Method for producing fusion polypeptide

Abstract

The present disclosure provides highly useful fusion polypeptides comprising an immunomodulatory moiety and a metal hydroxide-binding moiety, as well as a variety of related technologies, including methods for making and using such fusion polypeptides.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 424,013, filed November 9, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Useful fusion polypeptides have been developed in which a metal-binding polypeptide is linked to an immunomodulatory domain (e.g., an IL-12 immunomodulatory domain) (see, e.g., published international patent application WO2020 / 263399). Particular uses for such fusion peptides include the treatment of certain medical conditions, such as cancer. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides, for example, particular compositions and formulations of phosphorylated forms of fusion polypeptides, including specifically phosphorylated forms of IL-12 fusion polypeptides (e.g., those described in published International Patent Application WO 2020 / 263399). In some embodiments, the provided technology provides particularly stable compositions and formulations of phosphorylated forms of such fusion polypeptides.

[0004] In particular, the present disclosure identifies the causes of and provides solutions to problems associated with certain compositions or formulations containing such phosphorylated fusion polypeptides. For example, the present disclosure notes that related phosphorylated fusion polypeptides are highly unstable in common formulations and compositions, creating challenges in manufacturing and / or distribution; for example, some standard formulations exhibit instabilities such as drug deamidation, susceptibility to oxidation, and the formation of visible particles upon shaking. Furthermore, without wishing to be bound by any particular theory, the present disclosure notes that phosphorylated IL-12 fusion polypeptides may exhibit a unique "stickiness" that causes the drug to adhere to the surfaces of the container containing the formulation in many standard formulations.

[0005] The present disclosure further provides solutions to these problems, providing desirable compositions of phosphorylated fusion polypeptides and / or, in some embodiments, formulations of phosphorylated fusion polypeptides complexed with metal hydroxides. Among other things, the present disclosure provides metal hydroxide-binding polypeptides and fusion polypeptides comprising metal hydroxide-binding polypeptides that exhibit high levels of adsorption to metal hydroxides.

[0006] Particularly useful compositions comprise a phosphorylated fusion polypeptide that is or comprises a phosphorylated IL-12 fusion polypeptide in a Tris-buffered formulation at a pH of about 6.5 to 8 (e.g., about 7.4). In some embodiments, compositions according to the present disclosure may include the addition of salt (e.g., NaCl) and / or L-methionine and / or sucrose and / or a surfactant (e.g., polysorbate). Without wishing to be bound by any particular theory, it is believed that L-methionine reduces the susceptibility of the IL-12 polypeptide fusion to oxidation, and the surfactant (e.g., polysorbate) reduces the formation of visible particles upon shaking the composition. The addition of salt is believed to stabilize the structure of the molecule through ionic interactions.

[0007] The present disclosure further provides useful formulations comprising a fusion polypeptide-metal hydroxide complex containing a phosphorylated fusion polypeptide (e.g., a phosphorylated IL-12 fusion polypeptide) in a Tris-buffered formulation at a pH of about 6.5 to 8 (e.g., about 7.4). Formulations according to the present disclosure may include the addition of salt and / or L-methionine and / or a surfactant (e.g., polysorbate).

[0008] In some aspects, the disclosure provides a composition comprising a phosphorylated form of a fusion polypeptide comprising (a) an immunomodulatory polypeptide comprising an interleukin-12 immunoagonist portion and (b) a metal hydroxide-binding polypeptide, the amino acid sequence of which comprises multiple phosphorylation sites such that the fusion polypeptide can exist in phosphorylated and unphosphorylated forms; Tris buffer; salt; sucrose; L-methionine; and a surfactant; wherein the pH of the composition is within the range of about 6.5 to about 8. In some embodiments, the phosphorylated fusion polypeptide complexes with a metal hydroxide upon exposure to the metal hydroxide. In some embodiments, the metal hydroxide is aluminum hydroxide.

[0009] In some aspects, the disclosure provides a pharmaceutical formulation comprising a phosphorylated form of a fusion polypeptide comprising (a) an immunomodulatory polypeptide comprising an interleukin-12 immune agonist portion and (b) a metal hydroxide-binding polypeptide, the amino acid sequence of which comprises multiple phosphorylation sites such that the fusion polypeptide can exist in phosphorylated and unphosphorylated forms; a fusion polypeptide metal-hydroxide complex comprising a metal hydroxide, Tris buffer, salt, sucrose, L-methionine, and a surfactant; wherein the pH of the composition is within the range of about 6.5 to about 8.

[0010] In some aspects, the present disclosure provides a method for treating a subject, the method comprising administering a pharmaceutical composition according to the present disclosure.

[0011] In some aspects, the present disclosure provides methods of making compositions and / or pharmaceutical formulations according to the present disclosure.

[0012] In some aspects, the present disclosure provides methods for characterizing a composition according to the present disclosure by assessing the degree of phosphorylation of the fusion polypeptide. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows an exemplary schematic diagram of a fusion polypeptide metal-hydroxide complex of the present disclosure, which, when administered to a subject, can result in improved retention and / or efficacy compared to a suitable reference standard. [Figure 2] FIG. 1 shows a diagram of an exemplary fusion polypeptide of the present disclosure comprising a first (p40) and a second (p35) IL12 immune agonist moiety and a metal hydroxide-binding polypeptide having multiple phosphorylation sites. [Figure 3] Purity by size exclusion chromatography is shown, where A indicates the main peak (IL-12 fusion polypeptide) and B indicates high molecular weight species (HMW). [Figure 4] Purity by size exclusion chromatography is shown, A indicates the main peak (IL-12 fusion polypeptide), and B indicates high molecular weight species (HMW). [Figure 5] The retention of alum over time is shown. [Figure 6] 1 shows the activity of IL-12 fusion polypeptide when formulated in TBS or in an IL-12 fusion polypeptide composition bound to alum or without metal hydroxide. [Figure 7A] Quantification of ATP to determine viability of PBMCs from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 7B]Quantification of ATP to determine viability of PBMCs from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 7C] Quantification of ATP to determine viability of PBMCs from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 7D] Quantification of ATP to determine viability of PBMCs from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). [Figure 7E] ATP quantification to determine the viability of PBMCs from Donor 1 is shown. PBMCs from Donor 1 were isolated, settled, and stimulated with an aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). Appropriate controls included a negative control (no stimulation), a positive control (soluble CD3 [5 μg / ml] + aqueous CD28 [2 μg / ml]), and a solvent control (formulation buffer [0.04%]). ATP was quantified on day 3 using the CellTiter-Glo® 2.0 Cell Viability Assay Kit. Each graph shows the mean ± SEM of triplicates. Lower solid line: no stimulation, dotted line: solvent, and upper solid line: positive. ATP = adenosine triphosphate, CD = cluster of differentiation, Conc = concentration, PBMC = peripheral blood mononuclear cells, SEM = standard error of the mean. ANK-101: IL-12 fusion polypeptide complexed with alum. IL-12-ABP: IL-12 fusion polypeptide. [Figure 8A]Quantification of ATP to determine viability of PBMCs from donor 2 is shown. PBMCs from donor 2 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 8B] Quantification of ATP to determine viability of PBMCs from donor 2 is shown. PBMCs from donor 2 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 8C] Quantification of ATP to determine viability of PBMCs from donor 2 is shown. PBMCs from donor 2 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 8D] ATP quantification to determine viability of PBMCs from donor 2 is shown. PBMCs from donor 2 were isolated, rested, and stimulated with aqueous anti-CD3 (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). [Figure 8E]ATP quantification to determine PBMC viability from donor 2 is shown. PBMCs from donor 2 were isolated, settled, and stimulated with an aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). Appropriate controls included a negative control (no stimulation), a positive control (soluble CD3 [5 μg / ml] + aqueous CD28 [2 μg / ml]), and a solvent control (formulation buffer [0.04%]). ATP was quantified on day 3 using the CellTiter-Glo® 2.0 Cell Viability Assay Kit. Each graph shows the mean ± SEM of triplicates. Lower solid line: no stimulation, dotted line: solvent, and upper solid line: positive. ATP = adenosine triphosphate, CD = cluster of differentiation, Conc = concentration, PBMC = peripheral blood mononuclear cells, SEM = standard error of the mean. ANK-101: IL-12 fusion polypeptide complexed with alum. IL-12-ABP: IL-12 fusion polypeptide. [Figure 9A] IFNγ accumulation in PBMC cultures from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 9B] IFNγ accumulation in PBMC cultures from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 (100 ng / ml) in the presence of 12 different concentrations of alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 9C] IFNγ accumulation in PBMC cultures from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 9D]Figure 1 shows IFNγ accumulation in PBMC cultures from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). [Figure 9E] Figure 1 shows IFNγ accumulation in PBMC cultures from donor 1. PBMCs were isolated from donor 1, rested, and stimulated with an aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). Appropriate controls included a negative control (no stimulation), a positive control (soluble CD3 [5 μg / ml] + aqueous CD28 [2 μg / ml]), and a solvent control (formulation buffer [0.04%]). Cell culture supernatants were collected on day 3 and analyzed by TR-FRET. Each graph shows the mean ± SEM of triplicates. Lower solid line: no stimulation, dotted line: solvent, and upper solid line: positive. CD = cluster of differentiation, Conc = concentration, EC50 = half-maximal effective concentration, IFNγ = interferon gamma, NA = not applicable, PBMC = peripheral blood mononuclear cells, SEM = standard error of the mean, TR-FRET = time-resolved fluorescence energy transfer. ANK-101: IL-12 fusion polypeptide complexed with alum. IL-12-ABP: IL-12 fusion polypeptide. [Figure 10A] IFNγ accumulation in PBMC cultures from donor 2. PBMCs from donor 2 were isolated, rested, and stimulated with aqueous anti-CD3 (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 10B] IFNγ accumulation in PBMC cultures from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 (100 ng / ml) in the presence of 12 different concentrations of alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 10C]IFNγ accumulation in PBMC cultures from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 (100 ng / ml) in the presence of 12 different concentrations of preincubated alum-complexed IL-12 fusion polypeptide (ANK-101). [Figure 10D] Figure 1 shows IFNγ accumulation in PBMC cultures from donor 1. PBMCs from donor 1 were isolated, rested, and stimulated with aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). [Figure 10E] Figure 1 shows IFNγ accumulation in PBMC cultures from donor 1. PBMCs were isolated from donor 1, rested, and stimulated with an aqueous anti-CD3 solution (100 ng / ml) in the presence of 12 different concentrations of pre-incubated alum-free IL-12 fusion polypeptide (IL-12-ABP). Appropriate controls included a negative control (no stimulation), a positive control (soluble CD3 [5 μg / ml] + aqueous CD28 [2 μg / ml]), and a solvent control (formulation buffer [0.04%]). Cell culture supernatants were collected on day 3 and analyzed by TR-FRET. Each graph shows the mean ± SEM of triplicates. Lower solid line: no stimulation, dotted line: solvent, and upper solid line: positive. CD = cluster of differentiation, Conc = concentration, EC50 = half maximal effective concentration, IFNγ = interferon gamma, NA = not applicable; PBMC = peripheral blood mononuclear cells, SEM = standard error of the mean, TR-FRET = time-resolved fluorescence energy transfer. DETAILED DESCRIPTION OF THE INVENTION

[0014] definition Administration: As used herein, the term "administration" generally refers to the application of a composition to a subject or system. Those of skill in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be systemic. In some embodiments, administration may be topical. In some embodiments, administration may be enteral. In some embodiments, administration may be parenteral. In some embodiments, administration may be by injection (e.g., intramuscular injection, intratumoral injection, intravenous injection, or subcutaneous injection). In some embodiments, injection may include bolus injection, infusion, perfusion, or infusion. In many embodiments, administration according to the present disclosure is by intratumoral injection.

[0015] Affinity: As is well known in the art, "affinity" is a measure of the strength with which two or more binding partners bind to one another. Those skilled in the art will recognize a variety of assays that can be used to assess affinity, as well as appropriate controls for such assays. In some embodiments, affinity is assessed by a quantitative assay. In some embodiments, affinity is assessed across multiple concentrations (e.g., of one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitors (e.g., that may be present in a relevant (e.g., physiological) situation). In some embodiments, affinity is assessed relative to a reference (e.g., a reference with a known affinity above a certain threshold (see "positive control") or a reference with a known affinity below a certain threshold (see "negative control"). In some embodiments, affinity can be assessed relative to a concurrent reference. In some embodiments, affinity can be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.

[0016] Agent: Generally, as used herein, the term "agent" refers to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex), a combination, mixture, or system (e.g., a cell, tissue, organism), or phenomenon (e.g., heat, an electric current or field, a magnetic force or field, etc.). In appropriate circumstances, as will be clear to one of skill in the art from the context, the term may refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as will be clear from the context, the term may refer to a natural product, in that it is found in nature and / or obtained from nature. In some instances, also as will be clear from the context, the term may refer to one or more entities that are man-made, in that it is designed, engineered, and / or manufactured by the hand of man, or is not found in nature. In some embodiments, an agent can be used in isolated or pure form. In some embodiments, an agent can be used in crude form. In some embodiments, potential agents may be provided as a collection or library, eg, which can be screened to identify or characterize active agents therein.

[0017] Agonist: Those skilled in the art will recognize that the term "agonist" can be used to refer to an agent, condition, or event whose presence, level, degree, type, or form correlates with an increase in the level or activity of another agent (agonized agent or target agent). Generally, an agonist can be or include an agent of any chemical class, such as, for example, a small molecule, polypeptide, nucleic acid, carbohydrate, lipid, metal, and / or any other entity that exhibits related activation activity. In some embodiments, an agonist can be direct (in which case the agonist exerts its effect directly on the target, e.g., by physically binding to the target). In some embodiments, an agonist can be indirect (in which case the agonist exerts its effect by altering the level and / or activity of the target other than by binding to the target, e.g., by interacting with a modulator of the target).

[0018] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to compounds and / or substances that can be incorporated into, are incorporated into, or are already incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or the amino-terminal amino acid of a polypeptide, can have structural modifications compared to the general structures above. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulatory half-life of a polypeptide comprising the modified amino acid compared to a polypeptide comprising an otherwise identical unmodified amino acid. In some embodiments, such modifications may not significantly alter, for example, the relevant activity of a polypeptide comprising the modified amino acid compared to one comprising an otherwise identical unmodified amino acid. As is clear from the context, in some embodiments, the term "amino acid" may refer to a free amino acid. In some embodiments, the term may also be used to refer to an amino acid residue of a polypeptide.

[0019] Animal: As used herein, the term "animal" refers to a member of the animal kingdom. In some embodiments, "animal" refers to a human of either sex and at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, horse, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0020] Binding: As used herein, the term "binding" will be understood to generally refer to a non-covalent bond between two or more entities. "Direct" binding involves physical contact between the entities or moieties, while indirect binding involves a physical interaction through 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 when the interacting entities or moieties are tested in isolation or in the context of a more complex system (e.g., covalently, electrostatically, or otherwise bound to a carrier entity and / or within a biological system or cell). Binding between two entities can be considered "specific" if, under the conditions being assessed, the associated entities bind more readily to each other than to other available binding partners.

[0021] Cancer: The terms "cancer," "malignancy," "neoplasia," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, such that the cells exhibit an aberrant growth phenotype characterized by a marked loss of control of cell proliferation. In some embodiments, tumors can be or include precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. This disclosure specifically identifies particular cancers to which the teachings may be particularly relevant. In some embodiments, the relevant cancer can be a solid tumor. In some embodiments, the relevant cancer can be a hematological cancer. In general, examples of different types of cancer known in the art include hematopoietic tumors, including, for example, leukemia, lymphoma (Hodgkin's disease and non-Hodgkin's disease), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinoma of solid tissue, squamous cell carcinoma of the mouth, pharynx, larynx, and lung, liver cancer, genitourinary cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, endometrial cancer, and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and cancers of the nervous system, benign lesions, such as papillomas, and the like.

[0022] Characteristic sequence element: As used herein, the phrase "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, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the 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 generally comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises 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., consecutively linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of contiguous monomers separated by one or more spacer regions that may or may not vary in length between polymers sharing the sequence element.

[0023] Chemotherapeutic Agent: As used herein, the term "chemotherapeutic agent" has its art-recognized meaning to refer to one or more proapoptotic, cytostatic, and / or cytotoxic agents, and specifically includes, for example, agents used and / or recommended for use in treating one or more diseases, disorders, or conditions involving unwanted cell proliferation. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent is or can include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule-targeting agents such as taxanes, maytanstine and their analogs), one or more epothilones, one or more histone deacetylase (HDAC) inhibitors, one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based compounds, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs (i.e., those sharing related antiproliferative activity) of one or more of the following: In some embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea The antibody-drug conjugate may be or may include one or more of: idarubicin, idarubicin, imatinib, irinotecan, maytansine and its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be used in conjunction with the antibody-drug conjugate.In some embodiments, the chemotherapeutic agent is 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, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, the chemotherapeutic agent may be one described as being used in an antibody-drug conjugate such as those described or discussed in one or more of Govindan et al., The Scientific World Journal 10:2070, 2010,-2089. In some embodiments, the chemotherapeutic agent may be or include one or more of farnesyl-thiosalicylic acid (FTS), (4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), δ-tocatrienol, salinomycin, or curcumin. Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more agents can be administered simultaneously.In some embodiments, such agents can be administered sequentially, hi some embodiments, such agents are administered in overlapping dosing regimens.

[0024] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more regimens can be administered simultaneously. In some embodiments, such regimens can be administered sequentially (e.g., all "doses" of a first regimen are administered before any doses of a second regimen). In some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy can include administration of one or more agent(s) or modality(s) to a subject to which the other agent(s) or modality(s) in the combination are administered. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even simultaneously, if necessary), although in some embodiments, two or more agents or active portions thereof may be administered together in a combination composition or even a combination compound (e.g., as part of a single chemical complex or covalent conjugate).

[0025] Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" can refer to a set (usually multiple) unit doses that are administered individually to a subject, usually at regular time intervals. In some embodiments, a particular therapeutic agent has a recommended dosing regimen that may use one or more administrations. In some embodiments, a dosing regimen includes multiple administrations, each spaced apart in time from the other administrations. In some embodiments, the individual administrations are spaced apart by the same amount of time from each other, and in some embodiments, a dosing regimen includes multiple administrations and at least two different time intervals separating the individual administrations. In some embodiments, all administrations within a dosing regimen are the same unit dose. In some embodiments, different administrations within a dosing regimen are of different amounts. In some embodiments, a dosing regimen includes a first administration at a first dosage amount, followed by one or more additional administrations at a second dosage amount that is different from the first dosage amount. In some embodiments, the dosing regimen comprises a first administration at a first dosage amount, followed by one or more additional administrations at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0026] Epitope: As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, 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 close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0027] Excipient: As used herein, refers to a non-therapeutic agent that can be included in a pharmaceutical composition to, for example, impart or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0028] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcription product. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) generation of an RNA template from the DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, etc.), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0029] Functional: As used herein, the term "functional" is used to refer to forms or fragments of an entity that exhibit a particular property and / or activity.

[0030] Fragment: A "fragment" of a substance or entity described herein comprises a discrete portion of the whole, but has a structure lacking one or more portions found in the whole. In some embodiments, the fragment consists of such a discrete portion. In some embodiments, the fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, the polymer fragment is 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 monomer units (e.g., residues). In some embodiments, 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 more of the monomer units (residues) found in the whole polymer. The whole substance or entity may, in some embodiments, be referred to as the "parent" of the fragment.

[0031] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene comprises non-coding sequence. In some particular embodiments, a gene may comprise both coding sequence (e.g., exon sequence) and non-coding sequence (e.g., intron sequence). In some embodiments, a gene may comprise one or more regulatory elements that can, for example, control or affect one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression, etc.).

[0032] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to the RNA (pre- and / or post-processing) transcribed from a gene or the polypeptide (pre- and / or post-processing) encoded by the RNA transcribed from a gene.

[0033] Genome: As used herein, the term "genome" refers to the entire genetic information possessed by an individual organism or cell, represented by the complete DNA sequence of its chromosomes.

[0034] Host cell: As used herein, refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art will understand, upon reading this disclosure, that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain changes may occur in subsequent 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. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or fusion cells, such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell selected from the following cells: CHO (e.g., CHO K1, DXB-1 1CHO, Vegge-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cell comprises one or more viral genes.

[0035] Host cell protein(s) or "HCP": As used herein, refers to proteins that may be present in a cell extract or preparation, e.g., by being produced by the host cell in which a fusion polypeptide (e.g., a phosphorylated or non-phosphorylated fusion polypeptide) described herein is produced, or otherwise contained within or on the host cell, but are not the fusion polypeptide. In some embodiments, provided technology (e.g., provided production methods, such as provided purification methods) removes or reduces HCPs from a preparation(s) of fusion polypeptide (e.g., from a preparation of a phosphorylated fusion polypeptide described herein). An "HCP-reduced preparation" refers to a preparation that contains reduced HCPs, e.g., compared to the amount present before application of the relevant purification step (e.g., provided herein) and / or compared to the amount achieved by a different purification technology. In some embodiments, provided technology provides for the production of fusion polypeptide preparations (e.g., preparations of phosphorylated fusion polypeptides) in which HCPs are undetectable, e.g., by use of an ELISA assay. In some embodiments, removal of the HCP from the host cell, which in some embodiments may be, for example, an engineered mammalian cell described herein (e.g., expressing a fusion polypeptide and a kinase that phosphorylates it at a ratio in the range of about 4:1 to 10:1, e.g., about an 8:1 ratio), can be monitored or assessed, for example, during or after purification of a fusion polypeptide described herein (e.g., a phosphorylated form thereof).

[0036] "Improved," "increased," or "reduced": As used herein, these terms, or grammatically equivalent comparative terms, refer to a value relative to a comparable reference measurement. For example, in some embodiments, a measurement achieved with an agent of interest may be "improved" compared to a measurement obtained with an equivalent reference agent. Alternatively or additionally, in some embodiments, a measurement achieved in a subject or system of interest may be "improved" compared to a measurement obtained in the same subject or system, under different conditions (e.g., before or after an event such as administration of the agent of interest), or in a different, comparable subject (e.g., a comparable subject or system that differs from the subject or system of interest in the presence of one or more indicators of a particular disease, disorder, or condition of interest, or in terms of prior exposure to a condition or agent). In some embodiments, comparative terms refer to a statistically relevant difference (e.g., a difference of a ratio and / or magnitude sufficient to demonstrate statistical relevance). One of skill in the art will recognize or be able to readily determine the magnitude and / or ratio of difference necessary or sufficient to demonstrate such statistical significance in a particular context.

[0037] As used herein, the term "in vitro" refers to events that take place not inside the body of a small organism, but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0038] In vivo: As used herein, refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0039] As used herein, the term "isolated" refers to a substance and / or entity that is (1) separated from at least some of the components with which it was associated when originally created (whether in nature and / or in an experimental setting) and / or (2) designed, created, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from at least 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 originally associated. In some embodiments, an isolated agent is 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 greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those of skill in the art, a substance may be considered "isolated" or even "pure" even after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the isolation or purity of a substance is calculated without including such carriers or excipients. By way of example only, in some embodiments, a biopolymer such as a naturally occurring polypeptide or polynucleotide is considered "isolated" if it: a) is free from association with some or all components that, by virtue of its origin or source, accompany it in its natural state in nature; b) is substantially free from other polypeptides or nucleic acids of the same species from the species that produces it in nature; and c) is expressed by or associated with components derived from cells or other expression systems other than those of the species that produces it in nature. Thus, for example, in some embodiments, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from that which produces the polypeptide in nature is considered an "isolated" polypeptide.Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification methods can be considered an "isolated" polypeptide to the extent that it has been separated from a) other components that naturally accompany the polypeptide, and / or b) other components that were associated with the polypeptide when it was originally produced.

[0040] Linker: As used herein, this term refers to a moiety that connects different elements of a substance that includes multiple elements. For example, those skilled in the art will recognize that polypeptides having structures that include two or more functional or organizational portions or domains often include stretches of amino acids between such portions or domains that connect them to each other. In some embodiments, polypeptides that include linker elements have an overall structure of the general formula S1-L-S2, where S1 and S2, which may be the same or different, represent two portions or domains joined together by the linker. In some embodiments, the polypeptide 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. In some embodiments, the linker is characterized by a tendency to not adopt a rigid three-dimensional structure, but rather to provide flexibility to the polypeptide. A variety of different linker elements are well known in the art that can be used appropriately in engineering polypeptides (e.g., fusion polypeptides) (e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444,1993; Poljak et al. Structure 2:1121,1994).

[0041] Modulator: The term "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 the level and / or nature of that activity compared to that observed in the absence of the modulator under otherwise equivalent conditions. In some embodiments, a modulator is an activator, in that activity is increased in its presence compared to the activity observed in the absence of the modulator under otherwise equivalent conditions. In some embodiments, a modulator is an antagonist or inhibitor, in that activity is decreased in its presence compared to the activity observed in the absence of the modulator under otherwise equivalent conditions. In some embodiments, a modulator interacts directly with a target entity whose activity is of interest. In some embodiments, a modulator interacts indirectly with a target entity whose activity is of interest (i.e., directly through an intermediate agent that interacts with the target entity). In some embodiments, a modulator affects the level of a target entity of interest. Alternatively or additionally, in some embodiments, a modulator affects the activity of a target entity without affecting the level of the target entity of interest. In some embodiments, a modulator affects both the level and activity of a target entity of interest, such that the observed difference in activity is not entirely explained by or disproportionate to the observed difference in level.

[0042] Moiety: Those skilled in the art will recognize that a "moiety" is a defined chemical group or entity having a particular structure and / or activity as described herein. Typically, a "moiety" is a portion of a whole molecule or entity, i.e., less than the whole.

[0043] Variant: As used herein, the term "variant" refers to an entity that exhibits 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 relative to the reference entity. In many embodiments, a variant also differs functionally from the reference entity. Generally, whether a particular substance is appropriately considered a "variant" of a reference entity is based on the degree of structural identity with the reference entity. As will be understood by those skilled in the art, any biological or chemical reference entity possesses certain characteristic structural elements. A variant, by definition, is another chemical entity that shares one or more such characteristic structural elements. To give just a few examples, a small molecule may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant moieties; thus, a variant of a small molecule is one that shares the core structural element and characteristic pendant moieties but differs in the other pendant moieties and bond types (single vs. double, E vs. Z, etc.) present within the core. A polypeptide may have a characteristic sequence element consisting of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to a specific biological function. Nucleic acids may have characteristic sequence elements consisting of multiple nucleotide residues with designated positions relative to each other in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide due to 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. In some embodiments, a variant polypeptide exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference polypeptide. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide.In some embodiments, the variant polypeptide lacks one or more of the biological activities of the reference polypeptide, hi some embodiments, the variant polypeptide exhibits a reduced level of one or more biological activities compared to the reference polypeptide.

[0044] Operably linked: As used herein, refers to a positional relationship in which the described components 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 obtained under conditions compatible with the control elements. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest. In some embodiments, the control element acts in trans or remotely on the functional element of interest.

[0045] Patient: As used herein, the term "patient" refers to any organism to which provided compositions are or can be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient has or is susceptible to one or more diseases or conditions. In some embodiments, the individual exhibits one or more symptoms of a disease or condition. In some embodiments, the patient has been diagnosed with one or more diseases or conditions. In some embodiments, the disease or condition is or includes cancer or includes the presence of one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular treatment to diagnose and / or treat the disease, disorder, or condition.

[0046] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those adapted for particular routes of administration, e.g., as described herein.

[0047] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to an agent or entity that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and / or animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0048] Pharmaceutically acceptable carrier: As used herein, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent encapsulating material, etc., used to carry or transport a compound of interest from one organ or site in the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of 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 cellulose derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; 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; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0049] Polypeptide: As used herein, refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered, in that it has been designed and / or created by the hand of man. In some embodiments, a polypeptide can comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can comprise only D-amino acids. In some embodiments, a polypeptide can comprise only L-amino acids. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications that modify or are attached to one or more amino acid side chains, for example, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications can 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 include a cyclic moiety. In some embodiments, a polypeptide is not cyclic and / or does not include any cyclic moieties. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, and in such cases, the term is used herein to refer to polypeptides that share a related activity or structure and therefore can be considered members of the same class or family of polypeptides.For each such class, the specification provides exemplary polypeptides within the class whose amino acid sequences and / or functions are known and / or would be recognized by one of skill in the art. In some embodiments, such exemplary polypeptides are reference polypeptides of that polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with reference polypeptides of that class, share common sequence motifs (e.g., characteristic sequence elements), and / or share common activities (in some embodiments, at similar levels or within a specified range), and in some embodiments, share these characteristics with all polypeptides in that class. For example, in some embodiments, a member polypeptide exhibits at least about 30-40% or more, often about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more overall sequence homology or identity with a reference polypeptide, and / or contains at least one region (e.g., a conserved region that, in some embodiments, is or includes a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically contains at least 3-4, and often up to 20 or more, amino acids, and in some embodiments, the conserved region contains 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. In some embodiments, the related polypeptide may comprise or consist of a fragment of a parent polypeptide.In some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each fragment being found in the same parent polypeptide in a different spatial arrangement from that 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 the fragments may be present in a different order in the polypeptide of interest than in the parent), such that the polypeptide of interest is a derivative of its parent polypeptide.

[0050] Predetermined: Predetermined means deliberately selected, as opposed to, for example, occurring or occurring randomly.

[0051] Pure: As used herein, a substance or entity is "pure" if it is substantially free of other components. For example, a preparation containing greater than about 90% of a particular agent or entity is generally considered to be a pure preparation. In some embodiments, the 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.

[0052] Recombinant: As used herein, is intended to mean a polypeptide that is designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise engineered to express a gene(s) or genetic element(s) encoding and / or directing the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or a polypeptide prepared, expressed, created, or isolated by any other means, including splicing or ligation of selected nucleic acid sequence elements together, chemical synthesis of selected sequence elements, and / or alternative generation of nucleic acids encoding and / or directing the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of the selected sequence elements are found in nature. In some embodiments, one or more of the selected sequence elements are designed in silico. In some embodiments, one or more of the selected sequence elements result from mutagenesis (e.g., in vivo or ex vivo) of known sequence elements from a natural or synthetic source, such as the germline of a source organism of interest (e.g., human, mouse, etc.).

[0053] Reference Standard: As used herein, refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally implemented in a tangible medium. Typically, as will be understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. Those of skill in the art will recognize when sufficient similarity exists to justify reliance on and / or comparison to a particular possible reference or control.

[0054] Specific binding: As used herein, the term "specific binding" refers to the ability to differentiate between possible binding partners in the environment in which binding occurs. A binding agent that interacts with a particular target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or measuring the degree of binding between the binding agent and its partner. In some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or measuring the ability of a binding agent to compete for an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations.

[0055] Specific: As used herein with respect to an active agent, the term "specific," as used herein with respect to an active agent, will be understood by those skilled in the art to mean that the agent differentiates between potential target entities or conditions. For example, in some embodiments, an agent is said to bind "specifically" to a target if it preferentially binds to the target in the presence of one or more competing alternative targets. In many embodiments, the specific interaction depends on the presence of a particular structural feature of the target entity (e.g., an epitope, cleft, binding site). It should be understood that specificity is not necessarily absolute. In some embodiments, specificity can be assessed relative to the specificity of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding agent. In some embodiments, specificity is assessed relative to the specificity of a reference non-specific binding agent. In some embodiments, the agent or entity does not detectably bind to competing alternative targets under conditions for binding to the target entity. In some embodiments, a binding agent binds its target entity with a higher on-rate constant, a lower off-rate constant, a higher affinity, a lower dissociation rate, and / or a higher stability compared to competing surrogate target(s).

[0056] Specificity: As is well 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.

[0057] Subject: As used herein, the term "subject" refers to an organism, generally a mammal (e.g., a human, including, in some embodiments, prenatal human forms). In some embodiments, the subject is afflicted with a relevant disease, disorder, or condition. In some embodiments, the subject is predisposed to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject possesses one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom and / or who is or has been diagnosed and / or treated.

[0058] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0059] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, therapeutic composition, and / or therapeutic formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be recognized by those of skill in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance being delivered, and the target cell or tissue. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose. In some embodiments, multiple unit doses are required to administer a therapeutically effective amount.

[0060] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, improves, alleviates, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of subjects who do not exhibit signs of the associated disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of subjects who exhibit established signs of one or more of the associated diseases, disorders, and / or conditions. In some embodiments, treatment may be of subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who are known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic. In some embodiments, treatment may be therapeutic.

[0061] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor can comprise precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a symptom of cancer. In some embodiments, a tumor can be a disseminated tumor or a liquid tumor. In some embodiments, a tumor can be a solid tumor.

[0062] Variant: As used herein in the context of a molecule, e.g., a nucleic acid, protein, or small molecule, the term "variant" refers to a molecule that, compared to a reference entity, exhibits significant structural identity with the reference molecule, e.g., the presence or absence of one or more chemical moieties, but is structurally distinct from the reference molecule. In some embodiments, a variant also differs functionally from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be recognized by those skilled in the art, any biological or chemical reference molecule possesses certain characteristic structural elements. A variant, by definition, is a molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. To name just a few examples, a polypeptide can have characteristic sequence elements consisting of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function, and a nucleic acid can have characteristic sequence elements consisting of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid due to 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 covalent elements of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities.In some embodiments, a variant polypeptide or nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference sequence except for a small number of sequence changes at specific positions. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to the reference. Often, the variant polypeptide or nucleic acid contains only a small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues responsible for a particular biological activity) relative to the reference. In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, the variant polypeptide or variant nucleic acid comprises less 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, usually less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or reference nucleic acid is one found in nature. In some embodiments, the reference polypeptide or reference nucleic acid is a human polypeptide or human nucleic acid.

[0063] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. 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. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0064] Viral inactivation or removal: As used herein, the term "viral inactivation or removal" refers to the inactivation or removal of viruses that may be contained in a sample, such as, for example, a cell extract or a fusion polypeptide preparation. In some embodiments, viruses present in a sample may originate from the source material (e.g., host cells). Alternatively or additionally, in some embodiments, viruses present in a sample may have been introduced, for example, during processing of such source material. Those skilled in the art will recognize various techniques for inactivating or removing viruses, such as, for example, by pH inactivation, chemical inactivation (e.g., by the use of chemicals such as detergents), and the like. Those skilled in the art will understand that "pH virus inactivation" includes exposing viruses (e.g., a sample containing viruses) to a pH that inactivates (e.g., has been established to inactivate) viruses.

[0065] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning and refers to a form of an entity (e.g., a polypeptide or nucleic acid) that has a structure and / or activity that is found in nature in a "normal" (as opposed to a mutation, disease, or alteration) state or context. In some embodiments, multiple "wild-type" forms of a particular polypeptide or nucleic acid may exist in nature, for example, as "alleles" of a particular gene or as common variants of a particular polypeptide. In some embodiments, the form(s) of a particular polypeptide or nucleic acid that are most commonly observed in a population (e.g., the human population) are the "wild-type" forms.

[0066] Fusion Polypeptides In some embodiments, fusion polypeptides according to the present disclosure can be in phosphorylated and unphosphorylated forms.

[0067] Immunomodulatory Polypeptides The fusion polypeptides of the present disclosure comprise at least one immunomodulatory polypeptide.

[0068] In some embodiments, the immunomodulatory polypeptide (e.g., immune agonist moiety) activates or inhibits the activity of a cell of the immune system (e.g., has signaling capabilities). In some embodiments, the immunomodulatory polypeptide (e.g., immune agonist moiety) is evaluated as part of a fusion polypeptide, e.g., as described herein.

[0069] For example, in some embodiments, signaling capability is characterized by the immune agonist portion(s) or functional fragments thereof exhibiting comparable binding to a reference standard (e.g., a wild-type polypeptide) when assessed for binding to a specific binding partner. For example, in some embodiments, signaling capability is characterized by the immune agonist portion(s) or functional fragments thereof exhibiting comparable biological effects to a reference standard (e.g., a wild-type polypeptide) when assessed for biological effects, e.g., in vitro or in vivo.

[0070] In some embodiments, the immunomodulatory polypeptide comprises an interleukin-12 (IL-12) immunomodulatory polypeptide (eg, an IL-12 immunoagonist moiety).

[0071] IL-12 is a proinflammatory cytokine that plays an important role in innate and adaptive immunity. Wild-type IL-12 is a heterodimeric protein containing two subunits, p35 (IL-12A, GenBank Gene ID: 3592) and p40 (IL-12B, GenBank Gene ID: 3593), linked by disulfide bonds. Binding of IL-12 to the IL-12 receptor complex (IL-12Rβ1 / IL-12Rβ2) on T cells and natural killer (NK) cells leads to signal transduction via signal transducer and activator of transcription 4 (STAT4) and subsequent production and secretion of interferon-γ (IFN-γ).

[0072] The IL-12 subunits, IL-12A and IL-12B, can also form heterodimers with other IL-12 family members. For example, IL-12A can also dimerize with Epstein-Barr virus-induced gene 3 (EBI3) to form the IL-12 family member IL-35, and IL-12B can dimerize with p19 monomers to form the IL-12 family member IL-23.

[0073] IL-12 plays an important role in innate and adaptive immune responses, and dysregulation of IL-12 has been linked to numerous pathologies. Exemplary pathologies include, but are not limited to, inflammatory bowel disease, psoriasis, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, cancer, lupus erythematosus, primarily biliary cholangitis, and Sjögren's syndrome (Ullrich et al. EXCLI journal vol. 19 1563-1589. 11 Dec. 2020). The use of IL-12 as a therapeutic modality has been extensively studied, including in the treatment of tumors (Nastala CL et al. J Immunol. 1994 Aug 15; Lasek et al. Cancer immunology, immunotherapy: CII vol. 63,5(2014): 419-35).

[0074] In some embodiments, an immunomodulatory polypeptide disclosed herein is or comprises an IL-12 immune agonist moiety. In some embodiments, an immunomodulatory polypeptide disclosed herein comprises multiple 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 moieties of the multiple (e.g., two) IL-12 moieties are the same moiety. In some such embodiments, the multiple (e.g., two) IL-12 moieties are different moieties. In some such embodiments, the IL-12 moiety comprises an IL-12A polypeptide or a functional fragment thereof. In some such embodiments, the IL-12 moiety comprises an IL-12B polypeptide or a functional fragment thereof.

[0075] In some embodiments, the IL-12B immune agonist moiety is located N-terminal to the IL-12A immune agonist moiety in the immunomodulatory polypeptide. In some embodiments, the IL-12A immune agonist moiety is located N-terminal to the IL-12B immune agonist moiety in the immunomodulatory polypeptide.

[0076] In some embodiments, immunomodulatory polypeptides comprising multiple (e.g., two) IL-12 moieties (e.g., IL-12A and / or IL-12B) are directly linked. In some embodiments, immunomodulatory polypeptides comprising multiple (e.g., two) IL-12 moieties (e.g., IL-12A and / or IL-12B) are linked via a first linker. Non-limiting examples of linkers are described elsewhere herein.

[0077] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immune agonist portion that comprises a variant. In some embodiments, the variant of the IL-12A and / or IL-12B immune agonist portion comprises a substitution, deletion, addition, and / or insertion relative to the wild-type IL-12A or IL-12B polynucleotide or amino acid sequence. In some embodiments, the IL-12A and / or IL-12B immune agonist portion comprises multiple variants. In some embodiments, the multiple variants comprise one or more of substitutions, deletions, additions, and / or insertions relative to the wild-type IL-12A or IL-12B. In some embodiments, the variants comprise substitutions that do not change the amino acid sequence relative to the wild-type IL-12A or IL-12B.

[0078] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise IL-12A and / or IL-12B immunoagonist portions that are functional fragments thereof (e.g., fragments capable of signaling). In some embodiments, the immunomodulatory polypeptide comprises a functional IL-12A fragment. In some embodiments, the immunomodulatory polypeptide comprises a functional IL-12B fragment. In some embodiments, the immunomodulatory polypeptide comprises full-length IL-12A and a functional IL-12B fragment. In some embodiments, the immunomodulatory polypeptide comprises full-length IL-12B and a functional IL-12A fragment.

[0079] In some embodiments, 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) found in wild-type IL-12A or IL-12B.

[0080] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immunoagonist moiety that is a human IL-12A and / or IL-12B immunoagonist moiety.

[0081] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12B immunoagonist portion having at least 80% sequence identity to SEQ ID NO: 3, e.g., at least 85%, such as at least 90%, for example, at least 91%, such as at least 92%, for example, at least 93%, such as at least 94%, for example, at least 95%, for example, at least 96%, such as at least 97%, for example, at least 98%, for example, at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A immunoagonist portion having at least 80% sequence identity to SEQ ID NO: 4, e.g., at least 85%, such as at least 90%, for example, at least 91%, such as at least 92%, for example, at least 93%, such as at least 94%, for example, at least 95%, for example, at least 96%, such as at least 97%, for example, at least 98%, for example, at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12B immune agonist portion having at least 80% sequence identity to SEQ ID NO:3 and an IL-12A immune agonist portion having at least 80% sequence identity to SEQ ID NO:4.

[0082] In some embodiments, the 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%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99% sequence identity to SEQ ID NO:5.

[0083] Without being bound by any particular theory, hydroxyl substitution (e.g., by a phosphate group) can increase the adsorption of polypeptides through ligand exchange with metal hydroxides (e.g., aluminum hydroxide), and can further improve the tumor retention and antitumor efficacy of such polypeptides (e.g., particularly of fusion polypeptides comprising an immunomodulatory polypeptide and a metal hydroxide-binding polypeptide in which such hydroxyl substitution has occurred).

[0084] In some embodiments, fusion polypeptides according to the present disclosure can be in phosphorylated and unphosphorylated forms. In some embodiments, the immunomodulatory polypeptide comprises at least one amino acid that can be phosphorylated. In some embodiments, the immunomodulatory polypeptide comprises at least one kinase target motif. In some embodiments, the immunomodulatory polypeptide does not comprise a kinase target motif. In these embodiments, the immunomodulatory polypeptide can still comprise an amino acid that can be phosphorylated. In some embodiments, immunomodulatory polypeptides comprising one or more phosphorylated amino acids contribute to strong metal binding with metal hydroxides, e.g., aluminum hydroxide. Table 1 lists exemplary serine residues that can be phosphorylated within the immunomodulatory domain (e.g., S43, S154, S168, S233, S365, S398, S481 of SEQ ID NO: 2). In some embodiments, the immunomodulatory polypeptide comprises at least one phosphorylated serine. In some embodiments, the immunomodulatory polypeptide comprises at least two phosphorylated serine residues. In some embodiments, the immunomodulatory polypeptide comprises at least three phosphorylated serine residues. In some embodiments, the immunomodulatory polypeptide comprises at least 4 phosphorylated serine residues, hi some embodiments, the immunomodulatory polypeptide comprises at least 5 phosphorylated serine residues, e.g., 6 serine residues, e.g., 7 serine residues, e.g., 8 serine residues, e.g., 9 serine residues, e.g., 10 serine residues.

[0085] Hydroxylated metal-binding polypeptides In some embodiments, a fusion polypeptide of the present disclosure comprises at least one metal-binding polypeptide.

[0086] The present disclosure provides metal hydroxide-binding polypeptides, and fusion polypeptides containing them, that exhibit high levels of adsorption to metal hydroxides and desirable manufacturing properties (e.g., one or more of reproducibility, consistency, and manufacturing of uniformly phosphorylated fusion polypeptides).

[0087] In some embodiments, a fusion polypeptide comprises two or more metal-binding polypeptides (e.g., two or more alum-binding polypeptides). In some embodiments, 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 according to the present disclosure are the same. In some such embodiments, a fusion polypeptide comprises two or more metal-binding polypeptides that are different from each other.

[0088] As described above, metal-binding peptides can be fused to immunomodulatory polypeptides to enable strong binding to metal hydroxides, such as aluminum hydroxide. Various immunomodulatory polypeptides can be fused to metal-binding peptides. Without being bound by theory, metal-binding polypeptides adsorbed to alum in serum can be used to retain proteins and peptides within tumors.

[0089] In some embodiments, the metal hydroxide-binding polypeptide comprises an amino acid sequence containing multiple phosphorylation sites, and thus can exist in both phosphorylated and unphosphorylated forms. In some embodiments, the metal hydroxide-binding polypeptide comprises at least one kinase target motif. The target kinase motif comprises an amino acid that is phosphorylated by a kinase. Typically, phosphorylated amino acids contain hydroxyls, such as serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y) residues. A kinase motif refers to an amino acid sequence immediately N-terminal and / or C-terminal to an amino acid residue that can be phosphorylated. While not wishing to be bound by any one theory, many kinases contain structural features that confer specificity for the kinase to phosphorylate specific amino acids (e.g., serine, threonine, or tyrosine) of a particular kinase target motif.

[0090] Recognized kinase target motifs vary widely depending on the specific kinase type. In some embodiments, the present disclosure provides metal hydroxide-binding polypeptides comprising one or more kinase target motifs of secretory pathway kinases. The secretory pathway is the pathway by which cells secrete proteins and / or other biomolecules into the extracellular space, and refers to the endoplasmic reticulum (ER), Golgi apparatus (Golgi), plasma 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., the ER, the Golgi, etc.) and function to phosphorylate secreted proteins (Sreelatha et al. Biochimica et bio Physicala Acta vol. 1854, 10 Pt B(2015): 1687-93).

[0091] In some embodiments, the relevant kinase is a native secretory pathway kinase (e.g., one that functions by endogenously targeting the secretory pathway). In some embodiments, the secretory pathway kinase comprises a signal sequence that targets the kinase to the secretory pathway. Native 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.

[0092] In some embodiments, the related kinase is a non-naturally occurring secretory pathway kinase, hi some embodiments, the non-naturally occurring kinase is generated by linking a secretory signal peptide to a kinase that is endogenously localized in a cellular compartment of the non-secretory pathway.

[0093] In some embodiments, the kinase target motif is a target kinase motif of a secretory pathway kinase. In some embodiments, the target kinase motif of a secretory pathway kinase comprises an SXE motif. For example, Fam20C has been shown to phosphorylate serine and target motifs containing the amino acid sequences Ser-X-Glu (e.g., SXE), Ser-X-pSer (e.g., SX-pS), and Ser-X-Gln-XX-Asp-Glu-Glu (SXQXXDEE), where X is any amino acid and pS is a phosphorylated serine (Mercier, et al (1981) Biochimie, 63: 1-17; Mercier et al (1971) Eur J. Biochem. 23:41-51; Lasa-Benito (1996) FEES Lett. 382:149; Brunati, et al (2000) 3:765, Tagliabracci, et al (2015) Cell 161:1619-1632; Tagliabracci, et al (2012) Science 336:1150-1153). In some embodiments, the target kinase motif comprises the amino acid sequence SEEE. In some embodiments, the target kinase motif comprises the amino acid sequence SEEA. In some embodiments, the target kinase motif comprises the amino acid sequence SEEQ. In some embodiments, the target kinase motif comprises the amino acid sequence SEE.

[0094] In some embodiments, the metal hydroxide-binding polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 SXE motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises more than four SXE motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SXE motifs. In some embodiments, the number of target kinase motifs (e.g., SXE motifs) contributes to the number of phosphorylated residues on the metal hydroxide-binding polypeptide. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SEE motifs.

[0095] In some embodiments, the metal hydroxide-binding polypeptide is a metal hydroxide-binding polypeptide whose amino acid sequence comprises multiple phosphorylation sites. In some embodiments, the multiple phosphorylation sites comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs. In some embodiments, the multiple phosphorylation sites comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 SXE motifs. In some embodiments, the multiple phosphorylation sites comprise more than four SXE motifs. In some embodiments, the multiple phosphorylation sites comprise more than eight SXE motifs. In some embodiments, the number of target kinase motifs (e.g., SXE motifs) contributes to the number of phosphorylated residues on the metal hydroxide-binding polypeptide.

[0096] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs (e.g., SXE motifs) are immediately adjacent to (e.g., linked to) the next target kinase (e.g., SXE motif). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs (e.g., SXE motifs) are separated from the next target kinase (e.g., SXE motif) by a spacer. In some embodiments, the spacer comprises at least one glycine residue. In some embodiments, the spacer comprises multiple glycine residues. In some embodiments, the spacer comprises three glycine residues. In some embodiments, the spacer comprises at least four glycine residues. In some embodiments, the spacer has a sequence comprising four glycine residues. In some embodiments, 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 that includes GGGG.

[0097] In some embodiments, the metal hydroxide-binding polypeptide comprises four SXE motifs and three spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises six SXE motifs and five spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SXE motifs and seven spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SXE motifs and eight spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight motifs having the amino acid sequence SEE and eight spacers comprising four glycine residues.

[0098] In some embodiments, the metal hydroxide-binding polypeptide comprises a terminal sequence (e.g., an amino acid sequence) at the C-terminus of the fusion polypeptide. In some embodiments, the terminal sequence comprises a plurality of amino acid residues. In some embodiments, the plurality of amino acid residues comprises GGGG. In some such embodiments, the terminal sequence comprises the amino acid sequence GGGGS.

[0099] In some embodiments, the desired (e.g., optimal) number of kinase target motifs and / or spacing of kinase motifs can be determined based on one or more of, for example, a desired phosphate content to achieve strong metal-hydroxide retention and / or avoidance of one or more manufacturing challenges (e.g., as the present disclosure recognizes may be associated with highly phosphorylated elements). In some embodiments, the desired (e.g., optimal) number and / or spacing of kinase motifs results in exposure of the polypeptide to a kinase such that a desired level of fusion polypeptide phosphorylation is achieved. In some embodiments, the improved fusion polypeptides described herein provide improved one or more of the following: reproducibility, consistency, and production of uniformly phosphorylated fusion polypeptides. For example, in some embodiments, the provided technology provides reproducible production of equivalent formulations (e.g., formulations that consistently fall within established parameters) of the fusion polypeptides (e.g., phosphorylated fusion polypeptides) and / or complexes described herein. For example, in some embodiments, the provided technology provides reduced immunogenicity compared to an appropriate reference standard.

[0100] In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 0.5 to 7, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, or 13 to 14. In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 3, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 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. Linker

[0101] In some embodiments, the fusion polypeptides described herein may include one or more linkers or spacers.

[0102] For example, in some embodiments, a fusion polypeptide comprises an immunomodulatory polypeptide comprising a first and a second immune agonist moiety, in some embodiments, the first immune agonist moiety and the second immune agonist moiety are linked via a first linker.

[0103] In some embodiments, a fusion polypeptide of the present disclosure comprises an immunomodulatory polypeptide and a metal hydroxide-binding polypeptide, hi some embodiments, the immunomodulatory polypeptide and the metal hydroxide-binding polypeptide are linked via a second linker.

[0104] In some embodiments, the first linker and / or the second linker is a polypeptide linker. In some embodiments, the polypeptide linker is synthetic. For example, a synthetic polypeptide linker can include a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide.

[0105] In some embodiments, a polypeptide linker of the present disclosure is at least one amino acid in length and can be any suitable number of amino acids, hi some embodiments, 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.

[0106] In some embodiments, the first linker comprises a polypeptide linker. In some embodiments, the first linker comprises or consists of a glycine-serine (Gly-Ser or GS linker). A Gly-Ser linker is a polypeptide linker consisting of glycine and serine residues. In some embodiments, a Gly-Ser linker is (Gly4Ser) n wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the Gly-Ser linker is (Gly4Ser)1. In some embodiments, the Gly-Ser linker is (Gly4Ser)2. In some embodiments, the Gly-Ser linker is (Gly4Ser)3. In some embodiments, the Gly-Ser linker is (Gly4Ser)4. In some embodiments, the Gly-Ser linker is (Gly4Ser)5. In some embodiments, the Gly-Ser linker is (Gly4Ser)6. In some embodiments, the Gly-Ser linker is (Gly4Ser)7. In some embodiments, the Gly-Ser linker is (Gly4Ser)8. In some embodiments, the Gly-Ser linker is (Gly4Ser)9. In some embodiments, the Gly-Ser linker is (Gly4Ser) 10 is.

[0107] In some embodiments, the second linker comprises a polypeptide linker. In some embodiments, the second linker comprises a plurality of glycine residues. In some embodiments, the second linker comprises a polypeptide linker having the amino acid sequence GGGGSGGGG. In some embodiments, the second linker comprises a polypeptide linker having the amino acid sequence GGGGEGGGG. variant

[0108] In some embodiments, an immunomodulatory polypeptide or metal hydroxide-binding polypeptide used in accordance with the present disclosure is a variant of a related reference polypeptide (e.g., a wild-type polypeptide or a functional portion thereof).

[0109] In some embodiments, a variant exhibits at least 70% identity to the reference polypeptide. In some such embodiments, a variant exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the reference polypeptide.

[0110] In some embodiments, a variant comprises one or more conservative or other non-perturbing modifications (e.g., substitutions, deletions, or additions) relative to its reference. In some embodiments, a variant does not comprise any perturbing modifications (e.g., substitutions, deletions, or additions), such that the immunomodulatory polypeptide maintains one or more functional properties of the reference. In some embodiments, maintaining means that the immunomodulatory polypeptide exhibits equivalent activity (e.g., signaling ability or binding) compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide). For example, in some such embodiments, the immunomodulatory polypeptide has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more activity compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide).

[0111] metal hydroxide In some embodiments, the present disclosure provides a phosphorylated fusion polypeptide comprising an immunomodulatory polypeptide and a metal hydroxide-binding polypeptide, wherein the phosphorylated fusion polypeptide complexes with the metal hydroxide upon exposure to the metal hydroxide. The complex is formed by adsorption of the phosphorylated fusion polypeptide to the metal hydroxide. Without wishing to be bound by any one theory, it is hypothesized that adsorption of the phosphorylated fusion polypeptide to the metal hydroxide occurs via ligand exchange. Ligand exchange is, for example, the replacement or exchange of surface hydroxyls with another ligand. In some embodiments, the replacement or exchange of surface hydroxyls occurs via hydroxyl substituents (e.g., phosphate groups).

[0112] In some embodiments, a metal hydroxide is a substance containing at least one hydroxyl group bound to a metal. According to the present disclosure, in some embodiments, the metal hydroxide is capable of adsorbing a fusion polypeptide containing a hydroxyl-substituted moiety. In some embodiments, the hydroxyl-substituted moiety is a phosphate group.

[0113] In some embodiments, the metal hydroxide is selected based on its inherent properties or characteristics. In some embodiments, the metal hydroxide is selected for its biocompatibility for use in a subject (e.g., a mammal, e.g., a human). In some embodiments, the metal hydroxide is aluminum hydroxide (e.g., alum). In some embodiments, the metal hydroxide is iron hydroxide. Those skilled in the art will recognize that any number of metal hydroxides can be successfully used in accordance with the present disclosure.

[0114] In some embodiments, the aluminum hydroxide is formulated in a gel (e.g., an aluminum hydroxide gel). In some embodiments, the aluminum hydroxide is formulated in water. In some embodiments, the concentration of the aluminum hydroxide original preparation is 10 mg / mL.

[0115] Production of phosphorylated fusion polypeptides In one aspect of the disclosure, the phosphorylated form of the fusion polypeptide described herein is produced by a method comprising the steps of: (1) producing the phosphorylated form of the fusion polypeptide in a host cell.

[0116] In one aspect of the present disclosure, a highly pure preparation of the phosphorylated form of the fusion polypeptide from a cell extract containing the phosphorylated form is obtained by a method comprising the step of (2) purifying the fusion polypeptide from the cell extract (e.g., from step (1)).

[0117] In one aspect of the present disclosure, a fusion polypeptide metal-hydroxide complex comprising a phosphorylated form of a fusion polypeptide described herein is produced by a method comprising the step of: (3) contacting a phosphorylated form of the fusion polypeptide (e.g., from step (2)) with a metal hydroxide.

[0118] Expression in host cells In some embodiments of the present disclosure, fusion polypeptides are produced by production in host cells, such as mammalian cells. Typically, such host cells (e.g., such mammalian cells) have been engineered to express the fusion polypeptide. Those skilled in the art will be familiar with various techniques for introducing and expressing exogenous gene sequences (e.g., encoding the fusion polypeptide and / or kinase) into host cells, e.g., mammalian host cells.

[0119] For example, in some embodiments, polynucleotides (e.g., DNA or RNA) encoding fusion polypeptides of the present disclosure can be prepared, e.g., for introduction into a host cell. For example, sequences encoding fusion polypeptides can be excised from DNA using restriction enzymes, amplified from a plasmid or genomic polynucleotide sequence using, e.g., the polymerase chain reaction, or synthesized using chemical synthesis methods. In some embodiments, a combination of known methods is used to prepare recombinant polynucleotides encoding fusion polypeptides of the present disclosure.

[0120] A recombinant polynucleotide encoding a fusion polypeptide of the present disclosure can be cloned into a vector capable of expressing the fusion polypeptide. Cloning can be performed according to various available methods (e.g., Gibson assembly, restriction digestion, and ligation, etc.). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a transposon.

[0121] In some embodiments, an expressible vector comprising a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure is operably linked to a sequence(s) controlling expression of the polynucleotide (e.g., a promoter, an initiation signal, a termination signal, a polyadenylation signal, an activator, a repressor, etc.). In some embodiments, one or more regulatory sequences controlling expression are selected to obtain a desired level of expression. In some embodiments, multiple sequences controlling expression (e.g., promoters) are used. In some embodiments, multiple sequences controlling expression (e.g., promoters) are used to obtain a desired level of expression of multiple recombinant polynucleotides encoding multiple polypeptides. In some embodiments, multiple recombinant polypeptides are expressed from the same vector (e.g., a bicistronic vector, a tricistronic vector, a multicistronic vector). In some embodiments, multiple recombinant polypeptides are expressed, each expressed from a separate vector.

[0122] In some embodiments, an expressible vector comprising a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure is used to express the fusion polypeptide in a host cell.

[0123] Host cells can be selected from a variety of available known host cells suitable for expressing the fusion polypeptides disclosed herein (e.g., human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese hamster ovary cells).

[0124] There are various methods for introducing a vector into a host cell. In some embodiments, the vector can be introduced into the host cell using transfection. In some embodiments, the transfection is completed using, for example, calcium phosphate transfection, lipofection, or polyethyleneimine-mediated transfection. In some embodiments, the vector can be introduced into the host cell using transduction.

[0125] In some embodiments, a host cell (eg, a producer cell) is used to produce a phosphorylated form of a fusion polypeptide.

[0126] In some embodiments, host cells expressing the fusion polypeptide and / or kinase are cultured in single-use bioreactors (e.g., 50 L to 4000 L) or stainless steel bioreactors (e.g., 50 L to 4000 L). In some embodiments, the host cells are cultured at a temperature ranging from 30°C to 40°C. In some embodiments, the temperature is lower (e.g., 33°C) during the production phase. In some embodiments, the cell extract is harvested by two- or three-stage filtration followed by a terminal sterilization 0.22 μm filtration.

[0127] In some embodiments, a nucleic acid encoding a fusion polypeptide is introduced into a host cell such that the fusion polypeptide is expressed by the host cell. Alternatively or additionally, in some embodiments, a nucleic acid encoding a kinase that phosphorylates the fusion polypeptide is introduced into the host cell such that the host cell expresses the kinase. In many embodiments, both a nucleic acid encoding a fusion polypeptide and a nucleic acid encoding a kinase that phosphorylates the fusion polypeptide are introduced into the same host cell, as described herein. In some such embodiments, a single nucleic acid molecule may encode both.

[0128] In some embodiments, the nucleic acid molecule introduced into the cell is RNA (e.g., mRNA), and in some such embodiments, the encoded polypeptide(s) (e.g., fusion polypeptide and / or kinase) are expressed from such RNA. Alternatively or additionally, in some embodiments, the nucleic acid molecule introduced into the cell is DNA (e.g., single-stranded DNA or double-stranded DNA). In some embodiments, the nucleic acid is introduced into the cell such that the coding sequence is integrated into the host cell (e.g., into its genome). In some such embodiments, the encoded polypeptide(s) (e.g., fusion polypeptide and / or kinase) are expressed from the host cell.

[0129] In some embodiments, nucleic acid molecules (e.g., nucleic acid molecules encoding fusion polypeptides and / or kinases) introduced into cells comprise one or more expression elements that can, for example, control expression of such encoded polypeptide(s). Alternatively or additionally, in some embodiments, nucleic acid molecules (e.g., nucleic acid molecules encoding fusion polypeptides and / or kinases) introduced into cells can be designed or intended to associate (e.g., by integration) with one or more regulatory elements in the host cell.

[0130] In some embodiments, vectors (e.g., transposons) containing sequences encoding the fusion polypeptides and / or kinases described herein are used to express the fusion polypeptides and / or kinases in host cells.

[0131] In some embodiments, the host cell can be selected from a variety of available known host cells suitable for expressing the fusion polypeptides disclosed herein (e.g., human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese hamster ovary cells). In some embodiments, the host cell is a mammalian cell.

[0132] There are various methods for introducing nucleic acids (e.g., vectors such as expression vectors) into host cells. In some embodiments, nucleic acids can be introduced into host cells using transfection. In some embodiments, transfection is completed using, for example, calcium phosphate transfection, lipofection, or polyethyleneimine-mediated transfection. In some embodiments, vectors can be introduced into host cells using transduction. In some embodiments, vectors can be introduced into host cells using electroporation. In some embodiments, nucleic acids can be introduced into host cells using particle delivery, for example, polymer particle delivery, lipid particle delivery, gold particle delivery, etc.

[0133] phosphorylation In some embodiments, the present disclosure provides methods for producing a phosphorylated form of a fusion polypeptide by contacting a fusion polypeptide disclosed herein with a kinase. In some embodiments, a nucleic acid, e.g., a nucleic acid encoding the fusion polypeptide and / or the kinase, is introduced into a host cell. In some embodiments, the fusion polypeptide is contacted with the kinase by co-expressing the fusion polypeptide with the kinase in the host cell. In some embodiments, co-expression is achieved by introducing into the host cell two vectors, one containing a recombinant polynucleotide encoding the fusion polypeptide and the other containing a recombinant polynucleotide encoding the kinase. In some embodiments, co-expression is achieved by introducing a single multicistronic (e.g., bicistronic) vector (e.g., a transposon) containing multiple recombinant polynucleotides. In some embodiments, one recombinant polynucleotide encodes the fusion polypeptide and one recombinant polynucleotide encodes the kinase. In some embodiments, the transformed host cell is cultured after introduction of the vector (e.g., a transposon) into the host cell. Without wishing to be bound by any one theory, upon co-expression of the fusion polypeptide and the kinase in the host cell, the kinase can contact and phosphorylate the fusion polypeptide.

[0134] In some embodiments, coexpression is achieved by introducing into a host cell two vectors, one containing a recombinant polynucleotide encoding a fusion polypeptide and the other containing a recombinant polynucleotide encoding a kinase. In some embodiments, the two vectors are introduced into the host cell at a ratio of vector encoding the fusion polypeptide to vector encoding the kinase that is optimized to achieve a desired relative level of expression of the fusion polypeptide to the kinase. In some embodiments, the ratio of vector encoding the fusion polypeptide to vector encoding the 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.

[0135] In some embodiments, co-expression is achieved by introducing into the host cell a single vector (e.g., a bicistronic vector) containing both a recombinant polynucleotide encoding the fusion polypeptide and a recombinant polypeptide encoding the kinase. In some embodiments, co-expression is achieved by introducing into the host cell a single transposon containing both a recombinant polynucleotide encoding the fusion polypeptide and a recombinant polypeptide encoding the kinase. In some embodiments, the transposon is a DNA transposon. In some embodiments, the transposon, or a portion thereof (e.g., containing the nucleotides encoding the fusion polypeptide and the nucleotides encoding the kinase), is integrated into the host cell genome by an integrase (i.e., by an integrase enzyme such as DDE / D integrase). In some embodiments, the integrase is delivered to the host cell as mRNA. In some embodiments, the integrase is a PiggyBac enzyme. In some embodiments, the integrase is a leap-in transposase. In some embodiments, the transposon, or a portion thereof, is not integrated into the genome by random integration. In some embodiments, a single copy of a polynucleotide encoding a fusion polypeptide of the present disclosure is integrated into specific multiple host cell genomic loci. In some embodiments, 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 can ensure stable integration, allowing for the generation of highly stable cell lines. In some embodiments, the recombinant polynucleotide encoding the fusion polypeptide and the recombinant polynucleotide encoding the kinase can be operably linked to sequence(s) that control expression (e.g., promoter, start signal, stop signal, polyadenylation signal, activator, repressor, etc.). In some embodiments, the sequence(s) that control expression are selected to obtain a desired expression level.In some embodiments, a desired ratio of fusion polypeptide to kinase is achieved by using multiple regulatory nucleotide sequences (e.g., promoters) to control expression. In some embodiments, the 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. In some embodiments, the regulatory nucleotide sequence is a promoter. In some embodiments, a particular ratio (e.g., 2:1, 4:1, 8:1, or 15:1) is achieved by using a single transposon containing two promoters to express the fusion polypeptide and the kinase. In some embodiments, a single separate transposon contains promoters of different strengths to provide a desired ratio (e.g., 8:1). In some embodiments, the promoter is a CMV promoter or an EF1a promoter. In some embodiments, the fusion polypeptide is under the control of a CMV promoter or an EF1a promoter. In some embodiments, the promoter is an SV40 promoter or a Ubc promoter. In some embodiments, the fusion polypeptide is under the control of an SV40 promoter or a Ubc promoter. In some embodiments, the ratio of fusion polypeptide to kinase is 8:1.

[0136] In some embodiments, 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, has been introduced) are cultured following such transformation, e.g., to allow for expression of the recombinant polynucleotide. In some embodiments, the transformed host cells are cultured for at least 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 hours, or more. In some embodiments, the transformed host cells are cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. The transformed host cells are cultured under growth conditions (e.g., temperature, carbon dioxide level, growth medium) according to the requirements of the selected host cells. Those of skill in the art will recognize that culture conditions for selected host cells are well known in the art. In some embodiments, the host cells secrete the phosphorylated form of the fusion polypeptide into the cell extract. In some embodiments, the host cells may exhibit increased secretion of the phosphorylated fusion polypeptide into the cell extract compared to the non-phosphorylated fusion polypeptide. In some embodiments, the host cells secrete comparable levels of the phosphorylated and non-phosphorylated fusion polypeptide into the cell extract. In some embodiments, the cell extract comprises the non-phosphorylated form of the fusion polypeptide. In some embodiments, the cell extract comprises the phosphorylated form of the fusion polypeptide. In some embodiments, the cell extract comprises a mixture of both the non-phosphorylated and phosphorylated forms of the fusion polypeptide. In some embodiments, the cell extract comprises more of the phosphorylated form of the fusion polypeptide than the non-phosphorylated form of the fusion polypeptide.

[0137] In some embodiments, the host cells secrete the phosphorylated form of the fusion polypeptide into cell extracts, but not the kinase, hi some embodiments, the host cells do not secrete the kinase, or only secrete small amounts of the kinase.

[0138] In some embodiments, the host cell extract comprises a phosphorylated form of the fusion polypeptide. In some embodiments, the host cell extract comprises host cell proteins and / or host cell nucleotides.

[0139] In some embodiments, the phosphorylated form of the fusion polypeptide is recovered from the transformed host cells and clarified by centrifugation.

[0140] In some embodiments the transformed host cell is characterized in that a culture thereof produces a titer of fusion protein of at least 200 mg / L, such as at least 250 mg / L, for example, at least 300 mg / L, such as at least 350 mg / L, for example, at least 400 mg / L, such as at least 450 mg / L, for example, at least 500 mg / L, such as at least 550 mg / L, for example, at least 600 mg / L, such as at least 650 mg / L, for example, at least 700 mg / L, such as at least 750 mg / L, for example, at least 800 mg / L, such as at least 850 mg / L, for example, at least 900 mg / L, such as at least 950 mg / L, for example, at least 1 g / L or more.

[0141] In some embodiments, one or more serine residues at positions 43, 281, 306, 311, 316, 365, or 481 of SEQ ID NO:2 are phosphorylated. In some embodiments, one or more serine residues at positions 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 positions 43, 154, 168, 281, 306, 311, 316, 365, or 481 of SEQ ID NO:2 are phosphorylated. In some embodiments, one or more serine residues at positions 43, 154, 168, 281, 306, 311, 316, 365, 406, or 481 of SEQ ID NO:2 are phosphorylated. In some embodiments, at least the serine residue at position 481 of SEQ ID NO:2 is phosphorylated.

[0142] purification In some embodiments, the disclosure provides purification methods, e.g., methods comprising one or more purification steps, or techniques (e.g., manufacturing techniques) comprising the same. In some embodiments, the phosphorylated form of the fusion protein is purified from extracts of the cells described herein.

[0143] In some embodiments, the purification step can include the removal of common abnormal products from the cell extract (eg, residual proteins, host cell contaminants (eg, host DNA and / or proteins, etc.)).

[0144] In some embodiments, the production method, including one or more purification steps, results in a highly pure preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the highly pure preparation of the phosphorylated form of the fusion polypeptide contains host cell protein reduced to the same extent as the cell extracts described hereinabove. In some embodiments, such highly pure preparations are free of host cell protein. In some embodiments, the highly pure preparations contain less than 100 ng / mg, e.g., less than 50 ng / mg, e.g., less than 40 ng / mg, e.g., less than 30 ng / mg, e.g., less than 20 ng / mg, e.g., less than 10 ng / mg, e.g., less than 9 ng / mg, e.g., less than 8.5 ng / mg of host cell protein. In some embodiments, the highly pure preparation of the phosphorylated form of the fusion polypeptide contains host cell DNA reduced to the same extent as the cell extracts described hereinabove. In some embodiments, such highly pure preparations are free of host cell DNA. In some embodiments a highly pure preparation comprises less than 10 pg / mg, such as less than 9 pg / mg, for example less than 8 pg / mg, such as less than 7 pg / mg, for example less than 6 pg / mg, such as less than 5 pg / mg, for example less than 4 pg / mg, such as less than 3 pg / mg, for example less than 2 pg / mg, such as less than 1.5 pg / mg, for example less than 1 pg / mg, such as less than 0.9 pg / mg, for example less than 0.8 pg / mg, for example less than 0.7 pg / mg host cell DNA.

[0145] In some embodiments, the high purity preparation contains low levels of in-process compounds. In some embodiments, the in-process compounds can be tropolone, pluronic, PDMS, octamethylcyclotetrasiloxane D4, TDAO, and / or Fam20. In some embodiments, the high purity preparation contains less than 1 mg / mL TDAO, for example, less than 0.9 mg / mL TDAO, for example, less than 0.8 mg / mL TDAO, for example, less than 0.7 mg / mL TDAO, for example, less than 0.6 mg / mL TDAO, for example, less than 0.5 mg / mL TDAO, for example, less than 0.4 mg / mL TDAO, for example, less than 0.3 mg / mL TDAO. In some embodiments, a highly pure preparation has less than 5000 ng Fam20C per mg of IL-12 fusion polypeptide, such as less than 4000 ng Fam20C per mg of IL-12 fusion polypeptide, e.g., less than 3000 ng Fam20C per mg of IL-12 fusion polypeptide, e.g., less than 2500 ng Fam20C per mg of IL-12 fusion polypeptide, e.g., less than 2000 ng Fam20C per mg of IL-12 fusion polypeptide, e.g., less than 1800 ng Fam20C per mg of IL-12 fusion polypeptide, e.g., less than 1000 ng Fam20C per mg of IL-12 fusion polypeptide. The IL-12 fusion polypeptide may comprise less than 750 ng of Fam20C per mg of IL-12 fusion polypeptide, for example, less than 500 ng of Fam20C per mg of IL-12 fusion polypeptide, for example, less than 300 ng of Fam20C per mg of IL-12 fusion polypeptide, for example, less than 200 ng of Fam20C per mg of IL-12 fusion polypeptide, for example, less than 100 ng of Fam20C per mg of IL-12 fusion polypeptide, for example, less than 80 ng of Fam20C per mg of IL-12 fusion polypeptide, for example, less than 70 ng of Fam20C per mg of IL-12 fusion polypeptide, for example, less than 60 ng of Fam20C per mg of IL-12 fusion polypeptide.

[0146] In some embodiments, the phosphorylated form of the fusion polypeptide can be purified by including one or more chromatographic purification steps. In some embodiments, one or more conventional chromatographic steps are used. In some embodiments, the conventional chromatographic steps use anion or cation exchange, hydrophobic interaction, or hydroxyapatite chromatography.

[0147] In some embodiments, the phosphorylated form of the fusion polypeptide can be purified by including one or more purification steps selected from an ionic chromatography step (e.g., an anionic chromatography step) and a hydrophobic interaction chromatography step, thus separating the phosphorylated fusion polypeptide from impurities.

[0148] Those skilled in the art will be familiar with the various purification (e.g., chromatography) matrices and their formats that can be used in accordance with the present disclosure. For example, in some embodiments, beads, particles, microspheres, resins, etc. can be used. In some embodiments, the matrix used for purification (e.g., chromatography), in accordance with the present disclosure, has properties that allow for a different retention time of the fusion polypeptide relative to any other undesired components in the preparation of the fusion polypeptide.

[0149] In some embodiments, the phosphorylated form of the fusion polypeptide is not purified by affinity-based purification methods, e.g., in some embodiments, the purification techniques provided do not employ affinity chromatography.

[0150] In some embodiments, the phosphorylated form of the fusion polypeptide can be eluted from the solid substrate. In some embodiments, elution can be accomplished using specific elution. For example, in some embodiments, specific elution is accomplished by loading the polypeptide-substrate complex with an agent(s) that complexes with either the substrate or the polypeptide, releasing the polypeptide into solution. In some embodiments, elution can be accomplished using non-specific elution. For example, in some embodiments, non-specific elution is accomplished by manipulating solvent or buffer conditions to decrease the binding rate constant (e.g., increasing the concentration of a buffer, e.g., an imidazole buffer), thereby dissociating the polypeptide from the substrate.

[0151] First Chromatography Step In some embodiments, the methods according to the present disclosure include at least one ion chromatography step (e.g., an anion chromatography step). In some embodiments, the methods of the present disclosure include at least one anion chromatography step.

[0152] In some embodiments, the first chromatography step is a capture step (e.g., an anion chromatography capture step). Without wishing to be bound by any theory, phosphorylation of a polypeptide imparts a variance in the charge of the polypeptide, allowing for the separation of highly phosphorylated polypeptides using ion exchange chromatography (e.g., anion exchange chromatography). Anion exchange chromatography is a form of ion exchange in which negatively charged biomolecules (e.g., phosphorylated forms of the fusion polypeptides disclosed herein) bind to a positively charged solid substrate (e.g., a resin). In this way, the positively charged solid substrate captures the negatively charged fusion polypeptide from the cell extract while simultaneously removing not only positively charged impurities but also fusion polypeptide aggregates from the cell extract, since the fusion polypeptide aggregates are likely to have a less negative charge.

[0153] In some embodiments, anion exchange chromatography can be used to separate polypeptides having 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 six phosphorylation sites). Thus, in some embodiments, a preparation having a high concentration of phosphorylated fusion polypeptide and a low concentration of positively charged impurities is generated by using an anion exchange chromatography step (e.g., as a first step).

[0154] In some embodiments, purifying the phosphorylated form of the fusion polypeptide from the cell extract comprises an anion chromatography capture step, hi some embodiments, the anion chromatography capture step is the first capture step.

[0155] In some embodiments, anion exchange chromatography uses an ion exchange resin to which positively charged groups, such as quaternary amino groups, are covalently attached. Commercially available anion exchange resins include Q Sepharose, DEAE Sepharose, TMAE, GidaCap Q 650M, and 650S. Binding a negatively charged biomolecule (e.g., a phosphorylated form of a fusion polypeptide) to an anion exchange material, in some embodiments, involves exposing the negatively charged biomolecule to the resin under appropriate conditions (e.g., pH / conductivity), thereby binding the biomolecule to the anion exchange resin via ionic interactions between the negatively charged biomolecule and the charged group(s) of the ion exchange material.

[0156] The wash step may involve passing an appropriate buffer through the chromatography resin to flush out unwanted materials, such as host cell proteins or host cell nucleotides. In some embodiments, the wash buffer may contain various conditions, such as pH, conductivity, etc., aimed at dissociating impurities that nonspecifically bind to the chromatography resin. In some embodiments, the wash step uses a mixture of an equilibration buffer and an elution buffer.

[0157] The phosphorylated form of the fusion polypeptide can be eluted from the solid substrate (e.g., a positively charged resin) using elution. In some embodiments, the negatively charged substance (e.g., the phosphorylated form of the fusion polypeptide) is eluted using a buffer that reduces the interaction between the anion exchange resin and the negatively charged substance (e.g., the phosphorylated fusion polypeptide). In some embodiments, such elution buffers can have a higher salt concentration and / or a different pH to facilitate dissociation of the negatively charged agent from the chromatography resin.

[0158] In some embodiments, a gradient elution buffer (e.g., a buffer of increasing salt concentration) is used to elute from an ion exchange (e.g., anion exchange) column. In some such embodiments, the use of such a gradient allows for the separation of differentially phosphorylated polypeptides (i.e., separation of different phosphorylated forms can be achieved).

[0159] In some embodiments, the buffer is a Tris buffer. In some embodiments, a linear gradient of Tris buffer is used. In some embodiments, the linear gradient of Tris buffer comprises a linear gradient from 20 mM Tris (pH 7.1) to 20 mM Tris, 1 mM NaCl (pH 7.1) over a predetermined time period. In some embodiments, the linear gradient is performed over 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 more. In some embodiments, a Tris buffer is used in the first anion chromatography capture step. In some embodiments, the first anion chromatography capture step is performed at a pH of about 6 to about 9, e.g., about 7 to about 8.

[0160] In some embodiments, the first anion chromatography capture step is carried out using capture beads. In some embodiments, the capture beads of the first step have a diameter of at least 50 μm, for example, at least 55 μm, for example, at least 60 μm, for example, at least 65 μm, for example, at least 70 μm, for example, at least 75 μm. In some embodiments, the first capture beads are GidaCap Q 650M.

[0161] In some embodiments, the first anion chromatography capture step includes an immobilization step (e.g., binding the phosphorylated fusion polypeptide to the chromatography column), a pre-elution wash step, and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads having an average particle size of about 50 to about 100 micrometers, e.g., 75 micrometers, are used to immobilize the phosphorylated fusion polypeptide. In some embodiments, an immobilization composition having a low salt concentration is used during immobilization of the phosphorylated fusion polypeptide to the chromatography column (e.g., 0 M sodium chloride). In some embodiments, a pre-elution composition having an intermediate salt concentration (e.g., 215 mM sodium chloride) equivalent to the salt concentrations of the immobilization and elution compositions is used during the pre-elution wash step. In some embodiments, an elution composition having a high salt concentration is used during elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 350 mM sodium chloride). In some embodiments, the first anion chromatography capture step is performed at a pH within the range of about 7 to about 8 (e.g., 7.4). In some embodiments, a flow rate of 200-400 cm / h (eg, 300 cm / h) is used.

[0162] Second Chromatography Step In some embodiments, the methods of the present disclosure include at least one hydrophobic interaction chromatography step. In some embodiments, the hydrophobic interaction step is performed after the first anion chromatography step. Without being bound by theory, the phosphorylated forms of fusion proteins according to the present disclosure have low hydrophobicity (e.g., due to their degree of phosphorylation and therefore their charge), which can be used to separate the phosphorylated forms from hydrophobic impurities. In some embodiments, hydrophobic host cell impurities (e.g., host cell proteins) and / or fusion polypeptide aggregates are separated from a preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the preparation (e.g., a high-purity preparation) contains less than 5% aggregated fusion polypeptide, e.g., less than 4%, e.g., less than 3%, e.g., less than 3%, e.g., less than 2%, e.g., less than 1%, e.g., less than 0.9%, e.g., less than 0.8% aggregated fusion polypeptide.

[0163] The hydrophobic interaction step, in some embodiments, can separate product or process-related impurities, such as host cell proteins or aggregation products (e.g., fusion polypeptide aggregates), from the phosphorylated form of the fusion polypeptide based on the differential hydrophobic interactions of the phosphorylated fusion polypeptide and the impurities with hydrophobic substances. Such a step, in some embodiments, can be referred to as a final purification step.

[0164] Examples of 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 of a negatively charged substance (e.g., a phosphorylated form of a fusion polypeptide) to a hydrophobic interaction resin is, in some embodiments, accomplished by exposing the biomolecule to the resin under appropriate conditions (pH / conductivity) such that the biomolecule is immobilized to the hydrophobic resin via hydrophobic interactions between the biomolecule and the nonpolar groups of the hydrophobic interaction material. Binding via hydrophobic interactions typically occurs at high salt concentrations (e.g., 1 to 1.8 M ammonium sulfate). In some embodiments, the phosphorylated form of the fusion polypeptide is immobilized at high salt concentrations (e.g., 1.4 M sodium sulfate). In some embodiments, the phosphorylated form of the fusion polypeptide is eluted with a linear gradient ranging from 1.4 M ammonium sulfate to 0 M ammonium sulfate.

[0165] The wash step may involve passing an appropriate buffer through the chromatography resin to flush out unwanted materials, such as less hydrophobic host cell proteins. In some embodiments, the wash buffers may have different pHs, which facilitate dissociation of less hydrophobic materials from impurities nonspecifically bound to the chromatography resin. In some embodiments, the wash step uses a mixture of an equilibration buffer and an elution buffer.

[0166] The phosphorylated form of the fusion polypeptide can be eluted from the solid substrate (e.g., a hydrophobic resin) using elution. In some embodiments, less hydrophobic substances (e.g., phosphorylated forms of the fusion polypeptide) are eluted from the hydrophobic resin using a buffer that reduces the interaction between the hydrophobic interaction resin and negatively charged substances (e.g., phosphorylated fusion polypeptides). In some embodiments, such elution buffers can have a lower salt concentration or a pH change that promotes dissociation of biomolecules from the chromatography resin. In some embodiments, the phosphorylated form of the fusion polypeptide is eluted with a low salt concentration (e.g., 750 mM sodium sulfate).

[0167] In some embodiments, the hydrophobic interaction chromatography step includes an immobilization step (e.g., binding the phosphorylated fusion polypeptide to a chromatography column) and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads having an average particle size of about 50 to about 100 micrometers, e.g., 75 micrometers, are used to immobilize the phosphorylated fusion polypeptide. In some embodiments, an immobilization composition having a high salt concentration is used to immobilize the phosphorylated fusion polypeptide to the chromatography column (e.g., 1.4 M ammonium sulfate). In some embodiments, an elution composition having a low salt concentration is used to elute the phosphorylated fusion polypeptide from the chromatography column (e.g., 740 mM ammonium sulfate). In some embodiments, the hydrophobic interaction chromatography step is performed at a pH in the range of about 7 to about 8 (e.g., 7.4). In some embodiments, a flow rate of 200 to 350 cm / h (e.g., 275 cm / h) is used.

[0168] Third Chromatography Step In some embodiments, the method according to the present disclosure comprises a first anion chromatography step and a second anion chromatography step. In some embodiments, the method according to the present disclosure comprises 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 final purification step. In some embodiments, the second anion chromatography step is performed after the first hydrophobic interaction chromatography step.

[0169] In some embodiments, a method according to the present disclosure comprises the steps of: i) a first anion chromatography step; ii) a hydrophobic interaction chromatography step, and iii) A second anion chromatography step.

[0170] In some embodiments, the second anion chromatography step is performed using capture beads. In some embodiments, the capture beads of the second step have a diameter of at most 50 μm, at most 45 μm, at most 40 μm, or at most 35 μm. In some embodiments, the first capture beads are GidaCap Q 650S.

[0171] In some embodiments, the second anion chromatography capture step includes an immobilization step (e.g., binding the phosphorylated fusion polypeptide to the chromatography column), a pre-elution wash step, and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads having an average particle size of about 10 to about 50 micrometers, e.g., 35 micrometers, are used to immobilize the phosphorylated fusion polypeptide. In some embodiments, an immobilization composition having a low salt concentration is used during immobilization of the phosphorylated fusion polypeptide to the chromatography column (e.g., 0 M sodium chloride). In some embodiments, a pre-elution composition having an intermediate salt concentration (e.g., 274 mM sodium chloride) equivalent to the salt concentrations of the immobilization composition and elution composition is used during the pre-elution wash step. In some embodiments, an elution composition having a high salt concentration is used during elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 355 mM sodium chloride). In some embodiments, the 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 (eg, 300 cm / h) is used.

[0172] Optional additional step(s) In some embodiments, the method according to the present disclosure includes a virus inactivation or virus removal step. In some embodiments, the virus inactivation or virus removal step is performed before or after any of the chromatography steps described hereinabove. In one embodiment, the virus inactivation or virus removal step is performed before the first chromatography step (e.g., the first anion chromatography step). In some embodiments, the virus inactivation step includes pH inactivation or chemical inactivation (e.g., by using a chemical agent such as a detergent). In some embodiments, the virus inactivation step includes, without being bound by theory, the use of a detergent because IL12-ABP is highly sensitive and may aggregate at low pH. In some embodiments, the virus inactivation step includes the use of a detergent selected from myristyldimethylamine N-oxide, TDAO, Triton X-100, or polysorbate. In some embodiments, the virus removal step includes a filtration step.

[0173] Preparation of fusion polypeptides In some embodiments, the present disclosure provides, among other things, a fusion polypeptide preparation. In some embodiments, the fusion polypeptide preparation comprises a phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation is a highly purified preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises a mixture of unphosphorylated and phosphorylated forms of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises more phosphorylated polypeptide than unphosphorylated fusion polypeptide.

[0174] In some embodiments, the phosphorylated fusion polypeptide preparation comprises fusion polypeptides with various degrees of phosphorylation as described herein above.

[0175] In some embodiments, the fusion polypeptide preparation comprises a buffer. In some embodiments, the fusion polypeptide preparation comprises a Tris buffer at about pH 7 to about pH 8. In some embodiments, the fusion polypeptide preparation comprises a salt (e.g., NaCl).

[0176] Fusion Polypeptide Compositions In some embodiments, the present disclosure provides, among other things, a fusion polypeptide composition. In some embodiments, the fusion polypeptide composition comprises a phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide composition is a highly purified composition of the phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide composition comprises a mixture of unphosphorylated and phosphorylated forms of the fusion polypeptide. In some embodiments, the fusion polypeptide composition comprises more phosphorylated polypeptide than unphosphorylated fusion polypeptide. In some embodiments, the fusion polypeptide composition comprises fusion polypeptides with various degrees of phosphorylation as described herein above.

[0177] In some embodiments, the fusion polypeptide composition comprises a buffer. In some embodiments, the buffer is a Tris buffer. Among other things, the present disclosure shows that Tris buffer(s) are particularly useful for the stability of the IL-12 fusion polypeptides described herein. For example, the present disclosure shows that Tris buffer can provide significant stability advantages over alternative buffer(s), such as histidine buffer(s). In some embodiments, the buffer is not a histidine buffer.

[0178] In some embodiments, the fusion polypeptide composition has a pH of about 6.5 to about 8. In some embodiments, the fusion polypeptide composition has a pH of at most 7.5, e.g., at most 7.4, e.g., at most 7.7. In some embodiments, the fusion polypeptide composition has a pH of at least 7, e.g., at least 7.1, e.g., at least 7.2, e.g., at least 7.3. Without wishing to be bound by any particular theory, high pH may result in deamidation of the IL-12 fusion polypeptide.

[0179] In some embodiments, the fusion polypeptide composition comprises a salt. Without wishing to be bound by any particular theory, it is believed that salt can act as a tonicity adjuster in the composition and / or formulation and aid in the stability of the IL-12 fusion polypeptide by stabilizing the structure of the molecule through ionic interactions. In some embodiments, the fusion polypeptide composition comprises a salt, and the salt concentration can be in the range of about 1 mM to about 750 mM, e.g., about 10 mM to about 500 mM, e.g., about 20 mM to about 100 mM, e.g., about 30 mM to about 60 mM, e.g., about 35 mM to about 55 mM. In some embodiments, the salt is NaCl or Na2SO4.

[0180] In some embodiments, the fusion polypeptide composition comprises a surfactant. In some embodiments, the fusion polypeptide composition comprises a hydrophilic surfactant. Hydrophilic surfactants may interact more with charged molecules, such as phosphorylated forms of IL-12 fusion polypeptides, compared to more hydrophobic surfactants. This interaction reduces the formation of visible particles upon shaking (i.e., susceptibility to agitation). In some embodiments, the fusion polypeptide composition comprises a polysorbate (e.g., polysorbate 20 or polysorbate 80). In some embodiments, the fusion polypeptide composition comprises polysorbate 20. In some embodiments, the fusion polypeptide composition comprises a surfactant (e.g., polysorbate 20), wherein the concentration of the surfactant is in the range of about 0.0005% w / v to about 1% w / v, about 0.005% w / v to about 0.1% w / v, about 0.01% w / v to about 0.05% w / v, or about 0.015% w / v to about 0.2% w / v.

[0181] In some embodiments, the fusion polypeptide composition comprises L-methionine. The addition of methionine is believed to positively affect the stability of the IL-12 fusion polypeptide, particularly by reducing the risk of oxidation and preventing the generation of high molecular weight species (HMWS). In some embodiments, the fusion polypeptide composition comprises L-methionine, and the concentration of L-methionine is in the range of about 1 mM to about 20 mM, e.g., in the range of about 5 mM to about 15 mM.

[0182] In some embodiments, the fusion polypeptide composition comprises a disaccharide (e.g., sucrose or trehalose). In some embodiments, the fusion polypeptide composition comprises sucrose, and the concentration of sucrose is in the range of about 100 mM to about 200 mM.

[0183] Pharmaceutical Composition In some embodiments, pharmaceutical formulations comprise a fusion polypeptide-metal hydroxide complex described herein. An exemplary pharmaceutical composition is shown in Example 8. Such pharmaceutical formulations can be prepared by mixing a fusion polypeptide composition described herein above with a metal hydroxide. In some embodiments, the fusion polypeptide-metal hydroxide complex described herein is produced by contacting the fusion polypeptide composition with a metal hydroxide (e.g., aluminum hydroxide). In some embodiments, the aluminum hydroxide is formulated in a gel. In some embodiments, the aluminum hydroxide is formulated in water. In some embodiments, the concentration of the aluminum hydroxide original preparation is 10 mg / mL.

[0184] In some embodiments, the ratio of fusion polypeptide to metal hydroxide is 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, and 20:1.

[0185] In some embodiments, the fusion polypeptide is contacted with the metal hydroxide for at least 10 minutes, such as at least 15 minutes, for example, at least 20 minutes, for example, at least 25 minutes, for example, at least 30 minutes, for example, at least 40 minutes.

[0186] In some embodiments, the fusion polypeptide is contacted with the metal hydroxide at a temperature in the range of about 15°C to about 30°C, for example, about 20°C to about 25°C.

[0187] In some embodiments, the pharmaceutical composition comprises the same components as the fusion polypeptide composition and the metal hydroxide. In some embodiments, the concentration of each component in the pharmaceutical formulation is similar to the concentration of each component in the fusion polypeptide composition. In some embodiments, the concentration of each component in the pharmaceutical formulation is lower than the concentration of each component in the fusion polypeptide composition.

[0188] In some embodiments, the pharmaceutical formulation comprises 0.25 mg / mL of the fusion polypeptide, 15 mM Tris buffer, 38 mM NaCl, 7.5 mM L-methionine, 0.015% polysorbate 20, and 113 mM sucrose, 2.5 mg / mL of aluminum hydroxide, and the pH of the composition is in the range of 6-8.

[0189] Characterization Among other things, in some embodiments, the present disclosure provides techniques for characterizing fusion polypeptides (e.g., phosphorylated or non-phosphorylated preparations thereof) and / or complexes comprising such fusion polypeptides and metal hydroxides. Characterization can occur during and / or after the manufacturing process. In some embodiments, a particular preparation process can be modified or terminated in light of the characterization (e.g., if a particular preparation does not meet one or more specifications). In some embodiments, such characterization can include evaluation of one or more of metal hydroxide retention, degree of phosphorylation, phosphorylation heterogeneity, signaling activity, and / or efficacy.

[0190] Exemplary characterization of phosphate content In some embodiments, the degree of phosphorylation (e.g., of a fusion polypeptide of the present disclosure) is characterized. Various methods are available for measuring the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide). For example, in some embodiments, the degree of phosphorylation can be determined by a colorimetric method. In some embodiments, the colorimetric method is or includes a malachite green assay. Without wishing to be bound by any theory, the malachite green assay is based on the 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).

[0191] In some embodiments, the degree of phosphorylation (eg, the average number of phosphate molecules per polypeptide) is about 4-12, about 5-11, about 6-10, about 7-9, or about 7.5-8.5. In some embodiments, the 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. In some embodiments, the 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.

[0192] In some embodiments, the phosphorylation heterogeneity of fusion polypeptides of the present disclosure and / or preparations thereof is characterized. In some embodiments, phosphorylation heterogeneity is a measure of the degree of phosphorylation within a particular preparation of the fusion polypeptide. In some embodiments, phosphorylation heterogeneity is a measure of the degree of phosphorylation across multiple preparations of the fusion polypeptide. In some embodiments, phosphorylation heterogeneity is a measure of the position of a particular phosphate group on a polypeptide within a particular preparation of the fusion polypeptide. In some embodiments, phosphorylation heterogeneity is a measure of the position of a particular phosphate group on a polypeptide across multiple preparations of the fusion polypeptide.

[0193] Various techniques are available for measuring phosphorylation heterogeneity. For example, in some embodiments, the degree of phosphorylation can be determined by chromatographic methods. In some embodiments, the chromatographic method includes ion exchange chromatography. In some embodiments, for example, the chromatographic method includes analytical anion exchange chromatography. Anion exchange chromatography is a form of ion exchange in which negatively charged biomolecules (e.g., phosphorylated forms of the fusion polypeptides disclosed herein) bind to a positively charged resin. In some embodiments, anion exchange chromatography can be used to separate polypeptides with different numbers of phosphorylated amino acid residues (e.g., differentially phosphorylated polypeptides). Without wishing to be bound by any theory, phosphorylation of a polypeptide imparts variation to the charge of the polypeptide, allowing for the separation of highly phosphorylated polypeptides using ion exchange chromatography (e.g., anion exchange chromatography). The use of a gradient elution buffer (e.g., a buffer with increasing salt concentration) to elute from an ion exchange (e.g., anion exchange) column allows for the separation of differentially phosphorylated polypeptides. In some embodiments, the buffer is a Tris buffer. In some embodiments, a linear gradient of Tris buffer is used. In some embodiments, the linear gradient of Tris buffer comprises a linear gradient from 20 mM Tris (pH 7.1) to 20 mM Tris, 525 mM NaCl (pH 7.1) over a predetermined time period, hi some embodiments, the linear gradient is performed over 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 more.

[0194] In some embodiments, the differentially phosphorylated polypeptide is dephosphorylated. In some embodiments, dephosphorylation involves the use of a phosphatase (e.g., lambda phosphatase). In some embodiments, the fusion polypeptide is incubated with the phosphatase for a time and temperature that allows for phosphatase activity and dephosphorylation of the fusion polypeptide. In some embodiments, dephosphorylation occurs at an incubation temperature of about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or higher. In some embodiments, dephosphorylation occurs over an incubation time of 25, 30, 35, 40, 45, 50, 55, 60, 65 minutes, or higher. In some embodiments, dephosphorylation occurs over 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.

[0195] In some embodiments, the differentially phosphorylated polypeptides are dephosphorylated prior to separation. In some embodiments, the differentially phosphorylated polypeptides of the disclosure are evaluated against an appropriate reference standard (e.g., the dephosphorylated and / or unphosphorylated form of the fusion polypeptide).

[0196] In some embodiments, the amount of each differentially phosphorylated polypeptide is measured after separation of the differentially phosphorylated polypeptides (e.g., by ion exchange chromatography). In some embodiments, the amount of each differentially phosphorylated polypeptide is measured according to various methods available in the art. In some embodiments, for example, but not limited to, the differentially phosphorylated polypeptides are measured using a malachite green assay, analytical ion exchange, a spectrophotometer, a colorimetric assay, and / or Western blot.

[0197] Exemplary characterization of metal hydroxide retention In some embodiments, a fusion polypeptide of the present disclosure complexes with a metal hydroxide (e.g., aluminum hydroxide) upon exposure to the metal hydroxide. In some embodiments, the retention of a fusion polypeptide of the present disclosure on the metal hydroxide (e.g., metal hydroxide retention) is characterized. Various methods are available for measuring metal hydroxide retention. In some embodiments, for example, but not limited to, metal hydroxide retention can be measured by ellipsometry, surface plasmon resonance, optical waveguide light-mode spectroscopy, attenuated total internal reflection-infrared spectroscopy, circular dichroism spectroscopy (CD), total internal reflection-infrared spectroscopy (TIRF), and other high-resolution microscopy.

[0198] In some embodiments, metal hydroxide retention is characterized using an in vitro assay. For example, a known concentration of fusion polypeptide is mixed with an excess amount of metal hydroxide. The concentration of free, uncomplexed fusion polypeptide is quantified and compared to a standard curve to determine the metal hydroxide retention. The concentration of free, uncomplexed fusion polypeptide can be assessed according to various methods known to those skilled in the art. For example, but not limited to, in some embodiments, free, uncomplexed fusion polypeptide is quantified by enzyme-linked immunosorbent assay (ELISA), Western blot, bicinchoninic acid assay, or Bradford assay.

[0199] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the fusion polypeptide is complexed (e.g., retained) with the metal hydroxide when mixed with the metal hydroxide.

[0200] Exemplary characterization of signaling activity In some embodiments, the fusion polypeptides (and / or complexes thereof) described herein are characterized for activity (e.g., signaling activity). In some embodiments, activity is characterized by assessing signaling activity (e.g., signaling ability) compared to a suitable reference standard. A suitable reference standard can be, for example, a wild-type polypeptide and / or a fusion polypeptide lacking a metal hydroxide-binding polypeptide.

[0201] A variety of methods are available for assessing signaling capacity. In some embodiments, for example, signaling capacity is assessed using in vitro- or in vivo-based activity assays.

[0202] In some embodiments, signaling activity is assessed in an in vitro activity assay. In some embodiments, the in vitro activity assay comprises measuring activation or inhibition of signaling downstream of the fusion polypeptide. In some embodiments, measuring activation or inhibition of downstream activity comprises the use of a reporter (e.g., a reporter assay). In some embodiments, the reporter assay measures activity using a detectable molecule (e.g., a reporter) that correlates with fusion polypeptide activity.

[0203] In some embodiments, reporters include fluorescent, bioluminescent, and / or other detectable probes known to those of skill in the art. In some embodiments, reporters include the use of genetic reporters. For example, genetic reporters can be activated upon signal transduction induced by a polypeptide. For example, upon activation of genetic reporter transcription, a detectable product or enzyme can be utilized that can be activated upon addition of a substrate to generate a detectable product and / or by-product. In some embodiments, enzymes useful in accordance with reporter assays are, for example, luciferase or alkaline phosphatase (e.g., secreted alkaline phosphatase, SEAP). In some such embodiments, a HEK-Blue-IL12 reporter assay is used.

[0204] In some embodiments, signaling activity is assessed in an in vivo activity assay. In some embodiments, the fusion polypeptide is administered to a subject (e.g., a mouse, a non-human primate, a human, etc.) and activity is assessed. In some embodiments, activity is assessed by measuring activation or inhibition of signaling downstream of the fusion polypeptide, for example, compared to an appropriate reference standard (e.g., the activity of the wild-type polypeptide). Various methods are available for measuring activation or inhibition of signaling downstream of the fusion polypeptide. For example, but not limited to, changes in differential gene expression, protein expression, and / or post-translational modifications induced by the fusion polypeptide can be measured.

[0205] Exemplary Efficacy Characterization In some embodiments, efficacy can be characterized according to a variety of available methods. In some embodiments, for example, a fusion polypeptide (or complex thereof) described herein is administered (e.g., by intratumoral or peritumoral injection) to a subject (e.g., a mouse, a non-human primate, a human, etc.) and efficacy is determined relative to an appropriate reference standard. A suitable reference standard can be, for example, a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide-binding polypeptide or having the metal hydroxide-binding polypeptide in an unbound (e.g., unphosphorylated) state.

[0206] In some embodiments, efficacy is determined preclinically in an animal model (e.g., in mice, rats, non-human primates, etc.). In some embodiments, the fusion polypeptide is administered to the animal model (e.g., by intratumoral or peritumoral injection). For example, in some embodiments, the animal model is a tumor-bearing animal model (e.g., an animal model of cancer). In some embodiments, the cancer animal model is generated by inoculating the above-described animal model with tumor cells. In some embodiments, the tumor cells are inoculated into the flank region of the animal model. In some embodiments, the animal model is inoculated with tumor cells in a clinically relevant region (e.g., the mammary fat pad).

[0207] In some embodiments, a fusion polypeptide of the present disclosure is administered to an animal model of cancer. In some embodiments, the 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). In some embodiments, various available predetermined measures of efficacy known in the art, such as tumor volume and / or survival rate, are assessed over time against a suitable reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide-binding polypeptide).

[0208] In some embodiments, the efficacy of the fusion polypeptide is determined clinically. In some embodiments, the fusion polypeptide is administered to a tumor-bearing subject (e.g., intratumorally, by peritumoral injection, or into a peritumoral lymph node). In some embodiments, various available predetermined measures of efficacy known in the art, such as tumor volume and / or survival rate, are assessed over time in tumor-bearing subjects administered a reference standard (e.g., a treatment known in the art with known efficacy and / or a placebo).

[0209] use Treatment method In one aspect, the present disclosure relates to methods of treating a subject having a medical condition. In some embodiments, the present disclosure relates to methods of treating a subject having cancer (e.g., a subject having a tumor). Generally, methods of treatment aim to reduce tumor volume, reduce and / or prevent metastasis, extend survival, and / or cure the condition. Suitable subjects or individuals to whom a fusion polypeptide or fusion polypeptide-metal hydroxide complex of the present disclosure may be administered include, for example, a human or other mammal (e.g., a mouse, rat, rabbit, dog, horse, cat, pig, or non-human primate) having a tumor (e.g., cancer).

[0210] In some embodiments, a method of treating a subject having a tumor comprises treating the subject with a complex comprising a metal hydroxide and a fusion polypeptide comprising an immunomodulatory polypeptide comprising an immune agonist moiety and a metal hydroxide-binding polypeptide. In some embodiments, a method of treating a subject having a tumor comprises administering a fusion polypeptide comprising an immunomodulatory polypeptide comprising an immune agonist moiety and a metal hydroxide-binding polypeptide, wherein the fusion polypeptide is formulated with the metal hydroxide.

[0211] In some embodiments, the complexes described herein are administered as monotherapy. In some embodiments, the complexes described herein are administered in combination with a second therapeutic agent. In some embodiments, the complexes described herein are administered to a subject who has undergone or is undergoing therapy with at least one additional therapeutic agent.

[0212] The fusion polypeptides and / or complexes thereof and / or compositions and / or formulations of the present disclosure are useful, inter alia, for treating subjects with tumors. Non-limiting examples of tumor-related diseases include cancers (e.g., carcinomas, sarcomas, metastatic diseases, or hematopoietic neoplastic diseases). Tumors, including metastatic tumors, can arise from multiple primary tumor types. For example, but not limited to, in some embodiments, tumors or metastatic tumors can arise from primary tumors of the kidney (e.g., renal cell carcinoma), head and neck (e.g., head and neck squamous cell carcinoma), prostate, breast (e.g., triple negative), colon, skin (e.g., melanoma, Merkel cell carcinoma, cutaneous T-cell lymphoma, cutaneous squamous cell carcinoma, basal cell carcinoma), lung (e.g., non-small cell lung carcinoma), and pancreas. Thus, the fusion polypeptides and formulations thereof disclosed herein, including fusion polypeptide metal-hydroxide complexes and preparations thereof, can be administered to subjects with cancer.

[0213] Those skilled in the art will understand that the amount of fusion polypeptide-metal hydroxide complex, fusion polypeptide, or formulation thereof sufficient to inhibit tumor growth and size, i.e., a therapeutically effective amount, will vary not only with the particular compound or formulation selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be determined at the discretion of the patient's physician or pharmacist and / or based on clinical guidelines. The duration over which the compounds used in the methods of the present invention are administered will vary from individual to individual and / or based on clinical guidelines.

[0214] In some embodiments, a method of treating a subject having a tumor (e.g., cancer) includes treating the subject with a complex comprising a fusion polypeptide comprising an immunomodulatory polypeptide comprising an immune agonist moiety and a metal hydroxide-binding polypeptide, and a metal hydroxide. In some embodiments, the fusion polypeptide and the metal hydroxide are formulated together. "Formulated together" includes, for example, a preformed complex of the fusion polypeptide and the metal hydroxide. In some embodiments, the fusion polypeptide and the metal hydroxide are mixed immediately prior to administration.

[0215] In some embodiments, a method of treating a subject having a tumor (e.g., cancer) comprises treating the subject with a complex, wherein the complex is administered by intratumoral injection. In some embodiments, a method of treating a subject having a tumor (e.g., cancer) comprises treating the subject with a complex, wherein the complex is administered by peritumoral injection. In some embodiments, a method of treating a subject having a tumor (e.g., cancer) comprises treating the subject with a complex, wherein the complex is administered to a peritumoral lymph node or lymph node.

[0216] The methods of the invention often involve the administration of a therapeutically effective amount of a particular agent. A therapeutically effective amount refers to an amount sufficient to produce a desired biological or medical response or therapeutic effect in a tissue, system, or subject (principally for subjects with comparable characteristics, such as species, body type, build, severity of disease or disorder, severity or type of symptoms, history of responsiveness, and / or overall health). For example, the desired response can include one or more of delaying or preventing the onset of a medical condition, disease, or disorder; slowing or halting the progression, worsening, or deterioration of symptoms of the condition; causing an improvement in symptoms of the condition; and curing the condition.

[0217] When a combination of therapeutic agents is administered, the amount of any individual agent required for the combination may differ from the amount required for that same agent alone to achieve a therapeutic effect. In some cases, a synergistic effect between the combined therapeutic agents may reduce the amount required, while in other cases, an inhibitory interaction may increase the amount required. Thus, in general, a therapeutically effective amount of a drug combination may use absolute amounts of each agent that differ from what constitutes a therapeutically effective amount of each agent individually.

[0218] Combination therapy In some embodiments, the fusion polypeptide metal-hydroxide complexes or formulations thereof disclosed herein are administered in combination with other treatments. For example, in some embodiments, the IL-12 complexes are combined with another immunotherapy. Exemplary immunotherapies include, but are not limited to, chimeric antigen receptor (CAR) T-cell therapy, antibodies targeting tumor-associated antigens, immune checkpoint inhibitors, and cancer vaccines. In some embodiments, an immune response is induced or stimulated by agonizing such immune checkpoint inhibitors. The following table provides a list of immune checkpoint inhibitors suitable for use in combination with the pharmaceutical compositions of the present disclosure.

[0219] The second therapeutic agent can be selected from a variety of available anti-tumor agents known in the art. In some embodiments, the second therapeutic agent is administered prior to administration of the fusion polypeptide metal-hydroxide complex. In some embodiments, the second therapeutic agent is administered simultaneously with the fusion polypeptide metal-hydroxide complex. In some embodiments, the second therapeutic agent is administered after administration of the fusion polypeptide metal-hydroxide complex.

[0220] For example, in some embodiments, the second therapeutic agent is radiation (e.g., ionizing radiation). In some embodiments, the amount of ionizing radiation administered is about 1 Gy to about 1,000 Gy, about 5 Gy to about 900 Gy, about 10 Gy to about 800 Gy, about 10 Gy to about 700 Gy, about 10 Gy to about 600 Gy, about 10 Gy to about 500 Gy, about 10 Gy to about 400 Gy, about 10 Gy to about 300 Gy, about 10 Gy to about 200 Gy, about 10 Gy to about 100 Gy, about 5 Gy and about 15 Gy, about 7.5 Gy to about 12 Gy, or about 10 Gy to about 12 Gy. In some embodiments, the amount of ionizing radiation administered is about 12 Gy. In some embodiments, the amount of ionizing radiation is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 Gy. In some embodiments, the amount of ionizing radiation is less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 Gy.

[0221] For example, in some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent may be a targeted therapy (e.g., a BRAF inhibitor, a MEK inhibitor, etc.). In some embodiments, the chemotherapeutic agent may be any approved chemotherapeutic agent. For example, but not limited to, the chemotherapeutic agent may be one or more of adriamycin, anastrozole, cyclophosphamide, docetaxel, doxifluridine, doxorubicin, erlotinib, fluorouracil, gemcitabine, imatinib, Iressa, letrozole, methotrexate, paclitaxel, tarceva, and trastuzumab. The chemotherapeutic agent may be administered according to any approved and / or art-known regimen.

[0222] For example, the second therapeutic agent is an anti-tumor antibody. In some embodiments, the anti-tumor antibody is an immunomodulator. In some embodiments, the immunomodulator is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an antibody, or a functional fragment thereof. In some embodiments, the antibody targets one or more of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, and / or LAG3. In some embodiments, the antibody targets PD-1 (e.g., pembrolizumab). The anti-tumor antibody may be administered according to any approved and / or art-known regimen.

[0223] For example, in some embodiments, the second therapeutic agent is surgical tumor resection. In some embodiments, the fusion polypeptide metal-hydroxide complex is administered prior to surgical tumor resection. In some embodiments, the fusion polypeptide metal-hydroxide complex is administered to tissue after tumor resection, where the tissue may contain, for example, residual tumor (e.g., tumor cells). In some embodiments, the fusion polypeptide metal-hydroxide complex is administered during resection to tissue that cannot be removed by surgical tumor resection or to tissue in the vicinity of the resection.

[0224] For example, in some embodiments, the second therapeutic agent is or includes a cell therapy. In some embodiments, the cell therapy is or includes natural killer (NK) cells. In some embodiments, the cell therapy is or includes tumor-infiltrating lymphocytes (TILs). In some embodiments, the cell therapy is or includes ex vivo expanded cells. In some embodiments, the cell therapy is or includes chimeric antigen receptor (CAR) effector cell therapy (e.g., CAR T cells). CARs are genetically engineered, artificial transmembrane receptors that confer selected specificity to immune effector cells (e.g., T cells, natural killer cells, or other immune cells) for a selected ligand, resulting in activation of the effector cell upon recognition and binding of the ligand. Often, such ligand specificity is achieved by genetically engineering the CAR with the antigen specificity of a monoclonal antibody, thereby targeting the CAR T cells to the antigen recognized by the antibody.

[0225] In some embodiments, chimeric antigen receptor-expressing effector cells (e.g., CAR-T cells) are derived (e.g., isolated) from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with any specificity for a ligand. The cells perform at least one effector function (e.g., cytokine induction) useful for treating the disease or condition in the same patient, stimulated or induced by specific binding of the ligand to the CAR. The effector cell can be a T cell (e.g., a cytotoxic T cell or a helper T cell). Upon reading this disclosure, one skilled in the art will understand that chimeric antigen receptor effector cells can include effector cells other than T cells, as in some embodiments, cells other than T cells (e.g., natural killer cells, stem cells, etc.) can be engineered to express a CAR. In some embodiments, the CAR effector cell is a T cell (e.g., a cytotoxic T cell). In some embodiments, such CAR-T cells exert their effector function (e.g., a cytotoxic T cell response) against a target or target cell (e.g., a cancer cell) upon contact with or proximity to the target or target cell (e.g., a cancer cell) (see, e.g., Chen (2017) Trends Mol Med 23(5):430-450). In some embodiments, the cell therapy (e.g., CAR effector cell therapy) uses tumor-infiltrating lymphocytes (TILs). The TILs target cancer cells. In some embodiments, the TILs are isolated from a subject with cancer and expanded ex vivo. In some such embodiments, the TILs are isolated after surgical resection of the tumor and expanded ex vivo. In some embodiments, prior to administration of TILs, the subject is treated with lymphoid cell-depleting conditioning (Rohaan, Maartje W et al. "Adoptive cellular therapies: the current landscape." Virclows Archiv:an International journal of pathology vol. 474,4 (2019): 449-461).

[0226] In some embodiments, cell therapy (e.g., CAR effector cell therapy) uses natural killer (NK) cells. NK cells are an essential part of tumor immune surveillance, as evidenced in mouse models and clinical trials by the increased cancer susceptibility and metastasis associated with reduced NK activity. In some embodiments, for example, by using an array of germline-encoded surface receptors, NK cells can recognize and rapidly act against malignant cells without prior sensitization (Iu, S., Galat, V., Galat4, Y. et al. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol 14, 7 (2021)).

[0227] In some embodiments, a fusion polypeptide metal-hydroxide complex or preparation thereof disclosed herein is administered to a subject who has undergone or is undergoing therapy with at least one additional therapeutic agent. The additional therapeutic agent can be selected from a variety of anti-tumor agents known in the art. In some embodiments, the additional therapeutic agent is administered prior to administration of the fusion polypeptide metal-hydroxide complex. In some embodiments, the additional therapeutic agent is administered simultaneously with the fusion polypeptide metal-hydroxide complex. In some embodiments, the additional therapeutic agent is administered after administration of the fusion polypeptide metal-hydroxide complex.

[0228] For example, in some embodiments, the additional therapeutic agent is radiation (e.g., ionizing radiation). In some embodiments, the amount of ionizing radiation administered is about 1 Gy to about 1,000 Gy, about 5 Gy to about 900 Gy, about 10 Gy to about 800 Gy, about 10 Gy to about 700 Gy, about 10 Gy to about 600 Gy, about 10 Gy to about 500 Gy, about 10 Gy to about 400 Gy, about 10 Gy to about 300 Gy, about 10 Gy to about 200 Gy, about 10 Gy to about 100 Gy, about 5 Gy and about 15 Gy, about 7.5 Gy to about 12 Gy, or about 10 Gy to about 12 Gy. In some embodiments, the amount of ionizing radiation administered is about 12 Gy. In some embodiments, the amount of ionizing radiation is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 Gy. In some embodiments, the amount of ionizing radiation is less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 Gy.

[0229] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is or includes a targeted therapy (e.g., a BRAF inhibitor, a MEK inhibitor, etc.). In some embodiments, the chemotherapeutic agent can be any approved chemotherapeutic agent. For example, but not limited to, the chemotherapeutic agent can be one or more of adriamycin, anastrozole, cyclophosphamide, docetaxel, doxifluridine, doxorubicin, erlotinib, fluorouracil, gemcitabine, imatinib, Iressa, letrozole, methotrexate, paclitaxel, Tarceva, and trastuzumab. The chemotherapeutic agent can be administered according to any approved and / or art-known regimen. In some embodiments, the additional therapeutic agent is an anti-tumor antibody. In some embodiments, the anti-tumor antibody is an immunomodulator. In some embodiments, the immunomodulator is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an antibody, or a functional fragment thereof. In some embodiments, the antibody targets one or more of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, and / or LAG3. In some embodiments, the antibody targets PD-1 (e.g., pembrolizumab). The anti-tumor antibody may be administered according to any approved and / or art-known regimen. For example, in some embodiments, the additional therapeutic agent is or includes a cell therapy. In some embodiments, the cell therapy is or includes a chimeric antigen receptor (CAR) effector cell therapy (e.g., CAR T cells). CARs are engineered, artificial transmembrane receptors that confer selected specificity to immune effector cells (e.g., T cells, natural killer cells, or other immune cells) for a selected ligand, resulting in activation of the effector cell upon recognition and binding of the ligand. Often, such ligand specificity is achieved by genetically engineering the CAR with the antigen specificity of a monoclonal antibody, thereby targeting the CAR T cells to the antigen recognized by the antibody.

[0230] In some embodiments, chimeric antigen receptor-expressing effector cells (e.g., CAR-T cells) are derived (e.g., isolated) from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with any specificity for a ligand. The cells perform at least one effector function (e.g., cytokine induction) useful for treating the disease or condition in the same patient, stimulated or induced by specific binding of the ligand to the CAR. The effector cell can be a T cell (e.g., a cytotoxic T cell or a helper T cell). Upon reading this disclosure, one skilled in the art will understand that chimeric antigen receptor effector cells can include effector cells other than T cells, as in some embodiments, cells other than T cells (e.g., natural killer cells, stem cells, etc.) can be engineered to express a CAR. In some embodiments, the CAR effector cell is a T cell (e.g., a cytotoxic T cell). In some embodiments, such CAR-T cells exert their effector function (e.g., a cytotoxic T cell response) against a target or target cell (e.g., a cancer cell) upon contact with or proximity to the target or target cell (e.g., a cancer cell) (see, e.g., Chen (2017) Trends Mol Med 23(5):430-450). In some embodiments, the cell therapy (e.g., CAR effector cell therapy) uses tumor-infiltrating lymphocytes (TILs). The TILs target cancer cells. In some embodiments, the TILs are isolated from a subject with cancer and expanded ex vivo. In some such embodiments, the TILs are isolated after surgical resection of the tumor and expanded ex vivo. In some embodiments, prior to administration of TILs, the subject is treated with lymphoid cell-depleting conditioning (Rohaan, Maartje W et al. "Adoptive cellular therapies: the current landscape." Virclows Archiv:an International journal of pathology vol. 474,4 (2019): 449-461).

[0231] In some embodiments, cell therapy (e.g., CAR effector cell therapy) uses natural killer (NK) cells. NK cells are an essential part of tumor immune surveillance, as evidenced in mouse models and clinical trials by the increased cancer susceptibility and metastasis associated with reduced NK activity. In some embodiments, for example, by using an array of germline-encoded surface receptors, NK cells can recognize and rapidly act against malignant cells without prior sensitization (Iu, S., Galat, V., Galat4, Y. et al. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol 14, 7 (2021)).

[0232] In some embodiments, cell therapy (e.g., CAR effector cell therapy) uses bone marrow cells. In some embodiments, the bone marrow cells are or include macrophages. Macrophages have been shown to take up alum.

[0233] Table 1 shows exemplary amino acid sequences of the polypeptides described herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 2-1] [Table 2-2] [Table 2-3]

[0234] Example Throughout the examples, NKY-001 is used interchangeably with IL-12 fusion polypeptide.

[0235] Example 1. Preparation of an exemplary fusion polypeptide stock This example demonstrates the preparation of exemplary fusion polypeptide stocks of phosphorylated forms of the fusion polypeptides described herein. Exemplary amino acid sequences of interleukin-12 fusion polypeptides are shown in Table 1, and exemplary nucleotide sequences are shown in Table 2.

[0236] Bulk purified fusogenic agent was provided at a concentration of approximately 11 g / L (see Table 3) in 10 mM Tris, approximately 500 mM NaCl, pH 7.4, and stored at -65°C or below until formulation. [Table 3]

[0237] The following chemicals and excipients were used (Table 4). [Table 4]

[0238] Various IL-12 fusion polypeptide compositions were formulated using different components (e.g., buffers, surfactants, excipients) and different pH conditions. Evaluation of such preparations is described herein below (see Examples 2-4). The compositions were stored for a period of time (e.g., 6 weeks or more, and in some cases 12 weeks or more). Examples 2-4 are described below.

[0239] Each of the evaluated compositions tested in Examples 2-4 contained an IL-12 fusion polypeptide at a concentration ranging from about 0.5 mg / mL to about 3.5 mg / mL (e.g., about 2 mg / mL). Without wishing to be bound by any particular theory, an IL-12 fusion polypeptide concentration of about 2 mg / mL is an appropriate concentration when a composition containing a phosphorylated form of the fusion polypeptide is subsequently mixed with a metal hydroxide (e.g., aluminum hydroxide), thereby forming a fusion polypeptide metal-hydroxide complex.

[0240] Example 2. Exemplary Fusion Polypeptide Stock Compositions This example demonstrates that the stability of IL-12 fusion polypeptides is affected by the pH level and / or type of buffer used in IL-12 fusion polypeptide compositions. For example, this example demonstrates that IL-12 fusion polypeptides are stable in Tris-buffered compositions at a pH of about 7-8 (e.g., about 7.3-7.4). This example also provides exemplary compositions for use in accordance with the present invention. Evaluated Compositions

[0241] Eight different buffer / pH conditions were evaluated with eight different compositions (F1-F8) (see Table 5). Tris buffer and His / HisHCl buffer were evaluated in the stability evaluation. When present, NaCl concentrations were 50 mM or 100 mM. [Table 5]

[0242] Each composition (F1-F8) listed in Table 5 was prepared by buffer exchange to achieve the target buffer concentration and pH. The protein concentration, osmolality, and pH of each composition were determined. All composition solutions were filtered using a 0.22 μm polyvinylidene fluoride (PVDF) membrane filter.

[0243] Primary packaging materials were prepared according to standard procedures, and each composition was manually transferred, using aseptic technique, into 2R / 13mm glass Type I vials with a target fill volume of 1.0mL, stoppered with a 13mm bromobutyl rubber stopper (injection stopper), and sealed with a 13mm aluminum flip-off seal. Samples of all compositions were labeled and stored at 5±3°C until dispensed for stability evaluation.

[0244] Test samples of each composition were dispensed in an upright position into stability chambers according to Table 6. Vials were obtained from the different compositions at the following time points: 0° C. (initial / T0 / frozen starting material), after 1 week (T1W), and after 2 weeks (T2W) at selected temperatures of 5° C., 25° C., and 40° C. See Table 4 for specific vial distribution. [Table 6]

[0245] At the initial and each subsequent time point, test samples of each composition were removed and evaluated as described below. Visible particles (black and white) at all stability points. Clarity and opacity (turbidity) of the solution at all stability points. Determination of pH at all stability time points. · T0 / osmolality due to freezing point depression at initial time. Protein content by SoloVPE at all stability time points. Purity by size exclusion-HPLC at all stability time points. Purity by reversed-phase HPLC (CR-HPLC) (reducing and non-reducing conditions) at all stability time points. · For F5 and F6, CE-SDS (chip-based) was performed at 25°C for 2 weeks.

[0246] Composition after preparation The pH, protein concentration, and osmolality of each composition after formulation and filtration were measured. The results are shown in Table 7. Protein concentration measured by UV spectrophotometer (A280) at the initial time point of the stability study is also included. The pH and protein concentration of each composition (F1-F8) were close to the desired values. The osmolality results show a wide range, as expected for each composition. All compositions were colorless and free of visible particles after formulation and filtration. [Table 7]

[0247] Stability testing No significant changes were observed over the two-week stability evaluation. No visible particles were observed, and the pH and protein concentration remained stable. Turbidity showed values ​​between 0 and 1.

[0248] When analyzed by RP-HPLC, the composition containing a low pH histidine buffer (F5, pH 5.5) showed a decrease in the main peak followed by an increase in HMWS of approximately 5% at 40°C (see Figures 3A-B). In contrast, the compositions containing higher pH Tris buffers (F1-F4) were stable over time and at different temperatures. Thus, the present disclosure demonstrates that Tris buffer can be used to effectively formulate the IL-12 fusion polypeptide compositions described herein, while other buffer systems (e.g., histidine buffer) may not be useful for this purpose.

[0249] More specifically, the present disclosure demonstrates that while no significant differences were observed between compositions by HPLC under non-reducing conditions, some loss of the main peak was observed in the low-pH histidine buffer compositions under reducing conditions. Specifically, after 2 weeks at 40°C, F5 (pH 5.5) showed a greater than 20% decrease in the main peak, and F6 (pH 6.0) showed an approximately 17% decrease in the main peak, leading to an increase in peak A(s) in both cases. Compositions F7 and F8 in histidine buffer at pH 6.5 showed a more moderate decrease (approximately 8%) in the main peak at 40°C. Compositions in Tris buffer (pH 7.4 and pH 8.0) showed little change over time (and / or under temperature stress conditions). Thus, the present disclosure demonstrates the unexpected stability of such Tris buffer compositions.

[0250] After 2 weeks at 25°C, only two samples, F5 and F6, were subjected to CE-SDS (chip-based). The non-reduced samples were indistinguishable (i.e., 100% intact) when compared to TO (initial / frozen starting material), and the reduction profile was very similar to TO.

[0251] Stability data for up to two weeks showed that the interleukin-12 metallo-binding polypeptide fusion agent was more stable in Tris buffer at pH 7.4 compared to histidine buffer (evaluated at pH 5.5-6.5).

[0252] conclusion A particularly stable composition was prepared in 20 mM Tris buffer, pH about 7-8 (eg, about 7.4).

[0253] Example 3. Effect of surfactants on stability This example demonstrates that IL-12 fusion polypeptide compositions containing polysorbate-20 as a specific polysorbate protect the IL-12 fusion polypeptide from instability caused by shaking stress (without polysorbate 20, the IL-12 fusion polypeptide is sensitive to agitation). This example specifically demonstrates that polysorbate-20 is significantly more effective than another polysorbate surfactant (i.e., polysorbate-80) in reducing the formation of visible particles upon shaking. This example provides exemplary IL-12 fusion polypeptides for use in accordance with the present invention.

[0254] Evaluated Compositions Five different compositions were evaluated: polysorbate 20 and 80 at two concentration levels (0.02% (w / v) and 0.04% (w / v)), as well as a fifth composition containing no polysorbate as a control (see compositions in Table 8).

[0255] Each composition evaluated contained 2 mg / ml of interleukin-12 metallopolypeptide fusion agent in 20 mM Tris buffer (pH 7.3), 50 mM NaCl for further stabilization, and 10 mM L-methionine to mitigate potential oxidation risk. [Table 8]

[0256] Each composition listed in Table 8 was prepared by buffer exchange to achieve the target buffer concentration and pH. The protein concentration, osmolality, and pH of each composition were determined. All composition solutions were filtered using a 0.22 μm polyvinylidene fluoride (PVDF) membrane filter.

[0257] Primary packaging materials were prepared according to standard procedures, and each composition was manually transferred, using aseptic technique, into 2R / 13mm glass Type I vials with a target fill volume of 1.0mL, stoppered with a 13mm bromobutyl rubber stopper (injection stopper), and sealed with a 13mm aluminum flip-off seal. Samples of all compositions were labeled and stored at 5±3°C until dispensed for stability evaluation.

[0258] Two test samples of each composition were subjected to shaking stress in a horizontal position in a reciprocating (horizontal) shaker at a target speed of 200 rpm under low (5°C) and room (25°C) conditions for 2 and 5 days, respectively. Additionally, two test samples of each composition were subjected to three and five freeze / thaw cycles in a vertical position from below -65°C to room temperature, respectively. See Table 9 for specific vial distribution. [Table 9]

[0259] At the initial and each subsequent time point, test samples of each composition were removed and evaluated as described below. Visible particles (black and white) at all stability points. Clarity and opacity (turbidity) of the solution at all stability points. Color measurements using a colorimeter at all stability time points. No visible particles by the low volume light obscuration (LO) method at all stable time points. Determination of pH at all stability time points. Freezing point depression osmolality at all stable times. Protein content by SoloVPE at all stability time points. Purity by size exclusion-HPLC at all stability time points. Determination of polysorbate 20 / 80 content by fluorescent micellar assay (HPLC) at all stability time points. Purity by reversed-phase HPLC (CR-HPLC) (reducing and non-reducing conditions) at all stability time points. CE-SDS (chip-based)

[0260] Composition after preparation The pH, protein concentration, and osmolality of each composition after formulation and filtration were measured, and the results are shown in Table 10. For completeness, the protein concentration measured by UV spectrophotometer (A280) at the early time points of the stability study is also included. The pH and protein concentration of each composition (F1-F5) were close to the desired values. The osmolality results show a wide range, as expected for each composition. All compositions were colorless and free of visible particles after formulation and filtration. [Table 10]

[0261] Freeze-thaw and shaking test All compositions were subjected to 3 and 5 freeze-thaw cycles (-65°C to room temperature) and shaking stress at ambient and low temperatures for 2 and 5 days, respectively.

[0262] No visible particles were observed in any of the polysorbate-containing compositions (F1–F4). However, F5 showed some reduction in the main peak (IL-12 fusion polypeptide) after shaking stress, leading to an increase in HMWS (approximately 8% after 2 days of shaking and 15% after 5 days of shaking) (Figures 4A–B). Accordingly, in F5 (without polysorbate), many particles were observed after 5 days of shaking at room temperature (at both temperatures) and after 2 days of shaking.

[0263] Freeze-thaw stress did not appear to affect the interleukin-12 metallopolypeptide fusion agents in any of the compositions (F1-F5), however, F2 (PS80 0.04%) exhibited a slightly higher HMWS response when compared to F1 (PS80 0.02%).

[0264] No changes in pH or protein concentration were observed throughout the study. Turbidity and color remained stable at 1 NTU (specific gravity turbidity unit) and B9, respectively. While some variation in subvisible particles was observed, these values ​​generally remained in the low range (even in the case of the surfactant-free control composition). Only minor variations were observed under non-reducing conditions by RP-HPLC. Under reducing conditions, a slight decrease in peak A was observed in the stressed samples, reflected in an increase in the main peak (approximately 0.5% to 1%). Measurement of polysorbate content by a standard fluorescent micelle assay (FMA) showed no relevant changes after stress conditions.

[0265] conclusion Overall, polysorbate protects the interleukin-12 metallopolypeptide fusion agent from instability that can be induced by shaking stress. High concentrations of polysorbate (0.04%) do not appear to be beneficial to the IL-12 fusion polypeptide compared to low concentrations (0.02%). When polysorbate was present in the composition, no substantial difference was observed between the two stress periods (2 days and 5 days).

[0266] Example 4. Effect of other composition ingredients on stability This example demonstrates that methionine positively impacts the stability of interleukin-12 metallopolypeptide fusion agents, particularly in preventing the generation of high molecular weight species (HMWS). This example demonstrates that the IL-12 fusion polypeptide compositions evaluated can be stored at 2-8°C. This example also provides exemplary IL-12 fusion polypeptides for use in accordance with the present invention.

[0267] Evaluated Compositions The effect of adding methionine and sucrose relative to trehalose was tested in compositions containing polysorbate 20 or 80 (0.02% (w / v)) (see each composition in Table 11). Detergent evaluation was performed using compositions containing 2 mg / ml interleukin-12 metallopolypeptide fusion agent in 20 mM Tris buffer (pH 7.0, 7.3, or 7.6). The buffer type, NaCl (50 mM), and L-methionine (10 mM) were kept constant for all candidate compositions. Six different compositions were tested (F1–F6). [Table 11]

[0268] Each composition listed in Table 11 was prepared by buffer exchange to achieve the target buffer concentration and pH. The protein concentration, osmolality, and pH of each composition were determined. All composition solutions were filtered using a 0.22 μm polyvinylidene fluoride (PVDF) membrane filter.

[0269] Primary packaging materials were prepared according to standard procedures, and each composition was manually transferred, using aseptic technique, into 2R / 13mm glass Type I vials with a target fill volume of 1.0mL, stoppered with a 13mm bromobutyl rubber stopper (injection stopper), and sealed with a 13mm aluminum flip-off seal. Samples of all compositions were labeled and stored at 5±3°C until dispensed for stability evaluation.

[0270] Test samples of each composition were subjected to shaking stress in a reciprocating (horizontal) shaker at a target speed of 200 rpm at room temperature (25°C) for approximately 5 days. Additionally, test samples of each composition were subjected to five freeze / thaw cycles in a vertical position from below -65°C to room temperature. Vials were maintained at selected temperatures of -20°C, 5°C, 25°C, and 40°C and evaluated at the following time points: 0 (initial / T0 / frozen starting material), 3 weeks (T3W), 6 weeks (T6W), and 12 weeks (T12W). See Table 12 for specific vial distribution. [Table 12]

[0271] At the initial and each subsequent time point, test samples of each composition were removed and evaluated as described below. Visible particles at all stability points. Clarity and opacity (turbidity) of the solution at all stability points. Color measurements using a colorimeter at all stability time points. No visible particles by the low volume light obscuration (LO) method at all stable time points. Determination of pH at all stability time points. Freezing point depression osmolality at all stable times. Protein content by SoloVPE at all stability time points. Purity by size exclusion-HPLC at all stability time points. Purity by anion exchange chromatography (AEX) at all stability time points except FT (freeze / thaw) and shaking. Determination of polysorbate 20 / 80 content by fluorescent micellar assay (HPLC) at all stability time points. Purity by reversed-phase HPLC (CR-HPLC) (reducing and non-reducing conditions) at all stability time points.

[0272] Additionally, for each baseline composition (stored at -65°C), a 200 μL aliquot was taken for the alum binding assay, a 100 μL aliquot for the potency assay, and a 100 μL aliquot for the ELISA.

[0273] Composition after preparation The pH, protein concentration, and osmolality of each composition after formulation and filtration were measured, and the results are shown in Table 13. For completeness, the protein concentration measured by UV spectrophotometer (A280) at the early time points of the stability study is also included. The pH and protein concentration of each composition (F1-F5) were close to the desired values. The osmolality results show a wide range, as expected for each composition. All compositions were colorless and free of visible particles after formulation and filtration. [Table 13]

[0274] Freeze-thaw and shaking test All compositions subjected to five freeze-thaw cycles (-65°C to room temperature) or shaking stress at ambient temperature showed no relevant changes by any analytical method compared to the initial unstressed samples, indicating that the compositions effectively stabilized the IL-12 polypeptide fusion agent against both freeze-thaw and shaking stress. Only F1 and F5 appeared to have slightly higher particle counts after 5 days of shaking stress. Short-term stability studies (heat stress): storage at -20°C, 5°C, 25°C, and 40°C

[0275] No visible particles were observed for any of the compositions, turbidity was stable (0-1 NTU), and color ranged from colorless to B9. Protein concentration and pH remained stable over time.

[0276] Sub-visible particles were generally few in number. Sub-visible particles larger than 25 μm were not detected in most samples (detected as only one or two particles).

[0277] For all compositions analyzed using the SEC method, no significant differences in the percentage of the main peak were observed over time at temperatures below 25°C. Some LMWS was observed after 6 weeks at 40°C (≤0.1%), and HMWS showed an increase of 0.3% compared to TO for F1 and up to 0.5% for F4 and F5.

[0278] By RP-HPLC, reducing conditions appeared to be more stable than non-reducing conditions. Some signs of degradation were observed at 40 °C, especially in F5 (no L-methionine) (loss of 5% of the main peak after 6 weeks), indicating that methionine stabilizes the fusion polypeptide.

[0279] In measuring the polysorbate content, only slight variations were observed in the samples containing PS20. Samples containing PS80 (F2 and F5) showed some degradation at 40°C.

[0280] At T12W, the candidate composition (F1) was collected and the intended storage conditions were determined (refrigerated and frozen at -20°C).

[0281] Long-term (12-week) stability study of candidate composition (F1) To support decisions regarding storage conditions, one additional time point analysis was performed on the candidate composition (F1) only.

[0282] After 12 weeks, no visible particles were observed at either storage temperature. No differences were observed in turbidity (maintained at 1 NTU), color, pH, and protein concentration. No more than 25 sub-visible particles with a diameter of 10 μm or greater were observed per mL of the composition, and no more than 3 sub-visible particles with a diameter of 10 μm or greater were observed per mL of the composition.

[0283] By SEC, only the 40°C sample showed some decrease in the main peak (1%), reflected in an increase in HMWS (approximately 1.0–1.2% compared to TO and the frozen analytical reference) and the appearance of 0.1% LWMS.

[0284] RP-HPLC under non-reducing conditions showed no relevant changes in composition. Under reducing conditions, a decrease in the main peak (approximately 10-13%) was observed upon storage at 40 °C, reflected by a corresponding increase in peak A (approximately 10-11%).

[0285] No decrease in polysorbate content was observed under any of the storage conditions.

[0286] Overall, no differences were observed between 5°C and -20°C storage conditions for all purity methods. Potential differences in SVP counts are likely related to method variability inherent in particle counting methods.

[0287] In conclusion, the tested interleukin-12 metallopolypeptide fusion agent compositions can be stored at 2-8°C.

[0288] Advanced methods for sample analysis using anion exchange chromatography (AEX) This method involves digestion with a phosphatase kit during sample preparation prior to analysis by HPLC. FT and shaking stress were not analyzed in AEX. Composition samples from short-term stability studies were frozen after collection and analyzed together, including frozen analytical controls, in sequential order around the collection time point of 12 weeks after F1. Some variability was observed when AEX was performed as duplicate injections.

[0289] In practice, the profiles obtained with the DPS were similar to those seen from the development assay, showing two peaks of similar height separated by a valley. Results were reported for one main peak and a second (earlier) peak, labeled as acidic peak 1. This profile was generally maintained over the course of formulation testing, with only minor differences between compositions. Samples stored at -20°C, 5°C, and 25°C produced similar profiles, potentially showing a slight shift in peak height toward the earlier peak (e.g., 6 weeks at 25°C). In contrast, samples stored at 40°C began to show a loss of peak resolution for all compositions at 3 weeks (two peaks, but the valley was no longer distinct), and by 6 weeks the signal could no longer be integrated as two separate peaks. In some samples, the maximum signal occurred at the retention time between the peak assignments, making it impossible to distinguish between the "main" and "acidic" peaks.

[0290] For F1 analyzed at 12 weeks, some loss of clarity in the valley between the two peaks was observed in the 5°C sample. The 40°C sample showed a change in profile toward a shoulder before the peak and a more distinct peak, with a retention time similar to that assigned to the main peak during the development method.

[0291] conclusion Temperature may affect the composition, as particularly observed in the decrease of the primary peak in RP-HPLC at 40°C. The addition of methionine improves the stability of the interleukin-12 metallopolypeptide fusion agent, particularly in terms of the occurrence of HMWS. All pH values ​​within the tested range were acceptable. Polysorbate 20 was found to be more suitable for IL-12 fusion polypeptides than polysorbate 80 (especially at 40°C). The provided composition was also demonstrated to be stable against freeze-thaw and shaking stress, demonstrating appropriate behavior for handling during manufacturing.

[0292] Example 5. Aluminum hydroxide (Alum) retention assay This example provides an exemplary pharmaceutical composition containing a fusion polypeptide metal-hydroxide complex. This example demonstrates that such a pharmaceutical composition is stable and that complexation with aluminum hydroxide does not affect the stability of the fusion polypeptide.

[0293] Binding and retention of IL-12 fusion polypeptides to aluminum hydroxide was tested in vitro. Human IL-12 fusion polypeptide composition (20 mM Tris, 150 mM sucrose, 50 mM NaCl, 10 mM L-methionine, 0.02% (w / v) polysorbate 20, pH 7.3) was mixed with aluminum hydroxide (Invivogen catalog no. alu-vac-250) to a final concentration of 250 μg / mL IL-12 fusion polypeptide and 2.5 mg / mL aluminum hydroxide, or composition buffer alone as a control in a final volume of 40 μL.

[0294] The IL-12 fusion polypeptide / alum mixture was thoroughly resuspended by pipetting and incubated at room temperature for 30 minutes. The IL-12 fusion polypeptide / alum mixture or the IL-12 fusion polypeptide-only control was then diluted 25-fold with elution buffer containing 1 mM phosphate and 40% human serum to a final volume of 1 mL. The diluted samples were incubated at 37°C for 2–24 hours with gentle rotation. At each time point, 50 μL of sample was removed and centrifuged at 18,000 × g for 10 minutes to pellet the aluminum hydroxide. The clear supernatant was transferred to a new tube and stored at 4°C until ready for analysis. The concentration of free IL-12 fusion polypeptide in each supernatant sample was quantified using a human IL12p70 ELISA using MAB219 (R&D Systems) as the capture reagent and antibody 508802 (Biolegend) as the detection reagent. All dilutions were prepared in TBS + 1% BSA + 0.1% Tween-20. Test agents were used at a top concentration of 0.5 ng / mL and a standard curve of 2-fold dilutions, and supernatant samples were diluted to a theoretical concentration of 0.25 ng / mL when all polypeptides were released.

[0295] Protein stock AK346B, IL-12 fusion polypeptide complexed with alum_B1, or IL-12 fusion polypeptide complexed with alum_B2: 0.333 mg / ml in TBS or F1. F1 is an exemplary fusion polypeptide containing 20 mM Tris, 150 mM sucrose, 50 mM NaCl, 10 mM L-methionine, 0.02% w / v polysorbate 20 at pH 7.3.

[0296] Total alum sample: 10 mg / mL (undiluted)

[0297] A-H, listed below, were eluted with TBS / PBS using 1 mM phosphate and 40% human serum. PBS: 1x PBS (pH 7.4, 11.8 mM PO), 20 mM TBS (pH 7.4) per CSH protocol. Gender-pooled human serum: BioVT, catalog number HUMAN SERM-0001255, lot number HMN749277. Time points: 2 and 24 hours of incubation at 37°C. B1 and B2 are two different preparations of purified IL-12 fusion polypeptide.

[0298] Experimental conditions, proteins, and concentrations: A: 10 μg / mL AK346B (TBS), 30 μL protein + 10 μL TBS - alum-free control B: 10 μg / mL ANK101_B1(F1), 30 μL protein + 10 μL F1 buffer - alum-free control C: 10 μg / mL ANK101_B2(F1), 30 μL protein + 10 μL TBS-alum-free control D: 10 μg / ml LAK346B (TBS), 30 μL protein + 10 μL alum (alum:protein ratio = 10:1) E: 10 μg / mL ANK101_B1(F1), 30 μL protein + 10 μL alum (alum:protein ratio = 10:1) F: 10 μg / mL ANK101_B2 (F1), 30 μL protein + 10 μL alum (alum:protein ratio = 10:1) G: 10 μg / mL ANK101_B1 (TBS), 30 μL protein + 10 μL alum (alum:protein ratio = 10:1) H: 10 μg / mL ANK101_B2 (TBS), 30 μL protein + 10 μL alum (alum:protein ratio = 10:1)

[0299] The IL-12 fusion polypeptide in formulation buffer complexed with aluminum hydroxide remained stable after incubation in 1 mM phosphate and 40% human serum, with only approximately 4% protein released at 2 hours and approximately 14% at 24 hours (Figure 5, conditions E / F), similar to the release of the IL-12 fusion polypeptide when formulated in TBS prior to aluminum hydroxide (Figure 5, conditions G / H).

[0300] Example 6. IL-12 signaling activity assay This example demonstrates that an IL-12 fusion polypeptide retains its biological activity when formulated in a pharmaceutical composition according to the present disclosure.

[0301] IL12 signaling activity was assessed in vitro using the Promega IL12 Bioassay Kit (JA2601) according to the manufacturer's instructions. The IL12 Bioassay uses human cells engineered to express the IL12 receptor and a luciferase reporter under the control of an IL12-inducible promoter. Promega IL12 reporter cells are supplied in a frozen, ready-to-use format, eliminating the need for cell culture.

[0302] IL-12 fusion polypeptide or optimized IL-12 fusion polypeptide composition formulated in TBS (20 mM Tris, 150 mM sucrose, 50 mM NaCl, 10 mM L-methionine, 0.02% w / v polysorbate 20, pH 7.3) was diluted into assay medium to prepare a titration series with a top concentration of 3 μg / mL and a 3-fold dilution ratio. For samples mixed with aluminum alloy, a final concentration of 250 μg / mL of fusion polypeptide was mixed with a 10-fold mass excess of aluminum hydroxide, defined by the mass of metal in the formulation buffer, and incubated with shaking at room temperature for 30 minutes before dilution in the same assay medium. 25 μL of each sample in the titration series was transferred to a 96-well plate and mixed with 50 μL of Promega cell suspension to achieve a final top concentration of 1 μg / mL of fusion polypeptide. The plate was then incubated at 37°C in 5% CO2 for 6 hours or overnight. 75 μL of Bio-Glo reagent was added to the sample wells, incubated for 10 minutes, and luminescence was measured.

[0303] The IL12 fusion polypeptide in the optimized IL12 fusion polypeptide composition induces strong signaling in the Promega IL12 reporter assay both alone and after complexation with Alhydrogel (Figure 6). The EC50 values ​​are very similar to those of the IL12 fusion polypeptide in TBS, suggesting that the formulation does not affect biological activity.

[0304] Example 7. Effect of other composition components on the stability of IL-12 fusion polypeptides This example demonstrates that IL-12 fusion polypeptides exhibit reduced oxidation and deamidation when formulated in a composition containing Tris buffer, sucrose, salt, L-methionine, and a surfactant at a pH of about 7 to 7.5. Specifically, this example demonstrates that the L-methionine in the composition protects methionine and tryptophan in the IL-12 fusion polypeptide from oxidation, and that the low pH (about 7 to 7.5) protects asparagine and glutamine from deamidation.

[0305] Compositions F1, F2, F4, and F5 were prepared as described in Example 4. Each composition was incubated at 40° C. for 6 weeks.

[0306] The oxidation rates (%) of methionine and tryptophan are shown in Table 14 below. [Table 14]

[0307] Composition F5 (without methionine) showed approximately 27% oxidized species, while F1, F2, and F4 (all containing methionine) showed approximately 18-20% oxidized species, indicating that L-methionine can reduce or prevent the oxidation of methionine and tryptophan in compositions F1, F2, and F4.

[0308] The deamination rates (%) of asparagine (N) and glutamine (Q) are shown in Table 15 below. [Table 15] No modifications were detected at other positions.

[0309] Table 15 shows the effect of pH on deamidation. The composition with the highest pH (pH 7.6) (F4) showed significantly higher levels of deamidation for N162, N323, and N472 compared to the compositions with lower pH (F1, F2, and F5, which have a pH of about 7.3).

[0310] Example 8. Development of formulations of IL-12 fusion polypeptide alone or complexed with aluminum hydroxide This example demonstrates that IL-12 fusion polypeptide formulations can be complexed with aluminum hydroxide (alum). This example also demonstrates that complexing IL-12 fusion polypeptides with alum does not significantly affect the potency of the IL-12 fusion polypeptide or the viability of PBMCs. Preparation of complexed IL-12 fusion polypeptides using a syringe does not result in loss of IL-12 fusion polypeptides.

[0311] Exemplary IL-12 Fusion Polypeptide Formulations The IL-12 fusion polypeptide formulation is available as a 1.5 mg / vial formulation manufactured directly from the fully formulated drug substance. The IL-12 fusion polypeptide formulation is a sterile formulation packaged in single-use vials, each nominally containing 1.5 mg.

[0312] The IL-12 fusion polypeptide drug substance (nominal concentration 2 mg / mL) is fully formulated in 20 mM Tris, 50 mM sodium chloride, 150 mM sucrose, 0.02% polysorbate 20 (w / v), 10 mM L-methionine at a target pH of 7.3. The IL-12 fusion polypeptide drug substance is the only active ingredient in this formulation (1.5 mg of IL-12 fusion polypeptide formulation per vial).

[0313] The qualitative and quantitative composition of the IL-12 fusion polypeptide formulation is the same as that of the IL-12 fusion polypeptide drug substance. Stability studies of both the drug substance and the formulation have shown that there is no incompatibility between the excipients and the active ingredient.

[0314] An IL-12 fusion polypeptide formulation was developed for clinical trials using intratumoral administration, and consisted of 1.5 mg of IL-12 fusion polypeptide per glass vial.

[0315] The suitability of this formulation and dosage form has been confirmed through early-stage formulation development testing, including agitation, freeze / thaw, and storage stability testing. The buffer and pH were selected to provide a stable solution for the protein while maintaining the pH during storage of the drug substance and formulation. Polysorbate 20 was added to reduce the possibility of aggregation due to agitation and / or freeze / thaw. Sucrose was added to adjust the osmolality of the formulation. The formulation is designed to be stable with respect to freeze / thaw cycles.

[0316] Administered ingredients and simulated use A simulated dosing study was conducted to evaluate the initial steps in the preparation of the dosage form, including the compatibility of the formulation with several components and contact materials expected to be used in preparing doses for clinical drug administration via the intratumoral route.

[0317] To establish the stability of the diluted formulation and determine the suitability of representative clinical dosage preparation materials, the compatibility of the diluted IL-12 fusion polypeptide formulation with the formulation's specific diluent was tested in Type 1 glass vials (6R). The IL-12 fusion polypeptide formulation (Lot 101) was obtained as a liquid in the following formulation: 20 mM Tris, 150 mM sucrose, 50 mM sodium chloride, 10 mM L-methionine, 0.02% (w / v) polysorbate 20, pH 7.3, at a nominal concentration of 2 mg / ml (a representative batch with a nominal fill volume of 1.0 mL in a 2R glass vial (Type I) with an overfill). The formulation-specific diluent was obtained as a liquid in the following formulation: 20 mM Tris, 150 mM sucrose, 50 mM sodium chloride, 10 mM L-methionine, 0.02% (w / v) polysorbate 20, pH 7.3 (representative batch with a nominal fill volume of 6 mL in 6R glass vials (type I)). The IL-12 fusion polypeptide formulation and formulation-specific diluent were transferred into empty, sealed, sterile 6R glass vials using commercially available siliconized syringes (1 mL or 2 mL) and needles (21 gauge).

[0318] IL-12 fusion polypeptide formulations were diluted with formulation-specific diluents in 6R vials to a target concentration of 0.25 mg / mL. Diluted formulations (0.25 mg / mL) were prepared in triplicate (n=3). Physicochemical analytical data support the physicochemical stability of diluted IL-12 fusion polypeptide formulation solutions in 6R sterile sealed vials when exposed to ambient storage conditions (ambient temperature with exposure to light) for up to 4 hours.

[0319] No significant changes were observed in physicochemical analysis tests (clarity, color), purity by size exclusion-high performance liquid chromatography, or activity throughout the study, indicating good compatibility with the selected materials. Samples tested at time 0 contained virtually no visible particles (one of three replicates contained one fiber-like particle). At the 4-hour time point (T4h), each sample was assigned a reported result of almost no visible particles (two of three replicates contained one fiber-like particle each). Further investigation by particle characterization revealed that these visible particles were non-proteinaceous and reported to be primarily cellulose fibers and some oleamide particles. Therefore, these visible particles were inherent to the dosage formulation. Considering an expected dose of less than 100 mL, subvisible particles well met Pharmacopeial requirements <United States Pharmacopeia 787> for both subvisible particle counts of 25 μm or greater (≤600 particles / container) and 10 μm or greater (≤6,000 particles / container).

[0320] The recoveries at T4h were generally high for all samples (all above 99%), indicating good compatibility with the contact materials.

[0321] Results of the cell-based assay of the IL-12 fusion polypeptide formulation at T0 and T4 showed 102% activity (T0) and 103% activity (T4).

[0322] These results demonstrate that the IL-12 fusion polypeptide formulation is stable in the formulation-specific diluent at the target concentration (0.25 mg / mL) in contact with the glass vial at ambient conditions for up to 4 hours. Potency and Strength of IL-12 Fusion Polypeptides Complexed with Alhydrogel® Upon Dose Preparation

[0323] Aluminum hydroxide (Alum), the chemical basis of Alhydrogel®.

[0324] The compatibility of the complexed IL-12 fusion polypeptide formulation / Alhydrogel® was investigated in a point-of-use compatibility study using the IL-12 fusion polypeptide formulation or bulk Alhydrogel® diluent, sterile empty vials (SEVs), and commonly available components (e.g., syringes, needles, etc.) to mimic clinical pharmacy dispensing. A bracketing design was employed to cover the intended dose range, including intermediate doses. Dose preparation is described below.

[0325] High dose: 0.25 mg / mL IL-12 fusion polypeptide complexed with 2.5 mg Alhydrogel® (dose group 6) 1) 3.75 mL of diluent formulation was added to 6R of SEV. 2) 0.75 mL of the IL-12 fusion polypeptide formulation / drug substance (2 mg / mL) was added to the 6R vial containing the diluent and gently swirled to ensure mixing. 3) The vial of Alhydrogel® was shaken well to ensure homogeneity. 4) 1.5 mL of Alhydrogel® (10 mg / mL) was added to the diluted solution of IL-12 fusion polypeptide formulation and gently swirled to ensure mixing. a. IL-12 fusion polypeptide formulation / drug substance concentration = 0.250 mg / mL b. Alhydrogel® concentration = 2.5 mg / mL c. A total of 6 mL of IL-12 fusion polypeptide was placed in a 6R vial. 5) The mixed preparation was incubated at room temperature for 30 minutes or 6 hours.

[0326] Intermediate dose: 0.02 mg / mL IL-12 fusion polypeptide complexed with 0.2 mg Alhydrogel® (dose group 3)

[0327] Starting with a pre-prepared solution of 0.250 mg / mL IL-12-fusion polypeptide / 2.5 mg / mL Alhydrogel® (Dose Group 6) from the initial mixing process described above, the following dilution scheme was performed: 1) The vials prepared for dose group 6 were mixed gently. 2) 5.52 mL of diluent formulation was added to 6R of SEV. 3) 0.48 mL of Dose Group 6 was added to the 6R vial containing diluent and gently swirled to ensure mixing. a. IL-12 fusion polypeptide formulation / drug substance concentration = 0.02 mg / mL b. Alhydrogel® concentration = 0.2 mg / mL 4) The mixed preparation was incubated at room temperature for 30 minutes or 6 hours.

[0328] Low dose: 0.002 mg / mL IL-12-ABP fusion polypeptide complexed with 0.02 mg Alhydrogel® (dose group 1)

[0329] Starting with a pre-prepared solution of 0.02 mg / mL IL-12-fusion polypeptide / 0.2 mg / mL Alhydrogel® (Dose Group 3) from the initial mixing process described above, the following dilution scheme was performed: 1) The vials prepared for dose group 3 were mixed gently. 2) 5.4 mL of diluent formulation was added to 6R of SEV. 3) 0.6 mL of Dose Group 3 was added to the 6R vial containing diluent and gently swirled to ensure mixing. a. IL-12 fusion polypeptide formulation / drug substance concentration = 0.002 mg / mL b. Alhydrogel® concentration = 0.02 mg / mL 4) The mixed preparation was incubated at room temperature for 30 minutes or 6 hours.

[0330] Interleukin-12 (IL-12) signals via the heterodimeric complex of IL-12Rβ1 and IL-12Rβ2 expressed on T cells and natural killer (NK) cells, inducing interferon-gamma (IFNγ) expression via phosphorylation and activation of STAT4. The efficacy of free IL-12-ABP protein and complexed IL-12 fusion polypeptides to induce IFNγ expression in activated primary human peripheral blood mononuclear cells (PBMCs) from healthy donors was evaluated compared with a human IL-12 control protein lacking alum-binding proteins. Cells were treated with titrations of human IL-12 (control), IL-12 fusion polypeptides, or IL-12 fusion polypeptides complexed with alum in the presence of 100 ng / mL soluble αCD3 antibody (clone OKT3). After 3 days, IFNγ concentrations in the supernatants were measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. The concentration of α-CD3 antibody was selected to result in suboptimal activation of immune cells, making them highly responsive to the IL-12 drug while minimizing the induction of IFNγ by the immune cells themselves.

[0331] In this study, human PBMCs were isolated from two healthy donors and plated in round-bottom 96-well plates at 5 × 10 per well. 5Cells were seeded. PBMCs were stimulated with anti-CD3 (100 ng / mL) in the presence of IL-12 fusion polypeptide drug substance (Good Manufacturing Practice lot 1205114). IL-12 fusion polypeptide was prepared by syringe (either throughout or only upon removal from the dose vial) or by pipetting throughout (ATXFTE-06 study). Appropriate controls included a negative control (unstimulated PBMCs), a positive control (soluble CD3 (5 μg / mL) + soluble CD28 (2 μg / mL)), and diluent (formulation buffer). After 72 hours of incubation, cell culture supernatants were collected and stored at -80°C until cytokine analysis for IFNγ by TR-FRET was completed. Cell viability was determined using the CellTiter-Glo® 2.0 cell viability assay. Studies were performed using PBMCs from two donors. Each experimental condition was tested in triplicate, and each immunoassay reading was performed in singlet (Table 16). [Table 16]

[0332] The CellTiter-Glo® 2.0 cell viability assay was used to determine the number of viable cells in culture by quantifying the amount of adenosine triphosphate (ATP) present, which was used as an indicator of the presence of metabolically active cells.

[0333] result Across both donors, stimulation with the positive control (anti-CD3 (5 μg / mL) + anti-CD28 (2 μg / mL)) increased ATP levels above the unstimulated condition (Figures 7 and 8). In the presence of alum-complexed IL-12 fusion polypeptide, ATP levels were consistent with the solvent control at most protein concentrations in all dose-adjusted groups, with a decrease in ATP levels observed at the highest dose of alum-complexed IL-12 fusion polypeptide. Test compound preparation and incubation methods did not appear to consistently affect PBMC viability.

[0334] Stimulation with 100 ng / mL anti-CD3 induced suboptimal stimulation of PBMCs, resulting in moderate IFNγ production. Alum-complexed IL-12 fusion polypeptides in all dose-prepared groups demonstrated a dose-dependent increase in IFNγ production (Figures 9 and 10). Differences in IFNγ production between syringe- and pipette-prepared groups were limited in both donors. The incubation method (static vs. rotating) did not appear to affect IFNγ production. Increasing the incubation time slightly reduced the potency of alum-complexed IL-12 fusion polypeptides under rotating conditions only, but the 50% effective concentration (EC50) was still consistent with that under non-rotating conditions at all three doses.

[0335] overview IL-12 is an important stimulator of T cells and NK cells and plays a key role in directing T cell differentiation toward a pro-inflammatory phenotype by inducing IFNγ production. To investigate the effects of the preparation and administration of alum-complexed IL-12 fusion polypeptides, we analyzed IFNγ production by activated primary human PBMCs and ECs. 50 The effect of preparation method on PBMC viability was also investigated. Three preparation methods (syringe, syringe only upon removal from the dose vial, and no syringe (pipetting)) and three incubation times (6 hours static, 6 hours rotating, and 30 minutes rotating) were compared in this study (preparation with or without syringe with rotating device). Preparation conditions with or without syringe were compared for three different dose groups (high, medium, or low).

[0336] Without rotation, a high level of precipitation of the complex was observed at the end of the incubation period. Each condition was thoroughly inverted back and forth to ensure homogeneity.

[0337] For both donors, PBMC cell viability, as measured by ATP, was consistent with a moderate decrease in cell viability at the highest dose concentration (donor 2), comparable to observations in previous studies. This decrease in cell viability was not consistently affected by the dose of alum-complexed IL-12 fusion polypeptide preparation. Overall, syringe preparation of alum-complexed IL-12 fusion polypeptide did not appear to consistently affect PBMC viability.

[0338] In both donors, IL-12 fusion polypeptide complexed with alum increased IFNγ production in a dose-dependent manner, with maximal responses exceeding those of the positive control (anti-CD3 + anti-CD28). In all dose preparation groups, there was limited difference in IFNγ production between the syringe-prepared and pipette-prepared groups. The method of dose preparation did not appear to affect the maximal response of the cells, with similar responses at all doses, regardless of whether they were prepared by syringe or pipette. Donor 1 did not achieve a maximal response, resulting in EC 50 Values ​​were calculated only for donor 2. Longer incubation times may decrease the potency of IL-12 fusion polypeptides complexed with alum under rotational conditions alone, but the EC 50 remained consistent with non-rotational conditions at all three doses. Potency was consistent across donor 2 dose groups, ranging from 0.0541 ng / mL to 0.129 ng / mL, consistent with previous data for IL-12 fusion polypeptides complexed with alum.

[0339] Overall, these data suggest that the dose preparation method (syringe / no syringe), incubation method (static / rotating), and incubation time (30 min vs. 6 h) did not significantly affect the potency of alum-complexed IL-12 fusion polypeptides or PBMC viability.

[0340] MicroBCA assay of complexed IL-12 fusion polypeptides The content (protein concentration) of the complexed IL-12 fusion polypeptide was determined by a point-of-use suitability test mimicking clinical pharmacy dispensing. The test used IL-12 fusion polypeptide drug substance (Lot P4130826), Alhydrogel® (Lot 152-001-001), diluent (Lot 152-002-001), and SEV, as well as commonly available ancillary components (e.g., syringes, needles). A bracketing design was employed to cover the intended dose range, including intermediate doses. The amount of IL-12 fusion polypeptide was measured at three different doses using a microBCA assay (MicroBCA Protein Assay Kit, Thermo Scientific).

[0341] Samples were prepared for dose group 6 (250 μg / mL), dose group 3 (20 μg / mL), and dose group 1 (2 μg / mL), as well as a dose control set in which all preparations were performed by pipetting.

[0342] The microBCA assay was used to quantify IL-12 fusion polypeptide bound to Alhydrogel® in a point-of-use bracketing study. The study included the highest potential patient dose, as well as the middle and lowest doses. While improved sample handling allowed for more accurate quantification of protein in the high and middle doses, low doses remain challenging. The measured concentrations for the dose preparation (same materials and procedures as described in the Pharmacy Manual) were not significantly different from the dose control, where each group was prepared by pipetting. The use of syringes does not appear to result in significant protein loss during preparation. Precision was good in all cases, suggesting that syringe fluid handling is sufficiently accurate to allow reproducible dispensing at all volumes.

[0343] Example 9. Stability Data for Bulk IL-12 Fusion Polypeptide Drug Substance This example demonstrates the high stability of a reference batch of IL-12 fusion polypeptide drug substance and a GMO batch of IL-12 fusion polypeptide drug substance.

[0344] The following two batches of IL-12 fusion polypeptide drug substance were tested: 500L scale bioreactor batch (analytical reference standard batch) (P4130826ARS); and 1000L scale bioreactor batch (Good Manufacturing Practice (GMP) batch) (1205114).

[0345] A summary of the stability of the IL-12 drug substance is shown in Table 17. [Table 17]

[0346] The stability protocol for the IL-12 fusion polypeptide reference batch is shown in Table 18. [Table 18]

[0347] The stability protocols for the IL-12 fusion polypeptide GMO batches are shown in Table 19. [Table 19]

[0348] Analysis method Exemplary stability testing methods are described in this section. An AEX-HPLC method was used to separate dephosphorylated IL-12 fusion polypeptide drug substance and its charge variants. An AEX-HPLC column was used to quantify the charge variants present in the IL-12 fusion polypeptide drug substance. Samples were dephosphorylated using phosphatase and injected onto the column. Charge variants were separated based on the difference in surface charge of different molecular species. Negatively charged acidic molecules elute later than positively charged basic molecules. For IL-12 fusion polypeptide drug substance, charged species can be determined through detection of elution peaks by fluorescence detection using an excitation wavelength of 280 nm and an emission wavelength of 320 nm based on their surface charge differences. Relative quantification of acidic and basic species of the IL-12 fusion polypeptide drug substance was performed by evaluating the relative area (%).

[0349] overview To date, one-month stability data has been collected for the IL-12 fusion polypeptide drug substance GMP batch at each storage condition, and nine-month stability data has been collected for the IL-12 fusion polypeptide drug substance analytical reference standard batch at each storage condition. All stability storage conditions (-70°C, +5°C, and +25°C) met the stability study specifications. All samples tested to date have met the current GMP release specifications. The drug substance analytical reference standard batch sample stored at +5°C appeared to have a slight decrease in the percentage of the main peak in reduced capillary electrophoresis-sodium dodecyl sulfate (CE SDS) analysis at nine months compared to the start of the study; however, this was within the expected intermediate precision CV percentage for the main peak (CV = 1% for reduced CE SDS).

[0350] Comparison of samples of IL-12 fusion polypeptide from the analytical reference batch stored at -70°C for 9 months in low-density polyethylene (LDPE) bags and high-density polyethylene (HDPE) bottles showed consistent results, confirming that the type of container does not significantly affect the product by either test method.

[0351] Only at stress conditions of 25°C and at 6 months (Analytical Reference Standard batch) is there a decrease in the % of the main peak as shown by the CE SDS using the Drug Substance Analytical Reference Standard batch.

[0352] The similar small decrease across all conditions at 1 month for the GMP batches as shown by AEX-HPLC is likely due to assay variability, which will be investigated during planned assay validation.

[0353] Example 10. Stability Data of IL-12 Fusion Polypeptide Formulations This example demonstrates the high stability of IL-12 fusion polypeptide formulations (reference batch and GMO batch).

[0354] A summary of the stable IL-12 fusion polypeptide formulation lots is shown in Table 20. Stability studies and associated conditions are listed in Tables 21 and 22 below. [Table 20] [Table 21] [Table 22]

[0355] Exemplary Analysis Methods An AEX-HPLC method was used to separate dephosphorylated IL-12 fusion polypeptide formulations and their charge variants. An AEX-HPLC column was used to quantify the charge variants present in the IL-12 fusion polypeptide drug substance. Samples were dephosphorylated using phosphatase and injected onto the column. Charge variants were separated based on the differences in surface charge of different molecular species. Negatively charged acidic molecules elute later than positively charged basic molecules. For IL-12 fusion polypeptide drug substance, charged species can be determined through detection of elution peaks by fluorescence detection using an excitation wavelength of 280 nm and an emission wavelength of 320 nm based on their surface charge differences. Relative quantification of acidic and basic species in IL-12 fusion polypeptide formulations was performed by evaluating the relative area (%).

[0356] A helium leak test was used to conduct the container integrity (CCI) test. A quantitative mass spectrometry-based helium leak physical container integrity method was used to test the microbiological tightness of the container closure system. The container closure system was placed in an airtight flange connected to a mass spectrometer, and a vacuum pump was used to create a pressure difference between the inside of the mass spectrometer and the inside of the container closure system, continuously supplying helium gas into the container closure system. The mass spectrometer quantified the helium gas flow in mbarL / s escaping from potential leaks in the container closure system.

[0357] conclusion Non-GMP lots of IL-12 fusion polypeptide formulations remained stable and met all release specifications at 9 months and at the intended storage temperature of 5°C ± 3°C. There was minimal increase in subvisible particles ≥ 2 μm in both upright and inverted vials. A similar profile was obtained when IL-12 fusion polypeptide formulations were stored frozen at -20°C ± 5°C. At an accelerated temperature of 25°C ± 2°C / 60% ± 5% relative humidity (RH) (inverted) and at 6 months, the IL-12 fusion polypeptide formulations showed a slight decrease in the main peak when tested by reduced reversed-phase high-performance liquid chromatography (RP-HPLC) and reduced capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) compared to the previous time points. The molecule showed little tendency to aggregate (size exclusion-high-performance liquid chromatography (SE-HPLC)) or form subvisible particles. Only under stress conditions of 40°C ± 2°C / 75% ± 5% RH (inverted) and at 3 months did the IL-12 fusion polypeptide formulation show a significant decrease in the main peak (RP-HPLC, CE-SDS). Under the same conditions, the IL-12 fusion polypeptide formulation also showed a slight decrease in the percent monomer when assayed by SE-HPLC. The GMP lot of the IL-12 fusion polypeptide formulation met all specifications at the time of lot release (0 months) and is comparable to the non-GMP lot of the IL-12 fusion polypeptide formulation (0 months). The GMP lot of the IL-12 fusion polypeptide formulation is expected to exhibit a stability profile similar to that of the non-GMP lot of the IL-12 fusion polypeptide formulation.

[0358] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not limited to the above description, but rather is as set forth in the following claims.

Claims

1. i) a phosphorylated form of the fusion polypeptide, a) an immunomodulatory polypeptide comprising an interleukin-12 immune agonist portion; and b) a metal hydroxide-binding polypeptide; a phosphorylated form of the fusion polypeptide, the amino acid sequence of which contains multiple phosphorylation sites, such that the fusion polypeptide can be in a phosphorylated form and an unphosphorylated form; ii) Tris buffer; iii) salt; and iv) sucrose; and v) L-methionine; vi) a surfactant, The composition, wherein the pH of the composition is within the range of about 6.5 to about 8.

2. 10. A composition according to any preceding claim, wherein the fusion polypeptide forms a complex with a metal hydroxide when exposed to the metal hydroxide.

3. 10. A composition according to any preceding claim, wherein the metal hydroxide is aluminium hydroxide.

4. 10. The composition of any preceding claim, wherein the pH is in the range of about 6.5 to about 7.

8.

5. 10. The composition of any preceding claim, wherein the pH is in the range of about 7 to about 7.

6.

10. The composition of any one of the preceding claims, wherein the pH is from about 7.3 to about 7.

4.

6. 10. The composition of any of the preceding claims, wherein the concentration of the fusion polypeptide is in the range of about 0.1 g / L to about 15 g / L.

7. 10. The composition of any preceding claim, wherein the concentration of the fusion polypeptide is in the range of about 0.5 g / L to about 5 g / L.

8. 10. The composition of any preceding claim, wherein the concentration of the fusion polypeptide is in the range of about 1 g / L to about 3 g / L.

9. 10. The composition of any one of the preceding claims, wherein the concentration of the fusion polypeptide is about 2 g / L.

10. 10. The composition of any preceding claim, wherein the concentration of Tris buffer is in the range of about 1 mM to about 50 mM.

11. 10. The composition of any preceding claim, wherein the concentration of Tris buffer is in the range of about 10 mM to about 25 mM.

12. 10. The composition of any preceding claim, wherein the concentration of salt is in the range of about 1 mM to about 750 mM.

13. 10. The composition of any preceding claim, wherein the concentration of salt is in the range of about 10 mM to about 100 mM.

14. 10. The composition of any of the preceding claims, wherein the salt is NaCl or Na2SO4.

15. 10. The composition of any preceding claim, wherein the concentration of L-methionine is in the range of about 1 mM to about 20 mM.

16. 10. The composition of any preceding claim, wherein the concentration of L-methionine is in the range of about 5 mM to about 15 mM.

17. 10. The composition of any of the preceding claims, wherein the concentration of L-methionine is 10 mM.

18. 10. A composition according to any preceding claim, wherein the surfactant is a polysorbate.

19. 10. The composition of any preceding claim, wherein the surfactant is polysorbate 20 or polysorbate 80.

20. 10. The composition of any of the preceding claims, wherein the surfactant is Polysorbate 20.

21. 10. The composition of any preceding claim, wherein the concentration of polysorbate is in the range of about 0.005% w / v to about 0.1% w / v.

22. 10. The composition of any preceding claim, wherein the concentration of polysorbate is in the range of about 0.01% w / v to about 0.05% w / v.

23. 10. A composition according to any preceding claim, wherein the concentration of polysorbate is about 0.02% w / v.

24. 10. The composition of any preceding claim, wherein the concentration of sucrose is in the range of about 100 mM to about 200 mM.

25. 10. A composition according to any preceding claim, wherein the concentration of sucrose is 150 mM.

26. 10. The composition of any of the preceding claims, comprising 2 mg / mL of the fusion polypeptide, 20 mM Tris buffer, 50 mM NaCl, 10 mM L-methionine, 0.02% polysorbate 20, and 150 mM sucrose, and having a pH in the range of 6 to 8.

27. 10. A composition according to any preceding claim, stored at a temperature of at most -50°C, such as at most -55°C, for example at most -60°C, such as at most -65°C.

28. 10. The composition of any preceding claim, stored for a period ranging from about 1 day to about 500 days.

29. 10. A composition according to any preceding claim, wherein the pH of the composition after storage is the same as the pH of the composition before storage.

30. 10. The composition of any preceding claim, wherein the concentration of said fusion polypeptide in said composition after storage is the same as the concentration of said fusion polypeptide in said composition before storage.

31. 10. The composition of any preceding claim, wherein the osmolality of the composition after storage is the same as the osmolality of the composition before storage.

32. 10. A composition according to any preceding claim, wherein the colour of the composition after storage is the same as the colour of the composition before storage.

33. 10. A composition according to any preceding claim, wherein the amount of visible particles in the composition is the same as the amount of visible particles in the composition before storage.

34. 10. A composition according to any preceding claim, wherein the formulation is a liquid composition.

35. 10. The composition of any preceding claim, wherein the formulation is a solid composition.

36. 10. A composition according to any preceding claim in a dry form.

37. 10. The composition of any preceding claim, wherein the formulation is a powder.

38. 10. A composition according to any preceding claim which is frozen.

39. 10. A composition according to any preceding claim in a vial.

40. 10. The composition of any preceding claim, wherein the vial is light protected.

41. i) a fusion polypeptide metal-hydroxide complex, a) a phosphorylated form of the fusion polypeptide, ai) an immunomodulatory polypeptide comprising an interleukin-12 immune agonist portion; aii) a metal hydroxide-binding polypeptide; a phosphorylated form of the fusion polypeptide, wherein the amino acid sequence of the fusion polypeptide metal-hydroxide complex contains multiple phosphorylation sites, such that the fusion polypeptide can be in a phosphorylated form and an unphosphorylated form; (b) a metal hydroxide; and ii) Tris buffer; iii) salt; and iv) sucrose; and v) L-methionine; vi) a surfactant, The pharmaceutical formulation, wherein the pH of the composition is within the range of about 6.5 to about 8.

42. 42. The pharmaceutical formulation of claim 41, wherein the fusion polypeptide is adsorbed to the metal hydroxide via the at least one phosphorylated amino acid of the metal hydroxide-binding peptide via ligand exchange, thereby forming a fusion polypeptide-metal hydroxide complex.

43. 43. The pharmaceutical formulation according to claim 41, wherein the metal hydroxide is aluminum hydroxide.

44. 44. The pharmaceutical formulation of claims 41-43, wherein the concentration of the aluminum hydroxide is in the range of about 0.5 mg / mL to about 10 mg / mL.

45. 45. The pharmaceutical formulation of claims 41-44, wherein the concentration of the aluminum hydroxide is in the range of about 1 mg / mL to about 5 mg / mL.

46. 46. ​​The pharmaceutical formulation according to claims 41 to 45, wherein the concentration of the aluminum hydroxide is 2.5 mg / mL.

47. 47. The pharmaceutical formulation of claims 41-46, wherein the pH is in the range of about 6.8 to about 7.

8.

48. 48. The pharmaceutical formulation of claims 41-47, wherein the pH is in the range of about 7 to about 7.

6.

49. 49. The pharmaceutical formulation of claims 41 to 48, wherein the pH is about 7.

3.

50. 50. The pharmaceutical formulation of claims 41-49, wherein the concentration of the fusion polypeptide is in the range of about 0.0025 mg / mL to about 1 mg / mL.

51. 51. The pharmaceutical formulation of claims 41-50, wherein the concentration of the fusion polypeptide is in the range of about 0.05 mg / mL to about 0.75 mg / mL.

52. 52. The pharmaceutical formulation of claims 41-51, wherein the concentration of the fusion polypeptide is in the range of about 0.1 mg / mL to about 0.5 mg / mL.

53. 53. The pharmaceutical formulation of claims 41 to 52, wherein the concentration of the fusion polypeptide is 0.25 mg / mL.

54. 54. The pharmaceutical formulation of claims 41 to 53, wherein the concentration of the Tris buffer is in the range of about 1 mM to about 50 mM.

55. 55. The pharmaceutical formulation of claims 41 to 54, wherein the concentration of Tris buffer is in the range of about 10 mM to about 40 mM.

56. 56. The pharmaceutical formulation of claims 41 to 55, wherein the concentration of Tris buffer is in the range of about 15 mM to about 20 mM.

57. 57. The pharmaceutical formulation of claims 41-56, wherein the concentration of the salt is in the range of about 1 mM to about 100 mM.

58. 58. The pharmaceutical formulation of claims 41-57, wherein the concentration of the salt is in the range of about 20 mM to about 60 mM.

59. 59. The pharmaceutical formulation of claims 41-58, wherein the concentration of the salt is in the range of about 38 mM to about 50 mM.

60. 60. The pharmaceutical formulation of claims 41 to 59, wherein the salt is NaCl or Na2SO4.

61. 61. The pharmaceutical preparation of claims 41-60, wherein the concentration of L-methionine is in the range of about 1 mM to about 20 mM.

62. 62. The pharmaceutical preparation of claims 41-61, wherein the concentration of L-methionine is in the range of about 5 mM to about 15 mM.

63. 63. The pharmaceutical preparation of claims 41-62, wherein the concentration of L-methionine is in the range of about 7.5 mM to about 10 mM.

64. 64. The pharmaceutical formulation of claims 41 to 63, wherein the surfactant is a polysorbate.

65. 65. The pharmaceutical formulation of claims 41 to 64, wherein the surfactant is polysorbate 20 or polysorbate 80.

66. 66. The pharmaceutical formulation of claims 41 to 65, wherein the surfactant is polysorbate 20.

67. 67. The pharmaceutical formulation of claims 41-66, wherein the concentration of polysorbate is in the range of about 0.005% w / v to about 0.1% w / v.

68. 68. The pharmaceutical formulation of claims 41-67, wherein the concentration of polysorbate is in the range of about 0.01% w / v to about 0.05% w / v.

69. 69. The pharmaceutical formulation of claims 41-68, wherein the concentration of polysorbate is about 0.015% w / v.

70. 70. The pharmaceutical formulation of claims 41-69, wherein the concentration of sucrose is in the range of about 100 mM to about 200 mM.

71. The pharmaceutical formulation according to claims 41 to 70, wherein the concentration of sucrose is 113 mM.

72. The pharmaceutical formulation according to claims 41 to 71, which is a liquid composition.

73. 73. The pharmaceutical formulation of claims 41 to 72, which is a solid composition.

74. The pharmaceutical formulation according to claims 41 to 73, which is in the form of a powder.

75. 75. The pharmaceutical formulation of claims 41-74, wherein the composition comprises 0.25 mg / mL of the fusion polypeptide, 15 mM Tris buffer, 38 mM NaCl, 7.5 mM L-methionine, 0.015% polysorbate 20, and 113 mM sucrose, 2.5 mg / mL of aluminum hydroxide, and the pH of the composition is in the range of 6-8.

76. The pharmaceutical formulation according to claims 41 to 75, which is a liquid formulation.

77. The pharmaceutical preparation of claims 41 to 76, which is formulated for parenteral administration.

78. 78. The pharmaceutical preparation of claims 41 to 77, formulated for intratumoral injection.

79. The pharmaceutical formulation of claims 41 to 79, which is in a vial.

80. 42. A method of treating a subject comprising administering the pharmaceutical composition of claim 41.

81. 81. The method of claim 80, wherein the subject has cancer.

82. 82. The method of claim 81, wherein the cancer is associated with a tumor.

83. 81. The method of claim 80, wherein the subject is a human.

84. 81. The method of claim 80, wherein the composition is administered parenterally.

85. 83. The method of claim 82, wherein the composition is administered by intratumoral injection.

86. 84. The method of claim 83, wherein the composition is administered by peritumoral injection.

87. 81. The method of claim 80, wherein the composition is administered in combination with a second therapy.

88. 88. The method of claim 87, wherein the second therapy is a checkpoint inhibitor.

89. 10. A method of making the composition of claim 1, comprising combining the phosphorylated form of the fusion polypeptide with Tris buffer, salt, sucrose, L-methionine, and a detergent.

90. 42. A method for preparing the pharmaceutical formulation of claim 41, comprising: i) contacting the composition of claim 1 with a metal hydroxide.

91. 91. The method of claim 90, wherein the contacting is for a period of from 1 minute to 60 minutes.

92. 92. The method of claim 91, wherein the contacting is performed at room temperature.

93. A method for characterizing the composition of claim 1 by assessing the degree of phosphorylation of the fusion polypeptide.

94. 94. The method of claim 93, wherein said characterizing comprises assessing the purity of said fusion polypeptide in said composition.

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