Results for IL-12 fusion polypeptide (ANK-101) Reference Standard (P4130826ARS) and GMP Drug Substance Batch (1205114). [0370] Three peaks were detected in both the samples. The total relative percentage of main peak 1, main peak 2 and acidic peaks were calculated.
• Example 11 Characterization of exemplary IL-12 fusion polypeptide drug substance [0376] This Example provides exemplary IL-12 drug substance characterization. The present Example demonstrate IL-12 having a degree of phosphorylation of 7.1 or 8. ESI-MS for Mass Analysis [0377] The relative molecular mass was determined through ESI-MS. IL-12 fusion polypeptide drug substance contains high heterogeneity with respect to product variants, due to the presence of multiple different sialylated glycans (N-linked and O-linked glycans) and phosphorylation sites.
• the mass of intact IL-12 fusion polypeptide drug substance could not be determined directly by ESI-MS. Instead, the heterogeneity had to be reduced by at least removing the glycans (deglycosylation) with an enzyme mixture containing N- glycosidase (PNGaseF), Sialidase (SialExo), O-gycanase (OglyZOR). This was performed under reducing and denaturing conditions to increase the exposure of these post-translational modifications to the enzymes. Deglycosylated product samples were prepared both with and without dephosphorylation by lambda phosphatase and analyzed by ESI-MS with on-line desalting.
• PNGaseF N- glycosidase
• SialExo Sialidase
• Ogycanase Ogycanase
• the predominant mass detected for deglycosylated IL-12 fusion polypeptide drug substance in-process samples for the reference batch and GMP batch was 64,151 Da and 64,152 Da, respectively (Species 3), which was consistent with the expected theoretical mass of deglycosylated IL-12 fusion polypeptide drug substance with nine phosphorylations (64,154 Da).
• the theoretical mass was based on the theoretical amino acid sequence for IL-12 fusion polypeptide drug substance with fully reduced disulphide bonds and conversion of asparagine to aspartic acid for all potential N-linked glycosylation sites as result of enzymatic deglycosylation.
• Masses were assigned for a total of five species with Page 120 of 144 11666457v1 Attorney Docket: 2013762-0017 masses consistent with varying degrees of phosphorylation (theoretical mass difference (delta mass) for phosphorylation is + 80 Da).
• the ESI-MS spectra were visually comparable confirming similar levels of phosphorylated species for GMP drug substance batch and reference standard batch (representative in-process, pre-excipient addition samples). This was consistent with the findings by the Malachite Green phosphoprotein phosphate estimation and RP HPLC tryptic peptide mapping.
• the mass of the deglycosylated and completely dephosphorylated IL-12 fusion polypeptide Drug Substance protein chain was detected at 63,442 Da and 63,441 Da (Species 1) for both in-process samples for reference batch and GMO batch, respectively (theoretical mass 63,434 Da).
• Species 3 was observed for both samples, which was consistent with deglycosylated IL-12 fusion polypeptide Drug Substance with two phosphorylations or a hexose sugar, which may be indicative of glycation (glycation was confirmed by RP HPLC reduced tryptic peptide mapping).
• the ESI-MS spectra were visually comparable between reference batch and GMO batch in-process samples.
• ESI-MS with on-line RP HPLC revealed a larger spectrum of phosphorylation of the product (5 to 12 phosphorylations; predominant species had 8 phosphorylations) than determined by ESI-MS with on-line desalting (7 to 11 phosphorylations; predominant species had 9 phosphorylations). This is likely due to the differences in resolution of species due to different mass spectrometers used for each method and differences in interference from sample matrices (better removal of interfering/ion-suppressing entities by RP HPLC compared to desalting alone). Overall, both approaches confirmed the masses of the batches were as expected and the profile of product variants was comparable between the two batches.
• RP HPLC-MS and MS/MS Reduced Tryptic Peptide Mapping Test samples were denatured in guanidine hydrochloride and reduced with tris-(2-carboxyethyl)-phosphine (TCEP). Samples were subsequently buffer exchanged into a urea/TCEP buffer for trypsin digestion to maintain both enzyme activity and solubility of the molecule. The samples were then digested using trypsin and separated using RP HPLC. A combination of mass spectrometric and ultraviolet (UV) detection ( ⁇ 210 nm and ⁇ 280 nm) was used. [0384] 210 nm, 280 nm and total ion count (TIC) profiles were generated.
• UV ultraviolet
• N-terminal sequence verification was performed for IL-12 fusion polypeptide reference standard and GMP drug substance batch using mass spectrometric and tandem mass spectrometric detection.
• the expected N-terminal peptide sequence (IWELKK, P1-2) was identified and confirmed by MS/MS. No N-terminal modifications were detected.
• C-terminal sequence verification for IL-12 fusion polypeptide Reference Standard and GMP Drug Substance batch was performed using mass spectrometric and tandem mass spectrometric detection.
• the C-terminal peptide (VMSYLNASGGGGEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEE GGGGSEEGGGGSEEGGGGS, P59) was detected in both samples under dephosphorylated conditions as the unmodified and monophosphorylated peptides.
• Post-Translational Modifications [0388] For IL-12 fusion polypeptide reference standard and GMP drug substance batch, an approximation of the amount of potential post-translational modifications were assigned by comparing the ion intensities from the modified peptides to the ion intensities from the unmodified peptides.
• deamidation prone peptides have a high incidence of both N and Q Page 123 of 144 11666457v1 Attorney Docket: 2013762-0017 residues flanked by serine (S) and glycine (G) residues which have been suggested to stabilize intermediates in the deamidation reaction.
• S serine
• G glycine
• the putative oxidized peptide has a molecular mass 16 Da higher than the corresponding intact peptide, assuming the methionine (M) or tryptophan (W) was oxidized.
• M methionine
• W tryptophan
• the theoretical amino acid sequence of the IL-12 fusion polypeptide drug substance showed seven sequence motifs typically associated with N-linked glycosylation (Asn-X-Ser/Thr, where X is any residue except for proline) at Asn103, Asn113, Asn200, Asn281, Asn392, Asn406 and Asn516.
• N-linked glycans were detected at five of the seven potential N-linked glycan sites at Asn103, Asn200, Asn281, Asn392, and Asn406. Glycosylation was not detected at potential N-linked glycosylation sites Asn113 and Asn516. There was also evidence for the attachment of O-linked glycans.
• O-linked glycans are usually attached via the side group (hydroxyl group) of serine and threonine residues
• the precise site of O-linked glycosylation could not be predicted from the sequence.
• One peptide that contained O-linked glycosylation was identified from the trypsin digest data.
• the N-linked and O-linked glycosylation site occupancy for GMP drug substance batch was comparable to reference standard.
• Page 124 of 144 11666457v1 Attorney Docket: 2013762-0017 Phosphorylation [0394]
• IL-12 fusion polypeptide drug substance is believed to be phosphorylated by the co-expressed hum a kinase Fam20C.
• Glycosylation [0397] More Glycation occurs most readily at the N-terminal alpha-amino group of a protein, or the epsilon-amino group of lysine residues throughout the protein sequence. [0398] A data search was performed for the reduced tryptic peptide mapping MS and MS/MS data to determine if there was evidence for the attachment of hexose sugars that would indicate glycation.
• N-linked oligosaccharides were released by incubation with the enzyme peptide-N-glycosidase F (PNGase F) under denatured and reduced conditions. Protein was removed and the released oligosaccharides prepared for neutral and charged profiling. The released glycans were labelled with the fluorophore 2-aminobenzamide (2-AB) and incubated with ⁇ -sialidase to remove terminal sialic acid.
• PNGase F peptide-N-glycosidase F
• N-linked oligosaccharides are released by incubation with recombinant peptide-N-glycosidase F Page 126 of 144 11666457v1 Attorney Docket: 2013762-0017 (PNGaseF) under native conditions.
• the proteins are removed by precipitation and the released oligosaccharides are hydrolysed.
• the oligosaccharides are labelled with the fluorophore 2-aminobenzamide (2-AB) and treated with ⁇ -sialidase prior to analysis.
• the glycan profiles and peaks are quantified by fluorescence peak areas and identified by LCMS.
• N-linked oligosaccharide profiles of IL-12 fusion polypeptide reference standard and GMP drug substance batch were determined using LC-MS of fluorescently labelled glycans released using PNGase F. Chromatogram is presented in Figure 25.
• Proposed oligosaccharide structures were assigned based on accurate mass measurement. Further confirmation was made by retention time alignment with N-glycan standards and the IgG IAC was used to aid identification, where appropriate.
• the N-glycans detected were consistent with predominantly core fucosylated, biantennary complex type glycan structures with varying levels of terminal galactosylation, as expected for a GS-CHO cell line.
• the predominant N-glycan detected was G2F (peak 14).
• Other major Nglycans detected were two isomers of G3F (peak 23 and 24), Man-9 (peak 27), two isomers of G4F (peak 29 and 30), Man-6 (peak 11), G0F (peak 4) and two isomers of G1F (peak 8 and 9).
• the results by N-linked oligosaccharide profiling were consistent with the results from peptide mapping.
• Sialidase treatment was also performed to confirm that charged groups are due to the presence of sialic acids.
• the charged N-linked oligosaccharide profiles of IL-12 fusion polypeptide reference standard and GMP drug substance batch were analyzed by hydrophilic interaction liquid chromatography (HILIC) of fluorescently-labelled N-linked glycans.
• HILIC hydrophilic interaction liquid chromatography
• Page 127 of 144 11666457v1 Attorney Docket: 2013762-0017 reported charged groups was determined by comparing the test samples treated with ⁇ - sialidase.
• IL-12 fusion polypeptide drug substance has remained comparable throughout the development of the manufacturing process.
• the purpose of this assay is to measure the kinetics and binding strength of IL- 12 fusion polypeptide drug substance (IL-12-ABP) on Alhydrogel (aluminum hydroxide particles; alum).
• IL-12-ABP proteins contain a C-terminal fusion to a phosphorylated alum- binding peptide (ABP) and is expected to form a stable complex with Alhydrogel through a ligand exchange reaction between phosphoserines in the peptide and surface hydroxyl groups on the Alhydrogel.
• IL-12-ABP IL-12 fusion polypeptide drug substance
• biotinylated mouse monoclonal anti-human IL-12 (p70) detection antibody is added followed by avidin-HRP reagent, followed by TMB substrate, which produces a blue color proportional to the concentration of IL-12 (p70) present.
• Each plate includes an 8-point free IL-12-ABP standard curve (each point in duplicates), and alum-bound IL-12-ABP conditions are included and analyzed in triplicates.
• the 5-minute, 10-minute, and 30-minute interval alum-bound protein samples are diluted in 1 mM phosphate and 40% human serum and incubated at 37°C for 24 hours.
• the Example demonstrates that unwanted process impurities (e.g., TDAO, Tropolone, Pluronic, PDMS, Octamethylcyclo tetrasiloxane D4, and Fam20) are cleared during the manufacturing process of IL-12 fusion polypeptide drug substance.
• Low molecular weight molecules e.g., Octamethylcyclo tetrasiloxane D4 and Tropolone
• High molecular weight compounds PDMS, Pluronic, TDAO
• the manufacturing process is also capable of removing Fam20.
• Tropolone is a lipophilic iron chelator that improves transport of iron into cells, enabling cell growth and sustaining cell viability.
• Pluronic Pluronic is a component of the base powder used to prepare the media for the inoculum and production stage bioreactors. The purpose of this chemical is the enhancement of cell membrane stability during cell culture.
• Polydimethylsiloxane (PDMS) and Octamethylcyclo tetrasiloxane D4 Polydimethylsiloxane (PDMS) and Octamethylcyclo tetrasiloxane D4 are components of Simethicone, the active ingredient in the Antifoam C emulsion.
• Antifoam emulsion is added routinely to the cell culture during manufacture to control or inhibit foaming in the production bioreactor. The addition is variable and is dependent on the degree of foaming.
• TDAO N,N-dimethyl tetradecylamine N-oxide
• TDAO N,N-dimethyl tetradecylamine N- oxide
• x Fam20C Human Fam20C is an endogenous Golgi localized serine/threonine kinase that phosphorylates serine and threonine motifs on proteins.
• Table 51 Calculation of Safety Margins for TDAO polypeptide Drug Substance, a number of logs of clearance is required. It is a good practice to demonstrate a 90% safety factor in the clearance, if possible, i.e., the amount of the compound of interest remaining is at 10% or less than the PDEIV. Moreover, the total number of whole logs of clearance required to ensure levels of TDAO are below both the PDEIV and the 90% safety factor are summarized in Table 52.
• Table 52 Minimum log clearance values for TDAO
• Quantification of TDAO was performed by the use of (reversed-phase liquid chromatography mass spectrometry) RP-LC-MS/MS detection, monitoring two MS/MS mass transitions.
• a calibration curve for LC-MS/MS determination of the detergent was established by measuring at least five levels of calibration solutions. The regression functions and correlation coefficients of the calibration curve were used to calculate the concentrations of the analyte samples.
• GMP Good Manufacturing Practice
• Fam20C amount was monitored by LC-MS/MS using a multiple standard addition approach. Fam20C was spiked at three levels in the analyte.
• Fam20C quantification in the GigaCap Q-650M Load was measured to be three order of magnitudes lower than the PDEIV (1000 ⁇ g/d) when accounting for the maximum dose (1.25 mg/d) of IL-12 fusion polypeptide Drug Substance.

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