Method for preparing fusion polypeptide

Abstract

The present disclosure provides surprisingly useful fusion polypeptides comprising an immunomodulatory moiety and a metal hydroxide binding moiety, as well as various related techniques, including methods of making and using such fusion polypeptides.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 424,013, filed on November 9, 2022, the entire content of which is incorporated herein by reference. Background of the Invention

[0003] Functional fusion polypeptides have been developed in which a metal - binding polypeptide is conjugated to an immunomodulatory domain (e.g., an IL - 12 immunomodulatory domain) (see, e.g., published International Patent Application WO2020 / 263399). Specific uses of such fusion peptides include treating certain medical disorders, such as cancer. Summary of the Invention

[0004] The present disclosure provides specific compositions and formulations of phosphorylated forms of fusion polypeptides, specifically including, for example, phosphorylated forms of IL - 12 fusion polypeptides (such as those described in published International Patent Application WO2020 / 263399). In some embodiments, the techniques provided offer particularly stable compositions and formulations of such phosphorylated fusion polypeptides.

[0005] The present disclosure in particular identifies the sources of problems with certain compositions or formulations containing such phosphorylated fusion polypeptides and provides solutions thereto. For example, the present disclosure proposes that the relevant phosphorylated fusion polypeptides are unusually unstable in typical formulations and compositions, posing challenges to production and / or distribution. For example, some standard formulations result in deamidation of the agent, susceptibility to oxidation, and instability, such as the formation of visible particles induced by agitation. Additionally, without wishing to be bound by any particular theory, the present disclosure proposes that phosphorylated IL - 12 fusion polypeptides may exhibit unusual "stickiness", such that in many standard formulations, the agent adheres to the surface of the container that holds it.

[0006] Furthermore, the present disclosure provides solutions to these problems and provides desirable compositions of the provided phosphorylated fusion polypeptides and / or, in some embodiments, provides formulations of phosphorylated fusion polypeptides complexed with metal hydroxides. The present disclosure in particular provides metal - hydroxide - binding polypeptides, and fusion polypeptides comprising them, which exhibit a high level of adsorption to metal hydroxides.

[0007] Certain available compositions comprise phosphorylated fusion polypeptides, which are phosphorylated IL-12 fusion polypeptides in a Tris buffer preparation having a pH of about 6.5 - 8 (e.g., about 7.4). In some embodiments, the compositions according to the present disclosure may further comprise added 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, L-methionine may mitigate the oxidative susceptibility of the IL-12 polypeptide fusion, and the surfactant (e.g., polysorbate) may reduce the formation of visible particles upon agitation of the composition. The added salt may stabilize the structure of the molecule via ionic interactions.

[0008] In addition, the present disclosure provides available preparations comprising a fusion polypeptide metal hydroxide complex, which comprises a phosphorylated fusion polypeptide (e.g., a phosphorylated IL-12 fusion polypeptide) in a Tris buffer preparation having a pH of about 6.5 - 8 (e.g., about 7.4). The preparations according to the present disclosure may further include added salt and / or L-methionine and / or a surfactant (e.g., polysorbate).

[0009] In some aspects, the present disclosure provides a composition comprising a phosphorylated form of a fusion polypeptide, a Tris buffer, a salt, sucrose, L-methionine, and a surfactant, wherein the fusion polypeptide comprises: (a) an immunomodulatory polypeptide comprising an interleukin-12 immunostimulatory moiety; and (b) a metal hydroxide-binding polypeptide having an amino acid sequence comprising a plurality of phosphorylation sites such that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms, wherein the pH of the composition ranges from about 6.5 to about 8. In some embodiments, the phosphorylated fusion polypeptide forms a complex with a metal hydroxide upon exposure thereto. In some embodiments, the metal hydroxide is aluminum hydroxide.

[0010] In some aspects, the present disclosure provides a pharmaceutical preparation comprising a fusion polypeptide metal hydroxide complex, a Tris buffer, a salt, sucrose, L-methionine, and a surfactant, wherein the fusion polypeptide metal hydroxide complex comprises a phosphorylated form of a fusion polypeptide and a metal hydroxide, and the fusion polypeptide comprises: (a) an immunomodulatory polypeptide comprising an interleukin-12 immunostimulatory moiety; and (b) a metal hydroxide-binding polypeptide having an amino acid sequence comprising a plurality of phosphorylation sites such that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms, wherein the pH of the composition ranges from about 6.5 to about 8.

[0011] 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.

[0012] In some aspects, the present disclosure provides methods of making the compositions and / or pharmaceutical formulations according to the present disclosure.

[0013] In some aspects, the present disclosure provides methods of characterizing the compositions according to the present disclosure by assessing the degree of phosphorylation of the fusion polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 An exemplary schematic of the fusion polypeptide metal hydroxide complex of the present disclosure is provided. The fusion polypeptide metal hydroxide complex can be administered to a subject and results in retention and / or enhanced efficacy compared to an appropriate reference standard.

[0015] Figure 2 A diagram of an exemplary fusion polypeptide of the present disclosure is provided that includes a first (p40) and second (p35) IL12 immunostimulatory moiety and a metal hydroxide binding polypeptide having multiple phosphorylation sites.

[0016] Figure 3A and Figure 3B Shows purity according to size exclusion chromatography, where A) shows the main peak (IL-12 fusion polypeptide) and B) shows high molecular weight material (HMW).

[0017] Figure 4A and Figure 4B Shows purity according to size exclusion chromatography, where A) shows the main peak (IL-12 fusion polypeptide) and B) shows high molecular weight material (HMW).

[0018] Figure 5 Shows alum retention over time.

[0019] Figure 6 Shows IL-12 fusion polypeptide activity when formulated in TBS or in an IL-12 fusion polypeptide composition conjugated to alum or free of metal hydroxide.

[0020] Figures 7A to 7E Shows quantification of ATP for determination of PBMC viability - donor 1. PBMCs were isolated from resting donor 1 and stimulated with an aqueous solution of anti-CD3 (100 ng / mL) in the presence of 12 different concentrations of pre-incubated IL-12 fusion polypeptide complexed with alum (ANK-101) (A - C) or not complexed with alum (IL-12-ABP) (D - E). Appropriate controls; negative control (unstimulated), positive control (soluble CD3 [5 μg / mL] + aqueous solution of CD28 [2 μg / mL]), and vehicle control (formulation buffer [0.04%]). On day 3, use 2.0 Cell Viability Assay Kit quantifies ATP. The schema shows the mean ± SEM of triplicates. Lower solid line: unstimulated; dotted line: vehicle; and upper solid line: positive. ATP = adenosine triphosphate; CD = cluster of differentiation; Conc = concentration; PBMC = peripheral blood mononuclear cell; SEM = standard error of the mean. ANK-101: IL-12 fusion polypeptide complexed with alum. IL-12-ABP: IL-12 fusion polypeptide.

[0021] Figures 8A to 8E Shows quantification of ATP for determination of PBMC viability - donor 2. PBMCs were isolated from resting donor 2 and stimulated with aqueous anti-CD3 (100 ng / mL) in the presence of pre-incubated IL-12 fusion polypeptide complexed with alum (ANK-101) (A - C) or without alum (IL-12-ABP) (D - E) at 12 different concentrations. Appropriate controls; negative control (unstimulated), positive control (soluble CD3 [5 μg / mL] + aqueous CD28 [2 μg / mL]), and vehicle control (formulation buffer [0.04%]). On day 3, the 2.0 Cell Viability Assay Kit was used to quantify ATP. The schema shows the mean ± SEM of triplicates. Lower solid line: unstimulated; dotted line: vehicle; and upper solid line: positive. ATP = adenosine triphosphate; CD = cluster of differentiation; Conc = concentration; PBMC = peripheral blood mononuclear cell; SEM = standard error of the mean. ANK-101: IL-12 fusion polypeptide complexed with alum. IL-12-ABP: IL-12 fusion polypeptide.

[0022] Figures 9A to 9EShows the accumulation of IFNγ in PBMC medium - Donor 1. PBMC were isolated from resting Donor 1 and stimulated with an aqueous anti - CD3 solution (100 ng / mL) in the presence of 12 different concentrations of pre - incubated IL - 12 fusion polypeptides complexed with alum (ANK - 101) (A - C) or not complexed with alum (IL - 12 - ABP) (D - E). Appropriate controls; negative control (unstimulated), positive control (soluble CD3 [5 μg / mL]+aqueous CD28 [2 μg / mL]), and vehicle control (formulation buffer [0.04%]). On day 3, cell culture supernatants were harvested and analyzed by TR - FRET. The schema shows the mean ± SEM of triplicates. Lower solid line: unstimulated; dotted line: vehicle; and upper solid line: positive. CD = cluster of differentiation; Conc = concentration; EC50 = half - maximal effective concentration; IFNγ = interferon γ; 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.

[0023] Figures 10A to 10E Shows the accumulation of IFNγ in PBMC medium - Donor 2. PBMC were isolated from resting Donor 2 and stimulated with an aqueous anti - CD3 solution (100 ng / mL) in the presence of 12 different concentrations of pre - incubated IL - 12 fusion polypeptides complexed with alum (ANK - 101) (A - C) or not complexed with alum (IL - 12 - ABP) (D - E). Appropriate controls; negative control (unstimulated), positive control (soluble CD3 [5 μg / mL]+aqueous CD28 [2 μg / mL]), and vehicle control (formulation buffer [0.04%]). On day 3, cell culture supernatants were harvested and analyzed by TR - FRET. The schema shows the mean ± SEM of triplicates. Lower solid line: unstimulated; dotted line: vehicle; and upper solid line: positive. CD = cluster of differentiation; Conc = concentration; EC50 = half - maximal effective concentration; IFNγ = interferon γ; NA = not applicable; PBMC = peripheral blood mononuclear cells; SEM = standard error of the mean; TR - FRET = time - resolved fluorescence energy transfer.

[0024] Definition

[0025] Administration: As used herein, the term "administration" generally refers to the application of a composition to a subject or system. Those skilled in the art will appreciate the various routes that may be used, as appropriate, to administer to a subject (e.g., a human). By way of example, in some embodiments, the administration may be systemic; in some embodiments, the administration may be topical. In some embodiments, the administration may be enteral; in some embodiments, the administration may be parenteral. In some embodiments, the administration may be effected by injection (e.g., intramuscular, intratumoral, intravenous, or subcutaneous injection). In some embodiments, the injection may involve bolus injection, infusion, perfusion, or transfusion. In many embodiments, administration according to the present disclosure is effected by intratumoral injection.

[0026] Affinity: As is known in the art, "affinity" is a measure of the tightness with which two or more binding partners associate with one another. Those skilled in the art are aware of the various assays that may be used to evaluate affinity and will further appreciate the appropriate controls for such assays. In some embodiments, affinity is evaluated in a quantitative assay. In some embodiments, affinity is evaluated at multiple concentrations (e.g., one binding partner at a time). In some embodiments, affinity is evaluated in the presence of one or more potential competing entities (e.g., competing entities that may be present in a relevant (e.g., physiological) environment). In some embodiments, affinity is evaluated relative to a reference (e.g., a reference with a known affinity above a particular threshold ["positive control" reference] or a reference with a known affinity below a particular threshold ["negative control" reference]). In some embodiments, affinity may be evaluated relative to a contemporaneous reference; in some embodiments, affinity may be evaluated relative to a historical reference. Generally, when affinity is evaluated relative to a reference, it is evaluated under similar conditions.

[0027] Agent: Generally, as used herein, the term "agent" is used to refer to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or a complex, combination, mixture, or system thereof [e.g., a cell, tissue, organism]), or a phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.). Where appropriate, as will be clear to those skilled in the art from the context, the term may be used to refer to an entity that is or comprises a cell or organism or a part, extract, or component thereof. Alternatively or additionally, as will be clear from the context, the term may be used to refer to a natural product found and / or obtained from nature. In some cases, again as will be clear from the context, the term may be used to refer to one or more entities that are man-made, as it is designed, engineered, and / or produced by a human action and / or not found in nature. In some embodiments, an agent may be used in isolated or pure form; in some embodiments, an agent may be used in crude form. In some embodiments, potential agents may be provided in the form of a collection or library, e.g., the collection or library may be screened to identify or characterize the active agents therein.

[0028] Agonist: Those skilled in the art will understand that the term "agonist" may be used to refer to an agent, condition, or event whose presence, level, degree, type, or form is associated with an increase in the level or activity of another agent (i.e., the agent being agonized or the target agent). Generally, an agonist may be or include an agent of any chemical class that exhibits the relevant activating activity, such as a small molecule, polypeptide, nucleic acid, carbohydrate, lipid, metal, and / or any other entity. In some embodiments, an agonist may be direct (in which case it directly exerts its effect on its target, e.g., by physically binding to such a target); in some embodiments, an agonist may be indirect (in which case it exerts its effect in a manner other than by binding to its target; e.g., by interacting with a modulator of the target, thereby altering the level and / or activity of the target).

[0029] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to compounds and / or substances that can be incorporated into, incorporated into, or have been incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. A "standard amino acid" refers to any one of the twenty standard L-amino acids that are typically found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, regardless of whether it is synthetically prepared or obtained from natural sources. In some embodiments, an amino acid, including the carboxy-terminal and / or amino-terminal amino acids in a polypeptide, may contain structural modifications compared to the above general structure. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, polyethylene glycolylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, carboxylic acid group, one or more protons, and / or hydroxyl groups) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to a polypeptide containing the unmodified amino acid that is otherwise identical. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to a polypeptide containing the unmodified amino acid that is otherwise identical. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to free amino acids; in some embodiments, it may be used to refer to the amino acid residues of a polypeptide.

[0030] 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.

[0031] Binding: It should be understood that, as used herein, the term "binding" generally refers to the non-covalent association between two or more entities. "Direct" binding involves physical contact between the entities or moieties; indirect binding involves physical interactions through physical contact with one or more intermediate entities. The binding between two or more entities can generally be evaluated in any of a variety of situations, including where the interacting entities or moieties are studied in isolation or in the context of a more complex system (e.g., when covalently, electrostatically, or otherwise associated with a carrier entity and / or in a biological system or cell). If, under the conditions being evaluated, two entities are more likely to associate with each other compared to other available binding partners, then the binding between the relevant entities can be considered "specific".

[0032] Cancer: The terms "cancer", "malignancy", "neoplasm", "tumor", and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or spontaneous growth, such that they exhibit an abnormal growth phenotype characterized by significantly uncontrolled cell proliferation. In some embodiments, the tumor can be or include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. The present disclosure specifically identifies certain cancers that may be particularly relevant to the teachings. In some embodiments, the relevant cancers can be characterized by solid tumors. In some embodiments, the relevant cancers can be characterized by hematological tumors. Generally, examples of different types of cancers known in the art include, for example, hematopoietic cancers, including leukemia, lymphoma (Hodgkin's and non-Hodgkin's lymphoma), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, solid tissue carcinomas, squamous cell carcinomas of the oral cavity, pharynx, larynx, and lungs, liver cancer, genitourinary cancers (such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer), and renal cell cancer, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine system cancers, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancers, benign lesions, such as papilloma, etc.

[0033] Signature sequence element: As used herein, the phrase "signature sequence element" refers to a sequence element found in a polymer (e.g., a polypeptide or nucleic acid) that represents a characteristic portion of the polymer. In some embodiments, the presence of a signature sequence element is associated with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a signature sequence element identifies a particular polymer as a member (or not a member) of a particular family or group of such polymers. A signature sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a signature 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., monomers linked in sequence). In some embodiments, a signature sequence element comprises at least a first and a second segment of contiguous monomers separated by one or more intervening regions, the length of which may or may not vary in polymers sharing the sequence element.

[0034] Chemotherapeutic agent: As used herein, the term "chemotherapeutic agent" has the meaning understood in the art and refers to one or more apoptosis-inducing agents, cell growth inhibitors, and / or cytotoxic agents, for example, specifically including agents used for and / or recommended for treating one or more diseases, disorders, or afflictions associated with unwanted cell proliferation. In many embodiments, the chemotherapeutic agent can be used to treat cancer. In some embodiments, the chemotherapeutic agent can be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeleton disruptors (e.g., microtubule-targeting agents such as taxanes, maytansines, and their analogs), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., analogs sharing related antiproliferative activity). In some specific embodiments, the chemotherapeutic agent can be or comprise one or more of the following: 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, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoid, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof.In some embodiments, chemotherapeutic agents can be used in the context of antibody-drug conjugates. In some embodiments, the chemotherapeutic agent is a chemotherapeutic agent found in an antibody-drug conjugate selected from the group consisting of: hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, 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, vorsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, the chemotherapeutic agent can be a chemotherapeutic agent described as being used in one or more of those described or discussed in Govindan et al., The Scientific World JOURNAL 10:2070, 2010, -2089. In some embodiments, the chemotherapeutic agent can be or comprise one or more of the following: farnesylthiosalicylic acid (FTS), 4-(4-chloro-2-methylphenoxy)-N-hydroxybutyramide (CMH), estradiol (E2), tetramethoxystilbene (TMS), delta-tocotrienol, salinomycin, or curcumin. Combination Therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents).In some embodiments, two or more agents can be administered simultaneously; in some embodiments, such agents can be administered sequentially; in some embodiments, such agents are administered in an overlapping dosing regimen.

[0035] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all "doses" of the first regimen are administered before any dose of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy can involve administering one or more additional agents or modalities to a subject who is receiving one or more agents or modalities in combination. For clarity, combination therapy does not require that the individual agents be administered together (or even necessarily simultaneously) in a single composition, although in some embodiments, two or more agents or their active moieties can be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0036] Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" can be used to refer to a set of unit doses (usually more than one unit dose) that are typically administered to a subject individually at intervals of one or more time periods. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which can involve one or more doses. In some embodiments, the dosing regimen comprises a plurality of doses, each of which is temporally separated from the other doses. In some embodiments, the individual doses are separated from each other by the same length of time period; in some embodiments, the dosing regimen comprises a plurality of doses and at least two different time periods separating the individual doses. In some embodiments, all doses within the dosing regimen have the same unit dose amount. In some embodiments, the different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose at a first dose amount, followed by one or more other doses at a second dose amount different from the first dose amount. In some embodiments, the dosing regimen comprises a first dose at a first dose amount, followed by one or more other doses at a second dose amount the same as the first dose amount. In some embodiments, the dosing regimen is related to a desired or beneficial outcome when administered in a relevant population (i.e., is a therapeutic dosing regimen).

[0037] Epitope: As used herein, the term "epitope" refers to the moiety specifically recognized by the binding component of an immunoglobulin (such as an antibody or a receptor). In some embodiments, an epitope comprises 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, when the antigen adopts an alternative conformation (such as being linearized), at least some of such chemical atoms and groups are physically separated from each other.

[0038] Excipient: As used herein refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0039] 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 may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (such as by transcription); (2) processing of the RNA transcript (such as 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.

[0040] Functionality: As used herein, the term "functionality" is used to refer to the form or fragment of an entity that exhibits a particular property and / or activity.

[0041] Fragment: A "fragment" of a material or entity as described herein has a structure that includes discrete parts of the whole but lacks one or more parts found in the whole. In some embodiments, a fragment consists of such discrete parts. In some embodiments, a fragment comprises or consists of characteristic structural elements or parts found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) found in the whole polymer or consists of such. 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 (e.g., residues) found in the whole polymer or consists of such. In some embodiments, the whole material or entity can be referred to as the "parent" of the fragment.

[0042] 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 includes a coding sequence (i.e., a sequence that encodes a specific product); in some embodiments, a gene includes non-coding sequences. In some particular embodiments, a gene can include both coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene can include one or more regulatory elements that can control or affect one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression, etc.).

[0043] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to RNA (before and / or after processing) transcribed from a gene or a polypeptide (before and / or after modification) encoded by RNA transcribed from a gene.

[0044] Genome: As used herein, the term "genome" refers to all the genetic information carried by an individual organism or cell, represented by the complete DNA sequence of its chromosomes.

[0045] 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 term refers not only to a particular subject cell, but also to progeny of such cell. Because certain modifications may occur in succeeding generations due to mutations or environmental influences, such progeny may not in fact be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, the host cell includes 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 those of prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of Escherichia coli (E. coli), Bacillus spp., Streptomyces spp., etc.), Mycobacterium cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe), Pichia pastoris (P. pastoris), Pichia methanolica (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 cell fusions, such as hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO Kl, DXB-1 1CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, podocytes, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells and cell lines derived from the above cells. In some embodiments, the cell contains one or more viral genes.

[0046] "Host cell protein" or "HCP": As used herein, refers to proteins that may be present in cell extracts or preparations, for example because they are produced by the host cell or otherwise contained in or on the host cell, in which the fusion polypeptides (e.g., phosphorylated or non-phosphorylated fusion polypeptides) described herein are produced, and the proteins are not the fusion polypeptides. In some embodiments, the techniques provided (e.g., the manufacturing methods provided, such as the purification methods provided) exclude or reduce HCP from preparations of the fusion polypeptides (e.g., preparations from phosphorylated fusion polypeptides as described herein). An "HCP-reduced preparation" describes a preparation containing reduced HCP relative to, for example, the amount present prior to application of relevant purification steps (e.g., relevant purification steps as provided herein) and / or relative to that achieved by different purification techniques. In some embodiments, the techniques provided enable the preparation of a fusion polypeptide preparation (e.g., a preparation of a phosphorylated fusion polypeptide) in which, for example, the HCP is undetectable using, for example, ELISA methods. In some embodiments, the removal of HCP can be monitored or evaluated, for example, during or after purification of the fusion polypeptides (e.g., its phosphorylated form) as described herein from the host cell, which in some embodiments can be an engineered mammalian cell as described herein (e.g., an engineered mammalian cell expressing the fusion polypeptide and the kinase that phosphorylates it at a ratio in the range of about 4:1 to 10:1, such as a ratio of about 8:1).

[0047] "Improve", "increase", or "decrease": As used herein, these terms or grammatically similar comparative terms indicate a value relative to a similar reference measurement. By way of example, in some embodiments, an evaluated value achieved using an agent of interest can be "improved" relative to a value obtained using a similar reference agent. Alternatively or additionally, in some embodiments, an evaluated value achieved in a subject or system of interest can be "improved" relative to a value 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 different similar subjects (e.g., in a different similar subject or system compared to the subject or system of interest in the presence of one or more indicators of a particular disease, disorder, or affliction of interest or following prior exposure to conditions or agents, etc.). In some embodiments, the comparative terms refer to a statistically relevant difference (e.g., a statistically relevant difference in incidence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will know or will be able to readily determine the degree and / or incidence of the difference required or sufficient to achieve such statistical significance in a given situation.

[0048] In vitro: The term "in vitro" as used herein refers to an event occurring in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0049] In vivo: As used herein, refers to an event occurring within a multicellular organism, such as within a human or non-human animal. In the context of a cell-based system, the term can be used to refer to an event occurring within a living cell (as opposed to, for example, an in vitro system).

[0050] Isolated: As used herein, means that a substance and / or entity has been (1) separated from at least some of the components with which it was associated when initially produced (in nature and / or in an experimental setting), and / or (2) artificially designed, produced, prepared, and / or manufactured. An isolated substance and / or entity can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which it was initially 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 more 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 skilled in the art, a substance can still be considered "isolated" or even "pure" after having been combined with certain other components such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage of isolation or purity of the substance is calculated without including such carriers or excipients. By way of example only, in some embodiments, a biopolymer such as a polypeptide or polynucleotide that exists in nature a) is not associated with some or all of the components that accompany it in its native state in nature due to its source or the source from which it is derived; b) is substantially free of other polypeptides or nucleic acids of the same species as the species from which it is produced in nature; c) is expressed by or otherwise associated with components from a cell or other expression system that is not of the species from which it is produced in nature is considered "isolated". Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell system in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has undergone one or more purification techniques can be considered an "isolated" polypeptide when it has reached a degree of separation from a) the components with which it was associated in nature; and / or b) the other components with which it was associated when initially produced.

[0051] Linker: As used herein, refers to the portion of a multi-component agent that connects different components to each other. By way of example, those skilled in the art will understand that a polypeptide structure that includes two or more functional or organizational parts or domains often includes a stretch of amino acids that connects such parts or domains to each other. In some embodiments, a polypeptide that includes a linker element has an overall structure of the general formula S1-L-S2, where S1 and S2 can be the same or different and represent two parts or domains that are associated with each other through the linker. In some embodiments, the length of 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 some embodiments, the linker is characterized by its tendency not to adopt a rigid three-dimensional structure, but rather to provide flexibility to the polypeptide. A variety of different linker elements that can be appropriately used when engineering polypeptides (such as fusion polypeptides) are known in the art (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444, 1993; Poljak et al., Structure 2:1121, 1994).

[0052] 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 being observed is associated with a change in the level and / or nature of that activity compared to what is observed in other similar conditions when the modulator is absent. In some embodiments, the modulator is an activator, as the activity is increased in its presence compared to what is observed in other similar conditions when the modulator is absent. In some embodiments, the modulator is an antagonist or inhibitor, as the activity is decreased in its presence compared to other similar conditions when the modulator is absent. In some embodiments, the modulator directly interacts with a target entity that has the activity of interest. In some embodiments, the modulator indirectly interacts with a target entity that has the activity of interest (i.e., directly with an intermediate agent that interacts with the target entity). In some embodiments, the modulator affects the level of the target entity of interest; or alternatively or additionally, in some embodiments, the modulator affects the activity of the target entity of interest without affecting the level of the target entity. In some embodiments, the modulator affects the level and activity of the target entity of interest such that the difference observed in the activity cannot be fully explained by or correlated with the difference observed in the level.

[0053] Moiety: As will be understood by those skilled in the art, a "moiety" is a defined chemical group or entity having a specific structure and / or activity as described herein. Generally, a "moiety" is a part that is less than the entire molecule or entity.

[0054] Mutant: As used herein, the term "mutant" refers to an entity that shows significant structural identity with a reference entity but is different from the reference entity in the presence or level of one or more chemical moieties in its structure. In many embodiments, the mutant is also functionally different from its reference entity. Generally, whether a particular entity is properly considered a "mutant" of a reference entity is based on the degree of its structural identity with the reference entity. As will be understood by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a mutant is a unique 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 such that mutants of the small molecule are mutants that share the core structural element and the characteristic pendant moieties but are different in other pendant moieties and / or the types of bonds present (single and double bonds, E and Z, etc.). Within the core, a polypeptide may have a characteristic sequence element consisting of a plurality of amino acids that have a specified position relative to each other in linear or three-dimensional space and / or contribute to a particular biological function, and a nucleic acid may have a characteristic sequence element consisting of a plurality of nucleotide residues that have a specified position relative to each other in linear or three-dimensional space. For example, a mutant polypeptide may differ from a reference polypeptide by one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the mutant polypeptide shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% total sequence identity with the reference polypeptide. Alternatively or additionally, in some embodiments, the mutant 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, the mutant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, the mutant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, the mutant polypeptide shows a reduced level of one or more biological activities compared to the reference polypeptide.

[0055] Operably linked: As used herein, refers to contiguousness in which the components described therein are in a relationship that permits them to function in their intended manner. A control element that is "operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, a control element that is "operably linked" is linked (e.g., covalently) to an encoding element of interest; in some embodiments, the control element acts in trans or otherwise at the functional element of interest.

[0056] Patient: As used herein, the term "patient" refers to any organism to which the provided composition is administered 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 a human. In some embodiments, the patient has or is susceptible to one or more disorders or diseases. In some embodiments, the patient exhibits one or more symptoms of a disorder or disease. In some embodiments, the patient has been diagnosed with one or more disorders or diseases. In some embodiments, the disorder or disease is or includes cancer, or there is one or more tumors. In some embodiments, the patient is receiving or has received certain therapies for diagnosing and / or treating a disease, disorder, or disease.

[0057] 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 dosage amount suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition can be specifically formulated for administration in solid or liquid form, including, for example, those pharmaceutical compositions suitable for a particular route of administration as described herein.

[0058] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" is used to refer to agents or entities that, within the scope of reasonable medical judgment, are suitable for contact with human and / or animal tissue without excessive toxicity, irritation, allergic response, or other problems or complications, and that meet a reasonable benefit / risk ratio.

[0059] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the compounds of the present invention from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; 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; diols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffering solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0060] Polypeptide: As used herein, it refers to a polymeric chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that exists in nature. In some embodiments, the polypeptide has an amino acid sequence that does not exist in nature. In some embodiments, the polypeptide has an engineered amino acid sequence as it is designed and / or produced by artificial means. In some embodiments, the polypeptide may comprise natural amino acids, unnatural amino acids, or both, or consist of them. In some embodiments, the polypeptide may comprise only natural amino acids or only unnatural amino acids, or consist of them. In some embodiments, the polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may comprise only D-amino acids. In some embodiments, the polypeptide may comprise only L-amino acids. In some embodiments, the polypeptide may have at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof, one or more pendant groups or other modifications, such as modifications to one or more amino acid side chains or attachment to one or more amino acid side chains. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, polyethylene glycolylation, etc., including combinations thereof. In some embodiments, the polypeptide may be cyclic and / or may comprise a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not comprise any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended with a reference to the name, activity, or structure of the polypeptide; in such cases, it is used herein to refer to polypeptides that share the relevant activity or structure and are thus considered members of the same polypeptide class or family. For each such class, the present specification provides and / or those skilled in the art will know exemplary polypeptides within the class for which the amino acid sequence and / or function are known; in some embodiments, such exemplary polypeptides are the reference polypeptides of the polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (in some embodiments, with all polypeptides within the class), share a common sequence motif (such as a characteristic sequence element) with the reference polypeptide of the class (in some embodiments, with all polypeptides within the class), and / or share a common activity (in some embodiments, at a similar level or within a specified range). By way of example, in some embodiments, the member polypeptides exhibit at least about 30 - 40% and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more total sequence homology or identity with the reference polypeptide and / or include at least one region (e.g., a conserved region that may be or comprise a characteristic sequence element in some embodiments) that exhibits very high sequence identity (often greater than 90% or even 95%, 96%, 97%, 98% or 99%).Such conservative regions typically encompass at least 3-4 and often up to 20 or more amino acids; in some embodiments, the conservative region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, the relevant polypeptide may comprise or consist of a fragment of the parent polypeptide. In some embodiments, the available polypeptide may comprise or consist of multiple fragments, each fragment present in the same parent polypeptide in a different spatial arrangement relative to each other compared to 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 compared to the parent), such that the polypeptide of interest is a derivative of its parent polypeptide.

[0061] Predetermined: Predetermined means intentionally selected, e.g., as opposed to occurring or being achieved randomly.

[0062] Pure: As used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a preparation containing more than about 90% of a particular agent or entity is generally considered 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.

[0063] Recombinant: As used herein, means a polypeptide designed, engineered, prepared, expressed, produced, manufactured and / or isolated by recombinant means, such as 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 or otherwise engineered to express one or more genes or gene components encoding and / or directing the expression of the polypeptide or one or more of its components, parts, elements or domains; and / or a polypeptide prepared, expressed, produced or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to each other, chemically synthesizing the selected sequence elements, and / or otherwise generating a nucleic acid encoding and / or directing the expression of the polypeptide or one or more of its components, parts, elements or domains. In some embodiments, one or more of such selected sequence elements exist in nature. In some embodiments, one or more of such selected sequence elements are computer-simulated designed. In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements from natural or synthetic sources, such as in the germline of the original organism of interest (e.g., human, mouse, etc.).

[0064] Reference standard: As used herein, describes a standard or control against which comparisons are 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 assayed substantially simultaneously with the test or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, as will be understood by those of skill in the art, the reference or control is assayed or characterized under conditions or circumstances similar to those in which the evaluation is being made. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular potential reference or control.

[0065] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish a possible binding partner in the environment where binding will occur. 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 evaluated by detecting or assaying the degree of association between the binding agent and its partner; in some embodiments, specific binding is evaluated by detecting or assaying the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is evaluated by detecting or assaying the ability of the binding agent to compete for the alternative interaction between its partner and another entity. In some embodiments, specific binding is evaluated by performing such detections or assays at a series of concentrations.

[0066] Specific: When used herein with reference to an agent having activity, the term "specific" is understood by those of skill in the art to mean that the agent distinguishes potential target entities or states. For example, in some embodiments, an agent is said to "specifically" bind to its target if it preferentially binds to its target in the presence of one or more competing alternative targets. In many embodiments, specific interactions depend on the presence of specific structural features (e.g., epitopes, clefts, binding sites) of the target entity. It should be understood that specificity need not be absolute. In some embodiments, specificity can be evaluated relative to the specificity of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to a reference specific binding agent. In some embodiments, specificity is evaluated relative to a reference non-specific binding agent. In some embodiments, an agent or entity does not detectably bind to a competing alternative target under conditions where it binds to its target entity. In some embodiments, the binding agent binds to its target entity with a higher rate of association, a lower rate of dissociation, increased affinity, decreased dissociation, and / or increased stability compared to one or more competing alternative targets.

[0067] Specificity: As is known in the art, "specificity" is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

[0068] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments a pre - natal human form). In some embodiments, the subject has a related 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 is an individual having one or more characteristics that are indicative of susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and / or therapy has been administered and / or is being administered.

[0069] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or elicits 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, improve, mitigate, inhibit, prevent one or more symptoms or characteristics of a disease, disorder, and / or condition, delay its onset, reduce its severity, and / or reduce its incidence.

[0070] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means the amount of a substance (e.g., a therapeutic agent, a composition, and / or a 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 a disease, disorder, and / or condition and / or delay its onset when administered to a subject having or predisposed to a disease, disorder, and / or condition. As will be understood by one of ordinary skill in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, mitigates, inhibits, prevents one or more symptoms or characteristics of a disease, disorder, and / or condition, delays its onset, reduces its severity, and / or reduces its incidence. In some embodiments, a therapeutically effective amount is administered as a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0071] Treatment: As used herein, the term "treatment" (also referred to as "treat" or "treating") refers to a therapy that administers to partially or fully relieve, improve, mitigate, inhibit one or more symptoms, features, and / or causes of a particular disease, disorder, and / or affliction, delay its onset, reduce its severity, and / or reduce its incidence. In some embodiments, such treatment can be for a subject who does not exhibit signs of the relevant disease, disorder, and / or affliction and / or for a subject who exhibits only early signs of the disease, disorder, and / or affliction. Alternatively or additionally, such treatment can be for a subject who exhibits one or more established signs of the relevant disease, disorder, and / or affliction. In some embodiments, the treatment can be for a subject who has been diagnosed with the relevant disease, disorder, and / or affliction. In some embodiments, the treatment can be for a subject who is known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or affliction. Thus, in some embodiments, the treatment can be prophylactic; in some embodiments, the treatment can be therapeutic.

[0072] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissues. In some embodiments, a tumor can comprise pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a manifestation 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.

[0073] Variant: As used herein, in the context of a molecule (such as a nucleic acid, protein, or small molecule), the term "variant" refers to a molecule that exhibits significant structural identity to a reference molecule but is structurally different from the reference molecule (e.g., in terms of the presence or absence or level of one or more chemical moieties compared to the reference entity). In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of its structural identity to the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a distinct molecule that shares one or more such characteristic structural elements but is different from the reference molecule in at least one aspect. To give just a few examples, a polypeptide may have a characteristic sequence element consisting of multiple amino acids that have specified positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element consisting of multiple nucleotide residues that have a specified position relative to another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from the reference polypeptide or nucleic acid by one or more differences in the amino acid or nucleotide sequence and / or by one or more differences in chemical moieties (such as carbohydrates, lipids, phosphate groups) that are covalently attached components of the polypeptide or nucleic acid (e.g., covalently attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% total sequence identity to the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, if a polypeptide or nucleic acid of interest has the same amino acid or nucleotide sequence as a reference but has minor sequence alterations at specific positions, it is considered a "variant" of the reference polypeptide or nucleic acid. Generally, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substitutions, insertions, or deletions 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 residue compared to the reference.Typically, relative to a reference, a variant polypeptide or nucleic acid contains a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a particular biological activity). In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and in some embodiments, contains no addition or deletion. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically fewer than about 5, about 4, about 3, or about 2 additions or deletions. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0074] Vector: As used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which other DNA segments can be ligated. Another type of vector is a viral vector, in which other DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells 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 integrate into the genome of the host cell after introduction into the host cell and thereby replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0075] Virus inactivation or removal: As used herein, the term "virus inactivation or removal" describes the inactivation or removal of viruses that may be contained in a sample (such as a cell extract or a fusion polypeptide preparation). In some embodiments, the viruses present in the sample may be derived from the source material (e.g., host cells); or alternatively or additionally, in some embodiments, the viruses present in the sample may be introduced, for example, during the processing of such source material. Those skilled in the art will be aware of various techniques for achieving virus inactivation or removal, such as inactivation by pH, chemical inactivation (e.g., by using chemical agents such as surfactants), etc. Those skilled in the art will understand that "pH virus inactivation" involves exposing the virus (e.g., a sample containing the virus) to a pH value that inactivates the virus (e.g., has been determined to inactivate it).

[0076] Wild-type: As used herein, the term "wild-type" has its meaning as understood in the art and refers to a form of an entity (e.g., a polypeptide or nucleic acid) that has the structure and / or activity as found in nature in a "normal" state (as contrasted with a mutant, diseased, altered state) or context. In some embodiments, more than one "wild-type" form of a particular polypeptide or nucleic acid may exist in nature, e.g., as "alleles" of a particular gene or normal variants of a particular polypeptide. In some embodiments, the form (or forms) of a particular polypeptide or nucleic acid that is most commonly observed in a population (e.g., a human population) is the "wild-type" form.

[0077] Detailed description of certain embodiments

[0078] Fusion polypeptide

[0079] In some embodiments, the fusion polypeptides according to the present disclosure can exist in phosphorylated and non-phosphorylated forms.

[0080] Immunomodulatory polypeptide

[0081] The fusion polypeptides of the present disclosure comprise at least one immunomodulatory polypeptide.

[0082] In some embodiments, the immunomodulatory polypeptide (e.g., an immune agonist moiety) activates or inhibits the activity of cells of the immune system (e.g., is signal transduction competent). In some embodiments, the immunomodulatory polypeptide (e.g., an immune agonist moiety) is evaluated, for example, as part of a fusion polypeptide as described herein.

[0083] By way of example, in some embodiments, signal transduction ability is characterized in that when binding to a specific binding partner is evaluated, one or more immune agonist moieties or functional fragments thereof exhibit binding similar to a reference standard (e.g., a wild-type polypeptide). By way of example, in some embodiments, signal transduction ability is characterized in that when a biological effect (e.g., in vitro or in vivo) is evaluated, one or more immune agonist moieties or functional fragments thereof exhibit the biological effect similar to a reference standard (e.g., a wild-type polypeptide).

[0084] In some embodiments, the immunomodulatory polypeptide comprises an interleukin-12 (IL-12) immunomodulatory polypeptide (e.g., an IL-12 immune agonist moiety).

[0085] IL-12 is a pro-inflammatory cytokine that plays an important role in innate and adaptive immunity. Wild-type IL-12 is a heterodimeric protein that consists of two subunits linked by a disulfide bond: p35 (IL-12A; GenBank GeneID: 3592) and p40 (IL-12B; GenBank GeneID: 3593). 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 interferon γ (IFN-γ) production and secretion.

[0086] The IL-12 subunits IL-12A and IL-12B can also form heterodimers with other IL-12 family members. For example, IL-12A can dimerize with Epstein-Barr virus-induced gene 3 (EBI3) to form the IL-12 family member IL-35, and IL-12B can dimerize with the p19 monomer to form the IL-12 family member IL-23.

[0087] IL-12 plays an important role in innate and adaptive immune responses, and dysregulation of IL-12 has been implicated in many disease states. Exemplary such disease states include, but are not limited to, inflammatory bowel disease, psoriasis, diabetes, multiple sclerosis, rheumatoid arthritis, cancer, lupus erythematosus, primary biliary cholangitis, and Sjogren syndrome ( syndrome) (Ullrich et al. EXCLI journal Vol. 19, 1563 - 1589. December 11, 2020). The use of IL-12 as a therapeutic modality has been extensively studied, including for the treatment of tumors (Nastala CL et al. J Immunol. August 15, 1994; Lasek et al. Cancer immunology, immunotherapy: CII Vol. 63, 5 (2014): 419 - 35).

[0088] In some embodiments, the immunomodulatory polypeptides disclosed herein are or comprise an IL-12 immunostimulatory moiety. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise multiple IL-12 immunostimulatory moieties. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise exactly two IL-12 immunostimulatory moieties. In some embodiments, two or more of the two or more (e.g., two) IL-12 moieties are the same moiety. In some such embodiments, the two or more (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 embodiments, the IL-12 moiety comprises an IL-12B polypeptide or a functional fragment thereof.

[0089] In some embodiments, the IL-12B immunostimulatory moiety is located N-terminal to the IL-12A immunostimulatory moiety in the immunomodulatory polypeptide. In some embodiments, the IL-12A immunostimulatory moiety is located N-terminal to the IL-12B immunostimulatory moiety in the immunomodulatory polypeptide.

[0090] In some embodiments, an immunomodulatory polypeptide comprising multiple (e.g., two) IL-12 moieties (e.g., IL-12A and / or IL-12B) is directly linked. In some embodiments, an immunomodulatory polypeptide comprising multiple (e.g., two) IL-12 moieties (e.g., IL-12A and / or IL-12B) is linked via a first linker. Non-limiting examples of linkers are discussed elsewhere herein.

[0091] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immunostimulatory moiety and comprise variants. In some embodiments, the IL-12A and / or IL-12B immunostimulatory moiety variants comprise substitutions, deletions, additions, and / or insertions 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 immunostimulatory moiety 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 variant comprises a substitution that does not alter the amino acid sequence relative to the wild-type IL-12A or IL-12B.

[0092] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immunostimulatory moiety as a functional fragment thereof (e.g., a signal-competent fragment). 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.

[0093] In some embodiments, the IL-12A or IL-12B fragment comprises 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 or consists of such.

[0094] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immunostimulatory moiety as a human IL-12A and / or IL-12B immunostimulatory moiety.

[0095] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12B immunostimulatory moiety having at least 80% sequence identity to SEQ ID NO:3 (such as having at least 85% sequence identity to SEQ ID NO:3, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity). In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A immunostimulatory moiety having at least 80% sequence identity to SEQ ID NO:4 (such as having at least 85% sequence identity to SEQ ID NO:4, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity). In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12B immunostimulatory moiety having at least 80% sequence identity to SEQ ID NO:3 and an IL-12A immunostimulatory moiety having at least 80% sequence identity to SEQ ID NO:4.

[0096] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5 (such as having at least 85% sequence identity to SEQ ID NO:5, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity).

[0097] Without being bound by a particular theory, hydroxy substitution (e.g., substitution with a phosphate group) can increase the adsorption of polypeptides via ligand exchange with metal hydroxides (e.g., aluminum hydroxide), and in addition can improve the tumor retention and anti-tumor efficacy of such polypeptides (e.g., fusion polypeptides comprising an immunomodulatory polypeptide and a metal hydroxide-binding polypeptide in which such hydroxy substitution has occurred).

[0098] In some embodiments, the immunomodulatory polypeptides according to the present invention can be in phosphorylated and non-phosphorylated 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 amino acids that can be phosphorylated. In some embodiments, an immunomodulatory polypeptide comprising one or more phosphorylated amino acids promotes strong binding of a metal to a metal hydroxide, such as aluminum hydroxide. Table 1 shows exemplary serine residues that can be phosphorylated in the immunomodulatory domain (e.g., S43, S154, S, 168, 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 four phosphorylated serine residues. In some embodiments, the immunomodulatory polypeptide comprises at least five phosphorylated serine residues, such as six serine residues, such as seven serine residues, such as eight serine residues, such as nine serine residues, such as ten serine residues.

[0099] Metal hydroxide-binding polypeptide

[0100] In some embodiments, the fusion polypeptides of the present disclosure comprise at least one metal-binding polypeptide.

[0101] The present disclosure provides metal hydroxide-binding polypeptides, as well as fusion polypeptides comprising metal hydroxide-binding polypeptides, which fusion polypeptides exhibit a high level of adsorption to metal hydroxides and further comprise desired manufacturing characteristics (such as one or more of the following: reproducibility, consistency, production of uniformly phosphorylated fusion polypeptides, etc.).

[0102] In some embodiments, the fusion polypeptide comprises two or more metal-binding polypeptides (such as two or more alum-binding polypeptides). In some embodiments, the fusion polypeptide comprises two or more identical metal-binding polypeptides; in some such embodiments, all of the metal-binding polypeptides in the fusion polypeptide according to the present disclosure are identical. In some such embodiments, the fusion polypeptide comprises two or more metal-binding polypeptides that are different from each other.

[0103] As discussed above, the metal-binding peptide can be fused to an immunomodulatory polypeptide, thereby allowing strong binding to metal hydroxides, such as aluminum hydroxide. A variety of immunomodulatory polypeptides can be fused to the metal-binding peptide. Without being limited by a particular theory, the metal-binding polypeptide adsorbed to alum in serum can be used to retain proteins and peptides in tumors.

[0104] In some embodiments, the metal hydroxide-binding polypeptide comprises an amino acid sequence comprising a plurality of phosphorylation sites such that it can exist in phosphorylated and non-phosphorylated forms. In some embodiments, the metal hydroxide-binding polypeptide comprises at least one kinase target motif. The target kinase motif comprises an amino acid phosphorylated by a kinase. Amino acids that are commonly phosphorylated include hydroxyl groups, such as serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y) residues. A kinase motif refers to the amino acid sequence N-terminal and / or C-terminal to an amino acid residue capable of being phosphorylated. Without wishing to be bound by any theory, many kinases contain structural features that confer specificity such that the kinase phosphorylates a specific amino acid (such as serine, threonine, or tyrosine) of a specific kinase target motif.

[0105] Depending on the specific type of kinase, the identified kinase target motifs are highly diverse. 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 a cell secretes proteins and / or other biomolecules into the extracellular space and refers to the endoplasmic reticulum (ER), Golgi apparatus (Golgi), cell membrane, and lysosomal storage compartments and the vesicles that move between them. Secretory pathway kinases are located throughout the secretory pathway (such as in the ER, Golgi, etc.) and have the function of phosphorylating proteins destined for secretion (Sreelatha et al., Biochimica et biophysica acta, Vol. 1854, 10 Pt B (2015): 1687-93).

[0106] In some embodiments, the relevant kinase is a naturally occurring secretory pathway kinase (e.g., endogenously targeted to the secretory pathway to function). In some embodiments, the secretory pathway kinase comprises a signal sequence that targets the kinase to the secretory pathway. Naturally occurring human secretory pathway kinases include, for example, tetraspanin kinase 1, Fam20A, Fam20B, Fam20C, vertebrate lonesome kinase (VLK), SGK196, and Fam69A, Fam69B, and Fam69C.

[0107] In some embodiments, the relevant kinase is a non-naturally occurring secretory pathway kinase. In some embodiments, the non-naturally occurring kinase is generated by linking a secretory signal peptide to a kinase that is endogenously localized to a non-secretory pathway cellular compartment.

[0108] In some embodiments, the kinase target motif is a target kinase motif of a secretory pathway kinase. In some embodiments, the secretory pathway kinase target kinase motif comprises an S-X-E motif. For example, Fam20C phosphorylates serine and has been shown to phosphorylate kinase target motifs comprising the following amino acid sequences: Ser-X-Glu (e.g., S-X-E), Ser-X-pSer (e.g., S-X-pS), and Ser-X-Gln-X-X-Asp-Glu-Glu (S-X-Q-X-X-D-E-E) where X is any amino acid and pS is 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.

[0109] In some embodiments, the metal hydroxide-binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises more than four S-X-E motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises eight S-X-E motifs. In some embodiments, the number of target kinase motifs (e.g., S-X-E motifs) contributes to the number of phosphorylated residues on the metal hydroxide-binding polypeptide. In some embodiments, the metal hydroxide-binding polypeptide comprises eight S-E-E motifs.

[0110] In some embodiments, the metal hydroxide-binding polypeptide is a metal hydroxide-binding polypeptide having an amino acid sequence comprising multiple phosphorylation sites. In some embodiments, the multiple phosphorylation sites comprise at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, the multiple phosphorylation sites comprise at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, the multiple phosphorylation sites comprise more than four S-X-E motifs. In some embodiments, the multiple phosphorylation sites comprise more than eight S-X-E motifs. In some embodiments, the number of target kinase motifs (e.g., S-X-E motifs) contributes to the number of phosphorylated residues on the metal hydroxide-binding polypeptide.

[0111] In some embodiments, at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs (such as the S-X-E motif) are directly adjacent (such as linked) to the next target kinase (such as the S-X-E motif). In some embodiments, at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs (such as the S-X-E motif) are separated (such as linked) from the next target kinase motif (such as the S-X-E 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 comprising GGGG.

[0112] In some embodiments, the metal hydroxide-binding polypeptide comprises four S-X-E motifs and three spacers each comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises six S-X-E motifs and five spacers each comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight S-X-E motifs and seven spacers each comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight S-X-E motifs and eight spacers each comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight motifs having the amino acid sequence SEE, and eight spacers each comprising four glycine residues.

[0113] In some embodiments, the metal hydroxide-binding polypeptide comprises a termination sequence (such as an amino acid sequence) at the C-terminus of the fusion polypeptide. In some embodiments, the termination sequence comprises multiple amino acid residues. In some embodiments, the multiple amino acid residues comprise GGGG. In some such embodiments, the termination sequence comprises the amino acid sequence GGGGS.

[0114] In some embodiments, the desired (e.g., optimal) number of kinase target motifs and / or the spacing of the kinase motifs can be determined based on, for example, one or more of the phosphate contents required to achieve strong metal hydroxide retention and / or avoid one or more manufacturing challenges (e.g., as understood by the present disclosure to be associated with highly phosphorylated elements). In some embodiments, the desired (e.g., optimal) number of kinase motifs and / or the spacing is such that the polypeptide is exposed to kinases to achieve the desired level of fusion polypeptide phosphorylation. In some embodiments, the improved fusion polypeptides as described herein result in one or more of the following: improved reproducibility, consistency, and / or production of uniformly phosphorylated fusion polypeptides. For example, in some embodiments, the techniques provided enable the reproducible manufacture of similar formulations (e.g., formulations that are always within defined parameters) of the fusion polypeptides (e.g., phosphorylated fusion polypeptides) and / or complexes as described herein. For example, in some embodiments, the techniques provided achieve reduced immunogenicity compared to an appropriate reference standard.

[0115] In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 0.5 - 7, 1 - 7, 2 - 7, 3 - 7, 4 - 7, 5 - 7, 6 - 7, 0.5 - 6, 0.5 - 5, 0.5 - 4, 1 - 6, 2 - 6, 3 - 6, 4 - 6, 5 - 6, 7 - 8, 8 - 9, 9 - 10, 10 - 11, 11 - 12, or 13 - 14. In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 3, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, or 12.

[0116] Linker

[0117] In some embodiments, the fusion polypeptides as described herein can include one or more linkers or spacers.

[0118] For example, in some embodiments, the fusion polypeptide comprises an immunomodulatory polypeptide that comprises first and second immunostimulatory moieties. In some embodiments, the first and second immunostimulatory moieties are linked via a first linker.

[0119] In some embodiments, the fusion polypeptides of the present disclosure comprise an immunomodulatory polypeptide and a metal hydroxide binding polypeptide. In some embodiments, the immunomodulatory polypeptide and the metal hydroxide binding polypeptide are linked via a second linker.

[0120] 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 may comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide.

[0121] In some embodiments, the polypeptide linker of the present disclosure has a length of at least one amino acid and can be any suitable number of amino acids. In some embodiments, the polypeptide linker has a length of 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.

[0122] In some embodiments, the first linker comprises a polypeptide linker. In some embodiments, the first linker comprises or consists of glycine-serine (Gly-Ser or G-S linker). A Gly-Ser linker is a polypeptide linker composed of glycine and serine residues. In some embodiments, the Gly-Ser linker comprises (Gly4Ser) n where 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, (Gly4Ser)8. In some embodiments, the Gly-Ser linker is (Gly4Ser)9. In some embodiments, the Gly-Ser linker is (Gly4Ser) 10 .

[0123] In some embodiments, the second linker comprises a polypeptide linker. In some embodiments, the second linker comprises multiple 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.

[0124] Variant

[0125] In some embodiments, the immunomodulatory polypeptide or metal hydroxide-binding polypeptide used in accordance with the present disclosure is a variant of a relevant reference polypeptide (e.g., a wild-type polypeptide or a functional portion thereof).

[0126] In some embodiments, the variant exhibits at least 70% identity to its reference polypeptide. In some such embodiments, the variant exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to its reference polypeptide.

[0127] In some embodiments, the variant contains one or more conservative or other non-destructive modifications (e.g., substitutions, deletions or additions) relative to its reference. In some embodiments, the variant does not contain any destructive modifications (e.g., substitutions, deletions or additions) such that the immunomodulatory polypeptide maintains one or more functional characteristics of the reference. In some embodiments, maintaining means that the immunomodulatory polypeptide exhibits similar activity (e.g., signal transduction ability or binding) compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide). By way of example, in some such embodiments, the immunomodulatory polypeptide maintains at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher activity compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide).

[0128] Metal hydroxide

[0129] 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 forms a complex therewith upon exposure to a metal hydroxide. The complex is formed by adsorption of the phosphorylated fusion polypeptide to the metal hydroxide. Without wishing to be bound by any theory, it is hypothesized that the adsorption of the phosphorylated fusion polypeptide to the metal hydroxide occurs by ligand exchange. By way of example, ligand exchange is the replacement or exchange of surface hydroxyl groups with another ligand. In some embodiments, the replacement or exchange of surface hydroxyl groups is carried out by a hydroxyl-displacing group (e.g., a phosphate group).

[0130] In some embodiments, the metal hydroxide is a substance comprising at least one hydroxyl group bound to a metal. According to the present disclosure, in some embodiments, the metal hydroxide can adsorb a fusion polypeptide comprising a hydroxyl-displacing moiety. In some embodiments, the hydroxyl-displacing moiety is a phosphate group.

[0131] In some embodiments, the metal hydroxide is selected based on its inherent qualities or characteristics. In some embodiments, the metal hydroxide is selected for use in a subject (such as a mammal, such as a human) because of its biocompatibility. In some embodiments, the metal hydroxide is aluminum hydroxide (such as alum). In some embodiments, the metal hydroxide is iron hydroxide. One of ordinary skill in the art will recognize that any number of metal hydroxides can be successfully used in accordance with the present disclosure.

[0132] In some embodiments, aluminum hydroxide is formulated in a gel (such as aluminum hydroxide gel). In some embodiments, aluminum hydroxide is formulated in water. In some embodiments, the concentration of the stock aluminum hydroxide preparation is 10 mg / mL.

[0133] Manufacturing phosphorylated fusion polypeptide

[0134] In one aspect of the present disclosure, a phosphorylated form of a fusion polypeptide as described herein is produced by a method comprising the step of (1) producing a phosphorylated form of the fusion polypeptide in a host cell.

[0135] In one aspect of the present disclosure, a high-purity preparation of the phosphorylated form of the fusion polypeptide from a cell extract is obtained by a method comprising the step of (2) purifying the fusion polypeptide from a cell extract (such as from step (1)) containing the phosphorylated species.

[0136] In one aspect of the present disclosure, a fusion polypeptide metal hydroxide complex comprising a phosphorylated form of a fusion polypeptide as described herein is produced by a method comprising the step of (3) contacting the phosphorylated form of the fusion polypeptide (such as from step (2)) with a metal hydroxide.

[0137] Expression in a host cell

[0138] In some embodiments of the present disclosure, the fusion polypeptide is manufactured by production in a host cell (such as a mammalian cell). Generally, such host cells (such as such mammalian cells) will have been engineered to express the fusion polypeptide. Those skilled in the art will be familiar with a variety of techniques for introducing exogenous gene sequences (such as encoding the fusion polypeptide, and / or a kinase) into a host cell (such as a mammalian host cell) for expression thereby.

[0139] For example, in some embodiments, a polynucleotide (e.g., DNA or RNA) encoding a fusion polypeptide of the present disclosure can be prepared, for example, for introduction into a host cell. For example, the sequence encoding the fusion polypeptide can be excised from DNA using restriction enzymes, amplified from a plasmid or genomic polynucleotide sequence using, for example, polymerase chain reaction, or synthesized using chemical synthesis techniques. In some embodiments, a combination of known methods is used to prepare a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure.

[0140] The recombinant polynucleotide encoding the fusion polypeptide of the present disclosure can be cloned into a vector capable of expressing the fusion polypeptide. Cloning can be carried out according to a variety of available methods (such as 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.

[0141] In some embodiments, an expressible vector contains a recombinant polynucleotide encoding the fusion polypeptide of the present disclosure, and the recombinant polynucleotide is operably linked to one or more sequences that regulate the expression of polynucleotides (such as promoters, start signals, stop signals, polyadenylation signals, activators, inhibitors, etc.). In some embodiments, one or more regulatory sequences controlling expression are selected to achieve the desired expression level. In some embodiments, more than one sequence controlling expression (such as a promoter) is used. In some embodiments, more than one sequence controlling expression (such as a promoter) is used to achieve the desired expression level of multiple recombinant polynucleotides encoding multiple polypeptides. In some embodiments, multiple recombinant polypeptides are expressed from the same vector (such as a bicistronic vector, a tricistronic vector, a polycistronic vector). In some embodiments, multiple recombinant polypeptides are expressed, each of which is expressed from a separate vector.

[0142] In some embodiments, the fusion polypeptide is expressed in a host cell using an expressible vector containing a recombinant polynucleotide encoding the fusion polypeptide of the present disclosure.

[0143] The host cell can be selected from a variety of available and known host cells suitable for expressing the fusion polypeptide disclosed herein (such as human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese hamster ovary cells).

[0144] A variety of methods can be used to introduce the vector into the host cell. In some embodiments, transfection can be used to introduce the vector into the host cell. In some embodiments, transfection is accomplished, for example, using calcium phosphate transfection, liposome transfection, or polyethyleneimine-mediated transfection. In some embodiments, transduction can be used to introduce the vector into the host cell.

[0145] In some embodiments, a host cell (e.g., a producer cell) is used to produce a phosphorylated form of a fusion polypeptide.

[0146] In some embodiments, a host cell expressing a fusion polypeptide and / or a kinase is cultured in a disposable bioreactor (e.g., 50 L to 4000 L) or a stainless-steel bioreactor (e.g., 50 L to 4000 L). In some embodiments, the host cell is cultured at a temperature in the range of 30°C to 40°C. In some embodiments, the temperature is lower (e.g., 33°C) during the preparation phase. In some embodiments, the cell extract is harvested through a 2- or 3-stage filter, followed by a final sterile 0.22 μm filtration.

[0147] In some embodiments, a nucleic acid encoding a fusion polypeptide is introduced into a host cell such that the host cell expresses the fusion polypeptide. 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, as described herein, both a nucleic acid encoding a fusion polypeptide and a nucleic acid encoding a kinase that phosphorylates it are introduced into the same host cell; in some such embodiments, a single nucleic acid molecule can encode both.

[0148] In some embodiments, the nucleic acid molecule introduced into the cell is RNA (e.g., mRNA); in some such embodiments, one or more encoded polypeptides (e.g., a fusion polypeptide and / or a 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 integrates into the host cell (e.g., its genome); in some such embodiments, one or more encoded polypeptides (e.g., a fusion polypeptide and / or a kinase) are expressed therefrom.

[0149] In some embodiments, the nucleic acid molecule introduced into the cell (e.g., a nucleic acid molecule encoding a fusion polypeptide and / or a kinase) includes one or more expression elements, such as expression elements that can regulate the expression of one or more such encoded polypeptides. Alternatively or additionally, in some embodiments, the nucleic acid molecule introduced into the cell (e.g., a nucleic acid molecule encoding a fusion polypeptide and / or a kinase) can be designed or intended to become associated with one or more regulatory elements in the host cell (e.g., by integration).

[0150] In some embodiments, a fusion polypeptide and / or a kinase are expressed in a host cell using a vector (e.g., a transposon) that includes a sequence encoding a fusion polypeptide and / or a kinase as described herein.

[0151] In some embodiments, the host cell can be selected from a variety of available and 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.

[0152] A variety of methods can be used to introduce nucleic acids (e.g., vectors, such as expression vectors) into host cells. In some embodiments, transfection can be used to introduce nucleic acids into host cells. In some embodiments, transfection is accomplished, for example, using calcium phosphate transfection, liposome transfection, or polyethyleneimine-mediated transfection. In some embodiments, transduction can be used to introduce nucleic acids into host cells. In some embodiments, electroporation can be used to introduce nucleic acids into host cells. In some embodiments, particle delivery (e.g., polymer particle delivery, lipid particle delivery, gold particle delivery, etc.) can be used to introduce nucleic acids into host cells.

[0153] Phosphorylation

[0154] In some embodiments, the present disclosure provides a method for producing a phosphorylated form of a fusion polypeptide disclosed herein by contacting the fusion polypeptide with a kinase. In some embodiments, nucleic acids (such as nucleic acids encoding the fusion polypeptide and / or the kinase) are introduced into a host cell. In some embodiments, the fusion polypeptide is contacted with the kinase by co-expressing the fusion polypeptide and the kinase in the host cell. In some embodiments, co-expression is achieved by introducing two vectors into the host cell, one vector containing a recombinant polynucleotide encoding the fusion polypeptide and one vector containing a recombinant polynucleotide encoding the kinase. In some embodiments, co-expression is achieved by introducing a single polycistronic (e.g., dicistronic) 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 introducing the vector (e.g., transposon) into the host cell. Without wishing to be bound by any theory, after the fusion polypeptide and the kinase are co-expressed in the host cell, the kinase can contact the fusion polypeptide and phosphorylate it.

[0155] In some embodiments, co-expression is achieved by introducing two vectors into a host cell, one vector comprising a recombinant polynucleotide encoding a fusion polypeptide and one vector comprising a recombinant polynucleotide encoding a kinase. In some embodiments, the two vectors are introduced at an optimized ratio of the vector encoding the fusion polypeptide to the vector encoding the kinase introduced into the host cell to achieve the desired relative expression levels of the fusion polypeptide and the kinase. In some embodiments, the ratio of the vector encoding the fusion polypeptide to the 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.

[0156] In some embodiments, co-expression is achieved by introducing into a host cell a vector (e.g., a dicistronic vector) that contains both a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase. In some embodiments, co-expression is achieved by introducing into a host cell a transposon that contains both a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase. In some embodiments, the transposon is a DNA transposon. In some embodiments, the transposon or a portion thereof (e.g., containing the nucleotide encoding the fusion polypeptide and the nucleotide encoding the kinase) is integrated into the host cell genome by an integration enzyme (i.e., by an integrase enzyme, such as a DDE / D integrase). In some embodiments, the integrase is delivered to the host cell as an 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 the polynucleotide encoding the fusion polypeptide of the present disclosure is integrated into a specific plurality of host cell genomic loci. In some embodiments, the integration of the polynucleotide encoding the fusion polypeptide of the present disclosure is irreversible. The irreversible integration of the fusion polypeptide into the host cell genome ensures stable integration. This allows for the generation of very stable cell lines. In some embodiments, the recombinant polynucleotide encoding the fusion polypeptide and the recombinant polynucleotide encoding the kinase are operably linked to one or more sequences that control expression (e.g., a promoter, an initiation signal, a stop signal, a polyadenylation signal, an activator, a repressor, etc.). In some embodiments, one or more sequences that control expression are selected to achieve a desired expression level. In some embodiments, multiple regulatory nucleotide sequences that control expression (e.g., promoters) are used to achieve the desired expression ratio of the fusion polypeptide to the kinase. 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 single transposon having two promoters for expressing the fusion polypeptide and the kinase is used to achieve a specific ratio (e.g., 2:1, 4:1, 8:1, or 15:1). In some embodiments, a single individual transposon contains promoters of different strengths to produce the desired ratio (e.g., 8:1). In some embodiments, the promoter is a CMV or 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 the SV40 or Ubc promoter. In some embodiments, the kinase is under the control of the SV40 promoter or Ubc promoter. In some embodiments, the ratio of the fusion polypeptide to the kinase is 8:1.

[0157] In some embodiments, after such transformation, the transformed host cell (i.e., the host cell into which a nucleic acid, such as a nucleic acid encoding the fusion polypeptide and / or the kinase, has been transformed) is cultured, for example, to allow expression of the recombinant polynucleotide. In some embodiments, the transformed host cell is cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. In some embodiments, the transformed host cell is cultured for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or longer. The transformed host cell is cultured under growth conditions (such as temperature, carbon dioxide level, growth medium) according to the requirements of the selected host cell. One of ordinary skill in the art will recognize that the culture conditions for the selected host cell are well known in the art. In some embodiments, the host cell secretes the phosphorylated form of the fusion polypeptide into the cell extract. In some embodiments, the host cell may show increased secretion of the phosphorylated fusion polypeptide into the cell extract compared to the non-phosphorylated fusion polypeptide. In some embodiments, the host cell secretes similar levels of the phosphorylated and non-phosphorylated fusion polypeptides into the cell extract. In some embodiments, the cell extract contains the non-phosphorylated form of the fusion polypeptide. In some embodiments, the cell extract contains the phosphorylated form of the fusion polypeptide. In some embodiments, the cell extract contains a mixture of the non-phosphorylated and phosphorylated forms of the fusion polypeptide. In some embodiments, the cell extract contains more of the phosphorylated form of the fusion polypeptide compared to the non-phosphorylated form of the fusion polypeptide.

[0158] In some embodiments, the host cell secretes the phosphorylated form of the fusion polypeptide into the cell extract but does not secrete the kinase. In some embodiments, the host cell does not secrete the kinase or secretes only a small amount of the kinase.

[0159] In some embodiments, the host cell extract contains the phosphorylated form of the fusion polypeptide. In some embodiments, the host cell extract contains host cell proteins and / or host cell nucleotides.

[0160] In some embodiments, the phosphorylated form of the fusion polypeptide is harvested from the transformed host cell and purified by centrifugation.

[0161] In some embodiments, the transformed host cell is characterized in that its culture produces a fusion protein with a titer of at least 200 mg / L (such as at least 250 mg / L, such as at least 300 mg / L, such as at least 350 mg / L, such as at least 400 mg / L, such as at least 450 mg / L, such as at least 500 mg / L, such as at least 550 mg / L, such as at least 600 mg / L, such as at least 650 mg / L, such as at least 700 mg / L, such as at least 750 mg / L, such as at least 800 mg / L, such as at least 850 mg / L, such as at least 900 mg / L, such as at least 950 mg / L, such as at least 1 g / l or more).

[0162] 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.

[0163] Purification

[0164] In some embodiments, the present disclosure provides a purification method (e.g., a method comprising one or more purification steps) or a technique (e.g., a manufacturing technique) comprising a purification method. In some embodiments, the phosphorylated form of the fusion protein is purified from the extract of the cells described herein.

[0165] In some embodiments, the purification step may include removing common abnormal products (e.g., residual proteins, host cell contaminants (e.g., host DNA and / or proteins, etc.)) from the cell extract.

[0166] In some embodiments, a manufacturing method including one or more purification steps yields a high-purity preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the high-purity preparation of the phosphorylated form of the fusion polypeptide contains reduced host cell proteins similar to the cell extracts described above herein. In some embodiments, such high-purity preparations do not contain any host cell proteins. In some embodiments, the high-purity preparation contains less than 100 ng of host cell protein per milligram, such as less than 50 ng per milligram, such as less than 40 ng per milligram, such as less than 30 ng per milligram, such as less than 20 ng per milligram, such as less than 10 ng per milligram, such as less than 9 ng per milligram, such as less than 8.5 ng of host cell protein per milligram. In some embodiments, the high-purity preparation of the phosphorylated form of the fusion polypeptide contains reduced host cell DNA similar to the cell extracts described above herein. In some embodiments, such high-purity preparations do not contain any host cell DNA. In some embodiments, the high-purity preparation contains less than 10 pg of host cell DNA per milligram, such as less than 9 pg per milligram, such as less than 8 pg per milligram, such as less than 7 pg per milligram, such as less than 6 pg per milligram, such as less than 5 pg per milligram, such as less than 4 pg per milligram, such as less than 3 pg per milligram, such as less than 2 pg per milligram, such as less than 1.5 pg per milligram, such as less than 1 pg per milligram, such as less than 0.9 pg per milligram, such as less than 0.8 pg per milligram, such as less than 0.7 pg of host cell DNA per milligram.

[0167] In some embodiments, the high-purity formulation comprises a low level of in-process blend. In some embodiments, the in-process blend can be tropolone, pluronic, PDMS, octamethylcyclotetrasiloxane D4, TDAO, and / or Fam20). In some embodiments, the high-purity formulation comprises less than 1 mg of TDAO per milliliter, such as less than 0.9 mg of TDAO per milliliter, such as less than 0.8 mg of TDAO per milliliter, such as less than 0.7 mg of TDAO per milliliter, such as less than 0.6 mg of TDAO per milliliter, such as less than 0.5 mg of TDAO per milliliter, such as less than 0.4 mg of TDAO per milliliter, such as less than 0.3 mg of TDAO per milliliter. In some embodiments, the high-purity formulation comprises less than 5000 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 4000 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 3000 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 2500 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 2000 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 1800 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 1000 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 750 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 500 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 300 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 200 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 100 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 80 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 70 ng of Fam20C per milligram of IL-12 fusion polypeptide, such as less than 60 ng of Fam20C per milligram of IL-12 fusion polypeptide.

[0168] 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, hydroxyapatite chromatography.

[0169] In some embodiments, the phosphorylated form of the fusion polypeptide can be purified by including one or more purification steps selected from ion chromatography steps (e.g., anion chromatography steps) and hydrophobic interaction chromatography steps. Thus, the phosphorylated fusion polypeptide is separated from impurities.

[0170] Those skilled in the art will be familiar with the various purification (e.g., chromatography) matrices and their modes 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 for purification (e.g., for chromatography) has properties such that the retention time of the fusion polypeptide is different from that of any other undesirable component in the fusion polypeptide preparation in accordance with the present disclosure.

[0171] In some embodiments, the phosphorylated form of the fusion polypeptide is not purified by affinity-based purification methods. For example, in some embodiments, the provided purification techniques do not use affinity chromatography.

[0172] In some embodiments, the phosphorylated form of the fusion polypeptide can be eluted from a solid matrix. In some embodiments, the elution can be performed using specific elution. For example, in some embodiments, specific elution is accomplished by challenging the polypeptide-matrix complex with one or more agents that will complete complexation with the matrix or the polypeptide, thereby releasing the polypeptide into solution. In some embodiments, the elution can be performed using non-specific elution. For example, in some embodiments, non-specific elution is accomplished by manipulating the solvent or buffer conditions (e.g., increasing the concentration of a buffer, such as imidazole buffer) to decrease the association rate constant such that the polypeptide dissociates from the matrix.

[0173] First chromatographic step

[0174] In some embodiments, the method according to the present disclosure includes at least one ion chromatography step (e.g., an anion chromatography step). In some embodiments, the method according to the present disclosure includes at least one anion chromatography step.

[0175] 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, polypeptide phosphorylation confers variability in the charge of the polypeptide, thereby allowing the separation of differentially 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., the phosphorylated form of the fusion polypeptide disclosed herein) bind to a positively charged solid matrix (e.g., a resin). The positively charged solid matrix thus captures the negatively charged fusion polypeptide from the cell extract and simultaneously removes positively charged impurities from the cell extract and the fusion polypeptide aggregates, as the aggregates appear to have less negative charge.

[0176] In some embodiments, anion exchange chromatography can be used to resolve polypeptides having different numbers of phosphorylated amino acid residues (e.g., differentially phosphorylated polypeptides). Anion exchange chromatography can enrich highly phosphorylated species of the fusion polypeptide preparation (e.g., fusion polypeptides having more than 6 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 produced by using an anion exchange chromatography step (e.g., as a first step).

[0177] In some embodiments, the step of purifying the phosphorylated form of the fusion polypeptide from the cell extract includes an anion chromatography capture step. In some embodiments, the anion chromatography capture step is the first capture step.

[0178] In some embodiments, anion exchange chromatography uses an ion exchange resin having covalently bound positively charged groups (such as quaternary amino groups). Commercially available anion exchange resins include Q Sepharose, DEAE Sepharose, TMAE, GigaCap Q 650M, and 650S. In some embodiments, binding a negatively charged biomolecule (e.g., the phosphorylated form of the fusion polypeptide) to the anion exchange material includes exposing the negatively charged biomolecule to the resin under appropriate conditions (e.g., pH / conductivity) such that the biomolecule is immobilized to the anion exchange resin through ionic interactions between the negatively charged biomolecule and one or more charged groups of the ion exchange material.

[0179] A wash step may involve passing an appropriate buffer through the chromatography resin to wash away unwanted materials, such as host cell proteins or host cell nucleotides. In some embodiments, the wash buffer can include altered conditions, such as pH value, conductivity, with the aim of dissociating impurities that non-specifically bind to the chromatography resin. In some embodiments, the wash step uses a mixture of equilibration and elution buffers.

[0180] Elution can be used to elute the phosphorylated form of the fusion polypeptide from a solid matrix (e.g., a positively charged resin). In some embodiments, a buffer that reduces the interaction between the anion exchange resin and the negatively charged agent (e.g., the phosphorylated fusion polypeptide) is used to elute the negatively charged agent (e.g., the phosphorylated form of the fusion polypeptide). In some embodiments, such elution buffers can have a higher salt concentration and / or a different pH value such that dissociation of the negatively charged agent from the chromatography resin is promoted.

[0181] In some embodiments, a gradient elution buffer (e.g., a buffer having an increasing salt concentration) is used to elute from an ion exchange (e.g., anion exchange) column. In some such embodiments, using such a gradient can allow for the separation of differentially phosphorylated polypeptides (i.e., separation of different phospho forms can be achieved).

[0182] In some embodiments, the buffer is, for example, 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 M NaCl, pH 7.1 over a predetermined period of time. In some embodiments, the linear gradient is carried out 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 longer. In some embodiments, the first anion exchange chromatography capture step uses Tris buffer. In some embodiments, the first anion exchange chromatography capture step is carried out at a pH value of about 6 to about 9 (such as about 7 to about 8).

[0183] In some embodiments, capture beads are used for the first anion exchange chromatography capture step. In some embodiments, the first step capture beads have a diameter of at least 50 μm (such as at least 55 μm, such as at least 60 μm, such as at least 65 μm, such as at least 70 μm, such as at least 75 μm). In some embodiments, the first capture beads are GigaCap Q 650M.

[0184] In some embodiments, the first anion exchange chromatography capture step comprises a immobilization step (e.g., binding the phosphorylated fusion polypeptide to a chromatography column), a pre-elution wash step, and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads with a particle size of about 50 to about 100 microns (average) (such as 75 microns (average)) are used to immobilize the phosphorylated fusion polypeptide. In some embodiments, an immobilization composition with a low salt concentration (e.g., using 0 M sodium chloride) is used during the immobilization of the phosphorylated fusion polypeptide to the chromatography column. In some embodiments, a pre-elution composition with an intermediate salt concentration similar to the salt concentration of the immobilization composition and the elution composition (e.g., using 215 mM sodium chloride) is used during the pre-elution wash step. In some embodiments, an elution composition with a high salt concentration (e.g., using 350 mM sodium chloride) is used during the elution of the phosphorylated fusion polypeptide from the chromatography column. In some embodiments, the first anion exchange chromatography capture step is carried out at a pH value in the range of about 7 to about 8 (e.g., 7.4). In some embodiments, a flow rate of 200 - 400 cm / h (e.g., 300 cm / h) is used.

[0185] Second chromatographic step

[0186] In some embodiments, the method according to the present disclosure comprises at least one hydrophobic interaction chromatography step. In some embodiments, the hydrophobic interaction step is performed after a first anion chromatography step. Without being limited by a particular theory, the phosphorylated form of the fusion protein according to the present disclosure has low hydrophobicity (e.g., due to its degree of phosphorylation and thus due to its charge), and the low hydrophobicity can be used to separate it 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) comprises less than 5% of aggregated fusion polypeptide, such as less than 4%, such as less than 3%, such as less than 3%, such as less than 2%, such as less than 1%, such as less than 0.9%, such as less than 0.8% of aggregated fusion polypeptide.

[0187] In some embodiments, the hydrophobic interaction step can separate product or process-related impurities (e.g., host cell proteins or aggregated product (e.g., fusion polypeptide aggregates)) from the phosphorylated form of the fusion polypeptide based on the difference in hydrophobic interaction between the phosphorylated fusion polypeptide and impurities with hydrophobic materials. In some embodiments, such a step can be referred to as a polishing step.

[0188] Examples of hydrophobic interaction resins include, but are not limited to, hydrophobic ligands, such as alkyl groups in the range of 2 to 8 carbon atoms, or aryl groups, such as phenyl groups. In some embodiments, binding a negatively charged agent (e.g., the phosphorylated form of the fusion polypeptide) to the hydrophobic interaction resin comprises exposing the biomolecule to the resin under appropriate conditions (pH / conductivity), whereby the biomolecule is immobilized to the hydrophobic resin through hydrophobic interaction between the biomolecule and the non-polar groups of the hydrophobic interaction material. Hydrophobic interaction binding typically occurs in the presence of a high concentration of salt (e.g., 1 to 1.8 M ammonium sulfate). In some embodiments, the phosphorylated form of the fusion polypeptide is immobilized at a high salt concentration (e.g., 1.4 M sodium sulfate). In some embodiments, the phosphorylated form of the fusion polypeptide is eluted with a linear gradient in the range of 1.4 M ammonium sulfate to 0 M ammonium sulfate.

[0189] The washing step may be required to pass an appropriate buffer through the chromatography resin to wash away unwanted materials, such as less hydrophobic host cell proteins. In some embodiments, the washing buffer can have a different pH value to facilitate the dissociation of low hydrophobicity agents from impurities non-specifically bound to the chromatography resin. In some embodiments, the washing step uses a mixture of equilibration and elution buffers.

[0190] The phosphorylated form of the fusion polypeptide can be eluted using elution from a solid matrix (e.g., a hydrophobic resin). In some embodiments, a buffer that reduces the interaction between the hydrophobic interaction resin and a negatively charged agent (e.g., the phosphorylated fusion polypeptide) is used to elute a low-hydrophobicity agent (e.g., the phosphorylated form of the fusion polypeptide) from the hydrophobic resin. In some embodiments, such an elution buffer may have a low salt concentration or a pH change to facilitate dissociation of the biomolecule from the chromatographic resin. In some embodiments, the phosphorylated form of the fusion polypeptide is eluted at a low salt concentration (e.g., 750 mM sodium sulfate).

[0191] In some embodiments, the hydrophobic interaction chromatography step includes a binding 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 with a particle size of about 50 to about 100 microns (average) (such as 75 microns (average)) are used to bind the phosphorylated fusion polypeptide. In some embodiments, a binding composition with a high salt concentration (e.g., using 1.4 M ammonium sulfate) is used during binding of the phosphorylated fusion polypeptide to the chromatography column. In some embodiments, an elution composition with a low salt concentration (e.g., using 740 mM ammonium sulfate) is used during elution of the phosphorylated fusion polypeptide from the chromatography column. In some embodiments, the hydrophobic interaction chromatography step is carried out at a pH value in the range of about 7 to about 8 (e.g., 7.4). In some embodiments, a flow rate of 200 - 350 cm / h (e.g., 275 cm / h) is used.

[0192] Third chromatographic step

[0193] In some embodiments, the method according to the present disclosure includes a first anion chromatography step and a second anion chromatography step. In some embodiments, the method according to the present disclosure includes a first anion chromatography step and a second anion chromatography step, wherein the first anion chromatography step is a capture step and the second anion chromatography step is a polishing step. In some embodiments, the second anion chromatography step is carried out after the hydrophobic interaction chromatography step.

[0194] In some embodiments, the method according to the present disclosure includes the following steps:

[0195] i) A first anion chromatography step;

[0196] ii) A hydrophobic interaction chromatography step; and

[0197] iii) A second anion chromatography step.

[0198] In some embodiments, capture beads are used for a second anion chromatography step. In some embodiments, the second step capture beads have a diameter of at most 50 μm, at most 45 μm, at most 40 μm, at most 35 μm. In some embodiments, the first capture beads are GigaCap Q 650S.

[0199] In some embodiments, the second anion chromatography capture step includes a capture step (e.g., binding the phosphorylated fusion polypeptide to a chromatography column), a pre-elution wash step, and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads having a particle size of about 10 to about 50 microns (average) (such as a particle size of 35 microns (average)) are used to capture the phosphorylated fusion polypeptide. In some embodiments, a capture composition having a low salt concentration (e.g., using 0 M sodium chloride) is used during binding of the phosphorylated fusion polypeptide to the chromatography column. In some embodiments, a pre-elution composition having an intermediate salt concentration similar to the salt concentrations of the capture composition and the elution composition (using, for example, 274 mM sodium chloride) is used during the pre-elution wash step. In some embodiments, an elution composition having a high salt concentration (e.g., using 355 mM sodium chloride) is used during elution of the phosphorylated fusion polypeptide from the chromatography column. In some embodiments, the first anion chromatography capture step is performed at a pH value in the range of about 7 to about 8 (e.g., 7.3). In some embodiments, a flow rate of 200 - 400 cm / h (e.g., 300 cm / h) is used.

[0200] One or more optional additional steps

[0201] In some embodiments, the methods according to the present disclosure include a virus inactivation step or a virus removal step. In some embodiments, the virus inactivation step or the virus removal step is performed before or after any of the chromatography steps described above herein. In one embodiment, the virus inactivation step or the 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 surfactant). In some embodiments, the virus inactivation step includes using a detergent because (without being limited by a particular theory) IL12 - ABP is sensitive and may aggregate at low pH values. In some embodiments, the virus inactivation step includes using a detergent selected from: myristyl dimethylamine N - oxide, TDAO, Triton X - 100, or polysorbate. In some embodiments, the virus removal step includes a filtration step.

[0202] Fusion polypeptide preparation

[0203] In some embodiments, the present disclosure particularly provides 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 high-purity preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises a mixture of both non-phosphorylated and phosphorylated forms of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises more phosphorylated fusion polypeptide than non-phosphorylated fusion polypeptide.

[0204] In some embodiments, the phosphorylated fusion polypeptide preparation comprises a fusion polypeptide having varying degrees of phosphorylation as described above herein.

[0205] In some embodiments, the fusion polypeptide preparation comprises a buffer. In some embodiments, the fusion polypeptide preparation comprises a Tris buffer having a pH of from about 7 to about 8. In some embodiments, the fusion polypeptide preparation comprises a salt (such as NaCl).

[0206] Fusion polypeptide composition

[0207] In some embodiments, the present disclosure particularly provides 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 high-purity composition of the phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide composition comprises a mixture of both non-phosphorylated and phosphorylated forms of the fusion polypeptide. In some embodiments, the fusion polypeptide composition comprises more phosphorylated fusion polypeptide than non-phosphorylated fusion polypeptide. In some embodiments, the fusion polypeptide composition comprises a fusion polypeptide having varying degrees of phosphorylation as described above herein.

[0208] In some embodiments, the fusion polypeptide composition comprises a buffer. In some embodiments, the buffer is a Tris buffer. The present disclosure particularly demonstrates that one or more Tris buffers are particularly useful for the stability of the IL-12 fusion polypeptide as described herein. By way of example, the present disclosure demonstrates that Tris buffers can provide surprising stability advantages relative to one or more alternative buffers (such as one or more histidine buffers). In some embodiments, the buffer is not a histidine buffer.

[0209] In some embodiments, the fusion polypeptide composition has a pH value of from about 6.5 to about 8. In some embodiments, the fusion polypeptide composition has a pH value of at most 7.5, such as at most 7.4, such as at most 7.7. In some embodiments, the fusion polypeptide composition has a pH value of at least 7, such as at least 7.1, such as at least 7.2, such as at least 7.3. Without wishing to be bound by any particular theory, a high pH value can cause deamidation of the IL-12 fusion polypeptide.

[0210] In some embodiments, the fusion polypeptide composition comprises a salt. Without wishing to be bound by any particular theory, the salt can act as a tonicity regulator in the composition and / or formulation and can help stabilize the IL-12 fusion polypeptide by stabilizing the structure of the molecule via ionic interactions. In some embodiments, the fusion polypeptide composition comprises a salt, wherein the concentration of the salt ranges from about 1 mM to about 750 mM, such as from about 10 mM to about 500 mM, such as from about 20 mM to about 100 mM, such as from about 30 mM to about 60 mM, such as from about 35 mM to about 55 mM. In some embodiments, the salt is NaCl or Na2SO4.

[0211] In some embodiments, the fusion polypeptide composition comprises a surfactant. In some embodiments, the fusion polypeptide composition comprises a hydrophilic surfactant. Compared to more hydrophobic surfactants, hydrophilic surfactants can interact more with charged molecules (such as the phosphorylated form of the IL-12 fusion polypeptide). This interaction may reduce the formation of visible particles after agitation, i.e., be more susceptible 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 ranges from about 0.0005% w / v to about 1% w / v, from about 0.005% w / v to about 0.1% w / v, from about 0.01% w / v to about 0.05% w / v, from about 0.015% w / v to about 0.2% w / v.

[0212] In some embodiments, the fusion polypeptide composition comprises L-methionine. The addition of methionine appears to have a positive effect on the stability of the IL-12 fusion polypeptide, significantly reducing the risk of oxidation and having a positive effect on preventing the production of high molecular weight substances (HMWS). In some embodiments, the fusion polypeptide composition comprises L-methionine, wherein the concentration of L-methionine ranges from about 1 mM to about 20 mM, such as from about 5 mM to about 15 mM.

[0213] In some embodiments, the fusion polypeptide composition comprises a disaccharide (e.g., sucrose or trehalose). In some embodiments, the fusion polypeptide composition comprises sucrose, wherein the concentration of sucrose ranges from about 100 mM to about 200 mM.

[0214] Pharmaceutical composition

[0215] In some embodiments, the pharmaceutical preparation comprises a fusion polypeptide-metal hydroxide complex as described herein. Exemplary pharmaceutical compositions are shown in Example 8. Such pharmaceutical preparations can be prepared by mixing a fusion polypeptide composition as described above herein with a metal hydroxide. In some embodiments, a fusion polypeptide-metal hydroxide complex as described herein is produced by contacting the fusion polypeptide composition with a metal hydroxide (such as aluminum hydroxide). In some embodiments, aluminum hydroxide is formulated in a gel. In some embodiments, aluminum hydroxide is formulated in water. In some embodiments, the concentration of the stock aluminum hydroxide preparation is 10 mg / mL.

[0216] In some embodiments, the ratio between the fusion polypeptide and the 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.

[0217] In some embodiments, the fusion polypeptide is contacted with the metal hydroxide for at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes, such as at least 25 minutes, such as at least 30 minutes, such as at least 40 minutes.

[0218] 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 (such as about 20 °C to about 25 °C).

[0219] 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 the components in the pharmaceutical preparation is similar to the concentration of the components in the fusion polypeptide composition. In some embodiments, the concentration of the components in the pharmaceutical preparation is lower compared to the concentration of the components in the fusion polypeptide composition.

[0220] In some embodiments, the pharmaceutical preparation comprises 0.25 mg / mL 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 aluminum hydroxide, and wherein the pH value of the composition is in the range of 6 to 8.

[0221] Characterization

[0222] In some embodiments, the present disclosure particularly provides techniques for characterizing fusion polypeptides (e.g., their phosphorylated or non-phosphorylated preparations) and / or complexes comprising such fusion polypeptides and metal hydroxides. Characterization can be performed during and / or after the preparation process. In some embodiments, a particular preparation process can be modified or terminated based on the characterization (e.g., if a particular preparation fails to meet one or more specifications). In some embodiments, such characterization can involve assessing one or more of the following: metal hydroxide retention, degree of phosphorylation, heterogeneity of phosphorylation, signaling activity, and / or efficacy.

[0223] Exemplary characterization of phosphate content

[0224] In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) of a fusion polypeptide (e.g., of the present disclosure) is characterized. A variety of methods can be used to measure 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 the malachite green assay. Without wishing to be bound by any theory, the malachite green assay is based on the quantification of the green complex formed between malachite green, molybdate, and free orthophosphate, which can be measured (e.g., using a spectrophotometer or plate reader).

[0225] In some embodiments, the degree of phosphorylation (e.g., 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.

[0226] In some embodiments, the phosphorylation heterogeneity of the fusion polypeptides and / or their formulations of the present disclosure is characterized. In some embodiments, the phosphorylation heterogeneity is a measure of the degree of phosphorylation within a given formulation of the fusion polypeptide. In some embodiments, the phosphorylation heterogeneity is a measure of the degree of phosphorylation of multiple formulations of the fusion polypeptide. In some embodiments, the phosphorylation heterogeneity is a measure of the position of specific phosphate groups on the polypeptide within a given formulation of the fusion polypeptide. In some embodiments, the phosphorylation heterogeneity is a measure of the position of specific phosphate groups on the polypeptide of multiple formulations of the fusion polypeptide.

[0227] A variety of techniques can be used to measure phosphorylation heterogeneity. For example, in some embodiments, the degree of phosphorylation can be determined by chromatographic methods. In some embodiments, the chromatographic methods include ion exchange chromatography. In some embodiments, for example, the chromatographic methods include analytical anion exchange chromatography. Anion exchange chromatography is a form of ion exchange in which negatively charged biomolecules (such as the phosphorylated forms of the fusion polypeptides disclosed herein) bind to a positively charged resin. In some embodiments, anion exchange chromatography can be used to resolve polypeptides with different numbers of phosphorylated amino acid residues (such as differentially phosphorylated polypeptides). Without wishing to be bound by any theory, polypeptide phosphorylation confers variability in the charge of the polypeptide, thus allowing the separation of differentially phosphorylated polypeptides using ion exchange chromatography (such as anion exchange chromatography). Elution from an ion exchange (such as anion exchange) column using a gradient elution buffer (such as a buffer with increasing salt concentration) allows the separation of differentially phosphorylated polypeptides. In some embodiments, the buffer is, for example, Tris buffer. In some embodiments, a linear gradient of Tris buffer is used. In some embodiments, the linear gradient of Tris buffer includes a linear gradient from 20 mM Tris, pH 7.1 to 20 mM Tris, 525 mM NaCl, pH 7.1 over a predetermined period of time. In some embodiments, the linear gradient is carried out for 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes or longer.

[0228] In some embodiments, the differentially phosphorylated polypeptide is dephosphorylated. In some embodiments, dephosphorylation includes using a phosphatase (e.g., λ phosphatase). In some embodiments, the fusion polypeptide is incubated with the phosphatase for a period of time at a temperature that permits the activity of the phosphatase and dephosphorylation of the fusion polypeptide. In some embodiments, dephosphorylation is carried out 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 is carried out for an incubation time of 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes or longer. In some embodiments, dephosphorylation is carried out for an incubation time of 25 - 65 minutes, 30 - 60 minutes, 35 - 55 minutes, 40 - 50 minutes, 30 - 65 minutes, 35 - 65 minutes, 40 - 65 minutes, 45 - 65 minutes, 50 - 65 minutes or 55 - 65 minutes.

[0229] In some embodiments, the differentially phosphorylated polypeptide is dephosphorylated prior to isolation. In some embodiments, the differentially phosphorylated polypeptides of the present disclosure are evaluated relative to an appropriate reference standard (e.g., the dephosphorylated and / or non - phosphorylated form of the fusion polypeptide).

[0230] In some embodiments, after isolating the differentially phosphorylated polypeptides (e.g., by ion - exchange chromatography), the amount of each differentially phosphorylated polypeptide is measured. In some embodiments, the amount of each differentially phosphorylated polypeptide is measured according to a variety of methods available in the art. In some embodiments, by way of example but not limitation, the differentially phosphorylated polypeptides are measured using malachite green assay, analytical ion - exchange, spectrophotometer, colorimetric assay, and / or western blotting.

[0231] Exemplary characterization of metal hydroxide retention

[0232] In some embodiments, the fusion polypeptides of the present disclosure form complexes with metal hydroxides (e.g., aluminum hydroxide) upon exposure. In some embodiments, the retention of the fusion polypeptides of the present disclosure on metal hydroxides (e.g., metal hydroxide retention) is characterized. A variety of methods can be used to measure metal hydroxide retention. In some embodiments, by way of example but not limitation, metal hydroxide retention can be measured by ellipsometry, surface plasmon resonance, waveguide lightmode spectroscopy, attenuated total internal reflection - infrared spectroscopy, circular dichroism spectroscopy (CD), total internal reflection - infrared spectroscopy (TIRF), and other high - resolution microscopy techniques.

[0233] In some embodiments, in vitro assays are used to characterize metal hydroxide retention. For example, a known concentration of a fusion polypeptide is mixed with an excess of metal hydroxide. The concentration of free, uncomplexed fusion polypeptide is quantified and compared to a standard curve to determine metal hydroxide retention. The concentration of free, uncomplexed fusion polypeptide can be evaluated by a variety of methods known to those of skill in the art. For example, but not limited to, in some embodiments, the free, uncomplexed fusion polypeptide is quantified by enzyme-linked immunosorbent assay (ELISA), Western blotting, bicinchoninic acid assay, or Bradford assay.

[0234] 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 forms a complex (e.g., is retained) with the metal hydroxide when mixed therewith.

[0235] Exemplary characterization of signaling activity

[0236] In some embodiments, the activity (e.g., signaling activity) of a fusion polypeptide (and / or its complex) as described herein is characterized. In some embodiments, the activity is characterized by evaluating the signaling activity (e.g., signaling ability) compared to an appropriate reference standard. An appropriate reference standard can be, for example, a wild-type polypeptide and / or a fusion polypeptide lacking a metal hydroxide-binding polypeptide.

[0237] A variety of methods can be used to evaluate signaling ability. In some embodiments, for example, in vitro- or in vivo-based activity assays are used to evaluate signaling ability.

[0238] In some embodiments, in vitro activity assays are used to evaluate signaling activity. In some embodiments, the in vitro activity assay includes measuring the activation or inhibition of downstream signaling of the fusion polypeptide. In some embodiments, measuring the activation or inhibition of downstream activity includes using a reporter (e.g., a reporter assay). In some embodiments, the reporter assay measures activity using a detectable molecule (e.g., a reporter) related to the activity of the fusion polypeptide.

[0239] In some embodiments, the reporter comprises a fluorescent, bioluminescent, and / or other detectable probes known to those skilled in the art. In some embodiments, the reporter comprises a genetic reporter. For example, the genetic reporter can be activated after signal transduction initiated from a polypeptide. For example, after activating the transcription of the genetic reporter, a detectable product or an enzyme can be used, which can be activated after adding a substrate to produce a detectable product and / or by-product. In some embodiments, the enzyme used according to the reporter assay is, for example, luciferase or alkaline phosphatase (e.g., secreted alkaline phosphatase, SEAP). In some such embodiments, the HEK-Blue-IL12 reporter assay is used.

[0240] In some embodiments, in vivo activity assays are used to evaluate signal transduction activity. In some embodiments, the fusion polypeptide is administered to a subject (e.g., a mouse, a non-human primate, a human, etc.) and the activity is evaluated. In some embodiments, for example, the activity is evaluated by measuring the activation or inhibition of the downstream signal transduction of the fusion polypeptide compared to an appropriate reference standard (e.g., the activity of the wild-type polypeptide). A variety of methods can be used to measure the activation or inhibition of the downstream signal transduction of the fusion polypeptide. For example but not limited to, differential gene expression, protein expression, and / or post-translational modification changes induced by the fusion polypeptide can be measured.

[0241] Exemplary efficacy characterization

[0242] In some embodiments, efficacy can be characterized according to a variety of available methods. In some embodiments, for example, the fusion polypeptide (or its complex) as described herein is administered to a subject (e.g., a mouse, a non-human primate, a human, etc.) (e.g., by intratumoral or peritumoral injection) and compared with an appropriate reference standard to determine efficacy. For example, an appropriate reference standard can be a wild-type polypeptide and / or a polypeptide lacking the metal hydroxide-binding polypeptide or having a non-binding (e.g., non-phosphorylated) state of the metal hydroxide-binding polypeptide.

[0243] In some embodiments, efficacy is determined preclinically in an animal model (e.g., in a mouse, a rat, a non-human primate, 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 an animal model with a tumor (e.g., a cancer animal model). In some embodiments, the cancer animal model is generated by inoculating the 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 tumor cells are inoculated into the clinically relevant region (e.g., the mammary fat pad) of the animal model.

[0244] In some embodiments, the fusion polypeptides of the present disclosure are administered to a cancer animal model. In some embodiments, a reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide-binding polypeptide) is administered to a cancer animal model. In some embodiments, various available predetermined efficacy measurements known in the art, such as tumor volume and / or percentage survival, are evaluated over time relative to an appropriate reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide-binding polypeptide).

[0245] In some embodiments, the efficacy of the fusion polypeptide is determined clinically. In some embodiments, the fusion polypeptide is administered to a subject having a tumor (e.g., by intratumoral, peritumoral injection, or administration into the tumor-draining lymph node). In some embodiments, various available predetermined efficacy measurements known in the art, such as tumor volume and / or percentage survival, are evaluated over time relative to a subject having a tumor who has received a reference standard (e.g., a treatment and / or placebo known in the art to have efficacy).

[0246] Use

[0247] Method of treatment

[0248] In one aspect, the present disclosure relates to methods of treating a subject having a medical disorder. In some embodiments, the present disclosure relates to methods of treating a subject having cancer (e.g., a subject having a tumor). Generally, the methods of treatment are intended to reduce tumor volume, reduce and / or prevent metastasis, prolong survival, and / or cure the disorder. Suitable subjects or individuals who receive the fusion polypeptides or fusion polypeptide-metal hydroxide complexes of the present disclosure include, for example, humans or other mammals (e.g., mice, rats, rabbits, dogs, horses, cats, pigs, or non-human primates) having a tumor (e.g., cancer).

[0249] In some embodiments, a method of treating a subject having a tumor comprises the steps of treating the subject with a complex comprising: a fusion polypeptide comprising an immunomodulatory polypeptide containing an immune agonist moiety, and a metal hydroxide-binding polypeptide; and a metal hydroxide. In some embodiments, a method of treating a subject having a tumor comprises administering a fusion polypeptide comprising: an immunomodulatory polypeptide containing an immune agonist moiety and a metal hydroxide-binding polypeptide, wherein the fusion polypeptide is formulated with a metal hydroxide.

[0250] In some embodiments, the complex as described herein is administered as a single therapy. In some embodiments, the complex as described herein is administered in combination with a second therapeutic agent. In some embodiments, the complex as described herein is administered to a subject who has received or is receiving therapy with at least one other therapeutic agent.

[0251] The fusion polypeptides and / or their complexes and / or compositions and / or formulations disclosed herein can be used, in particular, for treating a subject having a tumor. Non-limiting examples of diseases associated with tumors include cancer (such as carcinoma, sarcoma, metastatic disease, or hematopoietic neoplastic disorders). Tumors (including metastatic tumors) can arise from a variety of primary tumor types. By way of example but not limitation, in some embodiments, the tumor or metastatic tumor can arise from a primary kidney tumor (such as renal cell carcinoma), head and neck cancer (such as head and neck squamous cell carcinoma), prostate cancer, breast cancer (such as triple-negative), colon cancer, skin cancer (such as melanoma, Merkel cell carcinoma, cutaneous T-cell lymphoma, cutaneous squamous cell carcinoma, basal cell carcinoma), lung cancer (such as non-small cell lung cancer), and pancreatic cancer. Accordingly, the fusion polypeptides and their formulations disclosed herein, including fusion polypeptide metal hydroxide complexes and their formulations, can be administered to a subject having cancer.

[0252] Those skilled in the art will understand that the amount or therapeutically effective amount of the fusion polypeptide-metal hydroxide complex, fusion polypeptide, or its formulation sufficient to reduce tumor growth and decrease tumor size will vary not only according to the particular compound or formulation selected, but also with the route of administration, the nature of the disorder being treated, and the age and condition of the patient, and will ultimately be determined by the patient's physician or pharmacist and / or based on clinical guidelines. The length of time for which the compounds used in the methods of the invention will be administered will vary according to the individual and / or according to clinical guidelines.

[0253] In some embodiments, a method of treating a subject having a tumor (such as cancer) includes the step of treating the subject with a complex comprising a fusion polypeptide and a metal hydroxide, the fusion polypeptide comprising an immunomodulatory polypeptide containing an immune agonist moiety and a metal hydroxide-binding polypeptide. In some embodiments, the fusion polypeptide and the metal hydroxide are formulated together. By way of example, formulating together includes pre-forming a 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.

[0254] In some embodiments, a method of treating a subject having a tumor (such as cancer) includes 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 (such as cancer) includes 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 (such as cancer) includes treating the subject with a complex, wherein the complex is administered to one or more tumor-draining lymph nodes.

[0255] The methods of the present invention often involve administering a therapeutically effective amount of a particular agent. A therapeutically effective amount is an amount sufficient to achieve a desired biological or medical response or therapeutic effect in a tissue, system, or subject (in principle, for subjects with similar characteristics, such as species, body size, dimensions, degree of disease or disorder, degree or type of symptoms, history of response, and / or general health). By way of example, the desired response may include one or more of the following: delaying or preventing the onset of a medical condition, disease, or disorder; slowing or halting the progression, exacerbation, or worsening of the symptoms of a condition; ameliorating the symptoms of a condition; and curing a condition.

[0256] When combinations of therapeutic agents are administered, the amount of any individual agent required in the combination may be different from the amount required for the same agent to achieve its therapeutic effect alone. In some cases, synergistic effects between the therapeutic agents used in the combination may result in a reduced amount being required; in other cases, inhibitory interactions may result in an increased amount being required. Thus, generally, the therapeutically effective amount of a combination of agents may use absolute amounts of the agents that are different from the therapeutically effective amounts of the agents that individually constitute the combination.

[0257] Combination therapy

[0258] In some embodiments, the fusion polypeptide metal hydroxide complex or a formulation thereof as disclosed herein is administered in combination with other therapies. By way of example, in some embodiments, the IL-12 complex is used in combination with another immunotherapy. Exemplary immunotherapies include, but are not limited to, chimeric antigen receptor (CAR) T cell therapy, tumor-associated antigen-targeting antibodies, immune checkpoint inhibitors, and cancer vaccines. In some embodiments, an immune response is induced or stimulated by antagonizing 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.

[0259] The second therapeutic agent may be selected from a variety of anti-tumor agents known and available in the art. In some embodiments, the second therapeutic agent is administered before the fusion polypeptide metal hydroxide complex. In some embodiments, the second therapeutic agent is administered in parallel with the fusion polypeptide metal hydroxide complex. In some embodiments, the second therapeutic agent is administered after the fusion polypeptide metal hydroxide complex.

[0260] 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 between about 1 Gy and about 1,000 Gy, about 5 Gy and 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, between about 5 Gy and about 15 Gy, between about 7.5 Gy and about 12 Gy, or between about 10 Gy and 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 1,000 Gy. In some embodiments, the amount of ionizing radiation is less than about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 Gy.

[0261] For example, in some embodiments, the second therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent can be a targeted therapy (e.g., BRAF inhibitor, MEK inhibitor, etc.). In some embodiments, the chemotherapeutic agent can be any approved chemotherapeutic agent. By way of example and not limitation, the chemotherapeutic agent can be one or more of the following: 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 known regimen in the art.

[0262] For example, in some embodiments, 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 the following: 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 can be administered according to any approved and / or known regimen in the art.

[0263] 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, which tissue may include, for example, residual tumor (e.g., tumor cells). In some embodiments, the fusion polypeptide metal hydroxide complex is administered to tissue that cannot be removed by surgical tumor resection or to tissue proximal to the resection site during the resection.

[0264] For example, in some embodiments, the second therapeutic agent is or comprises cell therapy. In some embodiments, the cell therapy is or comprises natural killer (NK) cells. In some embodiments, the cell therapy is or comprises tumor infiltrating lymphocytes (TIL). In some embodiments, the cell therapy is or comprises cells expanded ex vivo. In some embodiments, the cell therapy is or comprises chimeric antigen receptor (CAR) effector cell therapy (e.g., CAR T cells). A CAR is a genetically engineered artificial transmembrane receptor that confers on an immune effector cell (e.g., a T cell, natural killer cell, or other immune cell) a selected specificity for a selected ligand and causes activation of the effector cell upon recognition and binding to the ligand. Typically, such ligand specificity is achieved by engineering the antigen specificity of a monoclonal antibody into the CAR such that the CAR T cells target the antigen recognized by the antibody.

[0265] In some embodiments, the effector cells expressing chimeric antigen receptors (e.g., CAR-T cells) are cells derived (e.g., isolated) from a patient having a disease or disorder and are genetically modified ex vivo to express at least one CAR having any specificity for a ligand. The cells perform at least one effector function (e.g., cytokine induction), which is stimulated or induced by ligand-specific binding to the CAR and can be used to treat the disease or disorder of the same patient. The effector cells can be T cells (e.g., cytotoxic T cells or helper T cells). After reading this disclosure, those skilled in the art will understand that in some embodiments, cells other than T cells (e.g., natural killer cells, stem cells, etc.) can be engineered to express CARs such that the chimeric antigen receptor effector cells can include effector cells other than T cells. In some embodiments, the CAR effector cells are T cells (e.g., cytotoxic T cells); in some embodiments, such CAR-T cells exert their effector function (e.g., cytotoxic T cell response) on target cells (e.g., cancer cells) when in contact with or in proximity to the target or target cells (see, e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450). In some embodiments, cell therapy (e.g., CAR effector cell therapy) uses tumor-infiltrating lymphocytes (TILs). TILs target cancer cells. In some embodiments, TILs are isolated from a subject having cancer and expanded ex vivo. In some such embodiments, TILs are isolated and expanded ex vivo after surgical resection of the tumor. In some embodiments, before administering the TILs, the subject is treated with a lymphodepleting conditioning regimen (Rohaan, Maartje W et al “Adoptive cellular therapies: the current landscape.” Virchows Archiv: an international journal of pathology Vol. 474, 4 (2019):449-461).

[0266] In some embodiments, cell therapy (e.g., CAR effector cell therapy) uses natural killer (NK) cells. Natural killer (NK) cells are an important part of tumor immune surveillance, as evidenced by higher cancer susceptibility and metastasis associated with reduced NK activity in mouse models and clinical studies. In some embodiments, for example, using a germline-encoded surface receptor array, NK cells are able to recognize and rapidly act on 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)).

[0267] In some embodiments, the fusion polypeptide metal hydroxide complex or a formulation thereof as disclosed herein is administered to a subject who has received or is receiving therapy with at least one other therapeutic agent. The other therapeutic agent can be selected from a variety of anti-tumor agents known in the art. In some embodiments, the other therapeutic agent is administered before the fusion polypeptide metal hydroxide complex. In some embodiments, the other therapeutic agent is administered in parallel with the fusion polypeptide metal hydroxide complex. In some embodiments, the other therapeutic agent is administered after the fusion polypeptide metal hydroxide complex.

[0268] For example, in some embodiments, the other therapeutic agent is radiation (e.g., ionizing radiation). In some embodiments, the amount of ionizing radiation administered is between about 1 Gy and about 1,000 Gy, about 5 Gy and 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, between about 5 Gy and about 15 Gy, between about 7.5 Gy and about 12 Gy, or between about 10 Gy and 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 1,000 Gy. In some embodiments, the amount of ionizing radiation is less than about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 Gy.

[0269] For example, in some embodiments, the other therapeutic agent is a chemotherapeutic agent. In some embodiments, the other therapeutic agent is or comprises a targeted therapy (e.g., BRAF inhibitor, MEK inhibitor, etc.). In some embodiments, the chemotherapeutic agent can be any approved chemotherapeutic agent. By way of example and not limitation, the chemotherapeutic agent can be one or more of the following: doxorubicin, anastrozole, cyclophosphamide, docetaxel, doxifluridine, adriamycin, erlotinib, fluorouracil, gemcitabine, imatinib, gefitinib, letrozole, methotrexate, paclitaxel, tarceva, and trastuzumab. The chemotherapeutic agent can be administered according to any approved and / or known protocol in the art. In some embodiments, the other 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 the following: 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 can be administered according to any approved and / or known protocol in the art. For example, in some embodiments, the other therapeutic agent is or comprises a cell therapy. In some embodiments, the cell therapy is or comprises chimeric antigen receptor (CAR) effector cell therapy (e.g., CAR T cells). A CAR is a genetically engineered artificial transmembrane receptor that confers on an immune effector cell (e.g., a T cell, natural killer cell, or other immune cell) a selected specificity for a selected ligand and causes the effector cell to become activated upon recognition and binding to the ligand. Generally, such ligand specificity is achieved by engineering the antigen specificity of a monoclonal antibody into the CAR such that the CAR T cells target the antigen recognized by the antibody.

[0270] In some embodiments, the effector cells expressing chimeric antigen receptors (e.g., CAR-T cells) are cells derived (e.g., isolated) from a patient having a disease or disorder and are genetically modified ex vivo to express at least one CAR having any specificity for a ligand. The cells perform at least one effector function (e.g., induce cytokines), which is stimulated or induced by ligand-specific binding to the CAR and can be used to treat the disease or disorder of the same patient. The effector cells can be T cells (e.g., cytotoxic T cells or helper T cells). After reading this disclosure, those skilled in the art will understand that in some embodiments, cells other than T cells (e.g., natural killer cells, stem cells, etc.) can be engineered to express a CAR such that the chimeric antigen receptor effector cells can include effector cells other than T cells. In some embodiments, the CAR effector cells are T cells (e.g., cytotoxic T cells); in some embodiments, such CAR-T cells exert their effector function (e.g., cytotoxic T cell response) on target cells (e.g., cancer cells) when in contact with or in proximity to the target or target cells (see, e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450). In some embodiments, cell therapy (e.g., CAR effector cell therapy) uses tumor-infiltrating lymphocytes (TILs). TILs target cancer cells. In some embodiments, TILs are isolated from a subject having cancer and expanded ex vivo. In some such embodiments, TILs are isolated and expanded ex vivo after surgical resection of the tumor. In some embodiments, prior to administration of TILs, the subject is treated with a lymphodepleting conditioning regimen (Rohaan, Maartje W et al “Adoptive cellular therapies: the current landscape.” Virchows Archiv: an international journal of pathology Vol 474, 4 (2019):449-461).

[0271] In some embodiments, cell therapy (e.g., CAR effector cell therapy) uses natural killer (NK) cells. Natural killer (NK) cells are an important part of tumor immune surveillance, as evidenced by higher cancer susceptibility and metastasis associated with reduced NK activity in mouse models and clinical studies. In some embodiments, for example, using a germline-encoded surface receptor array, NK cells are able to recognize and rapidly act on 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)).

[0272] In some embodiments, cell therapy (e.g., CAR effector cell therapy) includes myeloid cells. In some embodiments, the myeloid cells are or include macrophages. Macrophages have been shown to take up alum.

[0273] Table 1 provides exemplary amino acid sequences of the polypeptides described herein.

[0274] Table 1: Exemplary Amino Acid Sequences

[0275]

[0276]

[0277]

[0278]

[0279] Table 2. Exemplary Nucleic Acid Sequences

[0280]

[0281]

[0282]

[0283]

[0284] Illustration

[0285] In an example, NKY-001 can be used interchangeably with the IL-12 fusion polypeptide.

[0286] Example 1: Exemplary Fusion Polypeptide Stock Preparation

[0287] This example presents an exemplary fusion polypeptide stock preparation of a phosphorylated form of a fusion polypeptide as described herein. Exemplary amino acid sequences of the interleukin-12 fusion polypeptide are shown in Table 1, and exemplary nucleotide sequences are shown in Table 2.

[0288] The bulk purified fusion agent is supplied in 10 mM Tris, approximately 500 mM NaCl, pH 7.4 and at a concentration of approximately 11 g / L, see Table 3 and stored at ≤ -65 °C until formulated.

[0289] Table 3. Bulk formulation of interleukin-12 fusion polypeptide.

[0290]

[0291] The following chemicals and excipients are used (Table 4).

[0292] Table 4. Chemicals and excipients

[0293]

[0294]

[0295] Various IL-12 fusion polypeptide compositions are formulated using different components (such as buffers, surfactants, excipients) and different pH conditions. The evaluation of such formulations is described below (see Examples 2-4). The compositions are stored for a period of time - for example, six (6) weeks or longer, and in some cases twelve (12) weeks or longer, as described in Examples 2-4 below.

[0296] Each evaluated composition tested in Examples 2-4 contains an IL-12 fusion polypeptide at a concentration in the range of approximately 0.5 mg / mL to approximately 3.5 mg / mL (for example, approximately 2 mg / mL). Without wishing to be bound by any particular theory, when a composition containing a phosphorylated form of the fusion polypeptide is later mixed with a metal hydroxide (such as aluminum hydroxide) and thereby forms a fusion polypeptide metal hydroxide complex, a concentration of approximately 2 mg / mL of the IL-12 fusion polypeptide is a suitable concentration.

[0297] Example 2: Exemplary fusion polypeptide stock composition

[0298] This example confirms that the degree of stability of the IL-12 fusion polypeptide is affected by the pH level and / or type of buffer used in the IL-12 fusion polypeptide composition. For example, this example confirms that the IL-12 fusion polypeptide is stable in a Tris buffer composition at a pH of approximately 7-8 (for example, approximately 7.3-7.4). This example also presents an exemplary composition used according to the invention.

[0299] Compositions to be evaluated

[0300] Eight different buffer / pH conditions were evaluated in eight different compositions (F1 - F8), see Table 5. Tris buffer and His / HisHCl buffer were evaluated in the stability assessment. The NaCl concentration (if present) was 50 mM or 100 mM.

[0301] Table 5. Formulations of compositions to be evaluated

[0302]

[0303] The compositions (F1 - F8) as described in Table 5 were prepared by buffer exchange to achieve the target buffer concentration and pH value. The protein concentration, osmotic pressure, and pH value of each composition were determined. All composition solutions were filtered using a 0.22 μm polyvinylidene fluoride (PVDF) membrane filter.

[0304] Primary packaging materials were prepared according to standard procedures, and each composition was manually (observing aseptic technique) transferred into 2R / 13 mm Type I glass vials with a target fill volume of 1.0 mL, stoppered with a 13 mm bromobutyl rubber stopper (injection stopper), and sealed with a 13 mm aluminum flip-off seal. Samples of all compositions were labeled and stored at 5 ± 3 °C until dispensed for stability assessment.

[0305] According to Table 6, test samples of each composition were dispensed into the stability chamber in a vertical position. Vials were obtained from different compositions at the following time points at the selected temperatures of 5 °C, 25 °C, and 40 °C: 0 (initial / T0 / frozen starting material), 1 week (T1W), and 2 weeks (T2W). See Table 4 for specific vial dispensing.

[0306] Table 6. Vial dispensing.

[0307]

[0308]

[0309] At the initial and subsequent time points, test samples of each composition were withdrawn and evaluated as follows:

[0310] · Visible particles (black and white) at all stability time points.

[0311] · Solution clarity and turbidity (turbidity) at all stability time points.

[0312] · pH value determination at all stability time points.

[0313] · Osmotic pressure determined by freezing point depression at T0 / initial.

[0314] ·All stability time points are based on the protein content of SoloVPE.

[0315] ·All stability time points are based on the purity of size exclusion-HPLC.

[0316] ·All stability time points are based on the purity of reverse phase-HPLC (CR-HPLC) (reduced and non-reduced conditions).

[0317] ·CE-SDS (chip-based) for F5 and F6, 2 weeks at 25 °C.

[0318] The composition after mixing

[0319] The pH value, protein concentration, and osmotic pressure of each composition after mixing and filtration were determined. The results are shown in Table 7. Also included is the protein concentration measured by UV spectrophotometer (A280) at the starting time point of the stability study.

[0320] ·The pH value and protein concentration of each composition (F1 - F8) were close to the desired values

[0321] ·As expected for each of the compositions, the osmotic pressure results showed a wide range

[0322] ·All compositions were colorless and free of visible particles after mixing and filtration.

[0323] Table 7. Characterization results of the compositions after mixing.

[0324]

[0325] a Values obtained from the T0 results;

[0326] Stability study

[0327] No significant changes were observed during the 2-week stability assessment; no visible particles were observed, and the pH value and protein concentration were stable. The turbidity showed values between 0 and 1.

[0328] When analyzed by RP-HPLC, the composition containing histidine buffer with a lower pH value (F5, pH 5.5) showed a decrease in the main peak, and subsequently an increase in HMWS of approximately 5% at 40 °C (see Figures 3A to 3B ). In contrast, the compositions containing Tris buffer with a higher pH value (F1 - F4) were stable over time and at different temperatures. Thus, the present disclosure confirms that Tris buffer can be used to effectively formulate IL-12 fusion polypeptide compositions as described herein, while other buffer systems (such as histidine buffer) may not be suitable for such purposes.

[0329] More specifically, the present disclosure has confirmed that while no significant differences were observed by HPLC between the compositions under non-reducing conditions, under reducing conditions, a certain loss of the main peak was observed for the histidine buffer compositions with lower pH values. Specifically, after 2 weeks at 40 °C, F5 (at pH 5.5) showed a loss of more than 20% of the main peak and F6 (pH 6.0) showed a loss of approximately 17% of the main peak, which caused an increase in peak A (group) in both cases. At 40 °C, compositions F7 and F8 in histidine buffer with a pH of 6.5 showed a more moderate decrease in the main peak (about 8%). The compositions in Tris buffer (pH 7.4 and pH 8.0) showed little change over time (and / or under conditions of temperature stress). Thus, the present disclosure has documented the surprising stability of such Tris buffer compositions.

[0330] After 2 weeks at 25 °C, CE-SDS (chip-based) was performed on only 2 samples: F5, F6. When compared to T0 (initial / frozen starting material) (i.e., 100% intact), the non-reduced samples showed no differences and the reduced form was also still very similar to T0.

[0331] The stability data up to 2 weeks indicate that the interleukin-12 metal-binding polypeptide fusion agent is more stable in Tris buffer at a pH of about 7.4 compared to histidine buffer (evaluated at pH 5.5 to 6.5).

[0332] Conclusion

[0333] Particularly stable compositions were produced in 20 mM Tris buffer at a pH of about 7 - 8 (e.g., about 7.4).

[0334] Example 3: Influence of surfactant on stability

[0335] This example has confirmed that an IL-12 fusion polypeptide composition containing a specific polysorbate (polysorbate-20) protects the IL-12 fusion polypeptide from instability triggered by agitation stress (without polysorbate 20, the IL-12 fusion polypeptide is susceptible to agitation). This example has specifically determined that polysorbate-20 is unexpectedly even more effective than another polysorbate surfactant (i.e., polysorbate-80) in alleviating the formation of visible particles after agitation. This example presents an exemplary IL-12 fusion polypeptide composition for use according to the present invention.

[0336] Compositions evaluated

[0337] Five different compositions were evaluated: polysorbates 20 and 80 at two concentration levels (0.02% (w / v) and 0.04% (w / v)), and a fifth composition without any polysorbate was evaluated as a control, see the compositions in Table 8.

[0338] The compositions evaluated included 2 mg / ml interleukin-12 metal-binding polypeptide fusion agent, 20 mM Tris buffer (pH 7.3). 50 mM NaCl was added to obtain additional stabilization and 10 mM L-methionine was added to reduce the risk of possible oxidation.

[0339] Table 8. Compositions evaluated

[0340]

[0341]

[0342] The compositions as described in Table 8 were prepared by buffer exchange to achieve the target buffer concentration and pH value. The protein concentration, osmotic pressure, and pH value of each composition were determined. All composition solutions were filtered using a 0.22 μm polyvinylidene fluoride (PVDF) membrane filter.

[0343] Primary packaging materials were prepared according to standard procedures, and each composition was manually (observing aseptic technique) transferred into 2R / 13 mm Type I glass vials with a target fill volume of 1.0 mL, stoppered with a 13 mm bromobutyl rubber stopper (injection stopper), and sealed with a 13 mm aluminum flip-off seal. Samples of all compositions were labeled and stored at 5 ± 3 °C until dispensed for stability evaluation.

[0344] At low temperature (5 °C) and room temperature (25 °C) conditions, in a reciprocating (horizontal) shaker at a target speed of 200 rpm, two test samples of each composition were subjected to shaking stress for 2 and 5 days respectively in the horizontal position. Additionally, two test samples of each composition were subjected to three and five freeze / thaw cycles from -65 °C or below to room temperature respectively in the vertical position. See Table 9 for specific vial dispensing.

[0345] Table 9. Vial dispensing

[0346]

[0347] At the initial and subsequent time points, test samples of each composition were withdrawn and evaluated as follows:

[0348] · Visible particles (black and white) at all stability time points.

[0349] · Solution clarity and turbidity (cloudiness) at all stability time points.

[0350] · Color measurements using a colorimeter were performed at all stability time points.

[0351] · Subvisible particles were determined at all stability time points according to the light obscuration (LO) low volume method.

[0352] · pH measurements were performed at all stability time points.

[0353] · Osmotic pressure determined from the freezing point depression was measured at all stability time points.

[0354] · Protein content according to SoloVPE was measured at all stability time points.

[0355] · Purity according to size exclusion - HPLC was measured at all stability time points.

[0356] · Determination of polysorbate 20 / 80 content by fluorescence micelle assay (HPLC) was performed at all stability time points.

[0357] · Purity according to reverse - phase - HPLC (CR - HPLC) (reducing and non - reducing conditions) was measured at all stability time points.

[0358] · CE - SDS (chip - based)

[0359] The composition after compounding

[0360] The pH value, protein concentration, and osmotic pressure of each composition after compounding and filtration were measured. The results are shown in Table 10. For completeness, the protein concentration measured by UV spectrophotometer (A280) at the starting time point of the stability study was also included.

[0361] · The pH value and protein concentration of each composition (F1 - F5) were close to the desired values

[0362] · As expected for each of the compositions, the osmotic pressure results showed a wide range

[0363] · All compositions were colorless and free of visible particles after compounding and filtration.

[0364] Table 10. Characterization results of the compositions after compounding.

[0365]

[0366] a Values obtained from the T0 results

[0367] Freeze - thaw and agitation studies

[0368] All compositions were subjected to 3 and 5 freeze - thaw cycles (-65 °C to room temperature), and subjected to oscillatory stress at ambient temperature and low temperature for 2 days and 5 days, respectively.

[0369] No visible particles were observed in the compositions containing polysorbate (F1 - F4). However, after oscillatory stress, F5 showed a certain degree of decrease in the main peak (IL - 12 fusion polypeptide), which translated into an increase in HMWS (an increase of about 8% after 2 days of oscillation and 15% after 5 days of oscillation) ( Figures 4A to 4B ). Thus, after 5 days of oscillatory stress (at both temperatures) and after 2 days of oscillation at room temperature, many particles were observed in F5 (without polysorbate).

[0370] For any of the compositions (F1 - F5), the freeze - thaw stress did not seem to have an effect on the interleukin - 12 metal - binding polypeptide fusion agent. However, when compared with F1 (PS80 0.02%), F2 (PS80 0.04%) showed a slightly higher HMWS response.

[0371] No changes in pH value and protein concentration were observed in the study. Turbidity and color remained stable at 1 nephelometric turbidity unit (NTU) and B9, respectively. For sub - visible particles, some variability was observed, but the values remained in an overall low range (even for the control composition without surfactant). In RP - HPLC, only small variability was observed under non - reducing conditions. Under reducing conditions, a small decrease in peak A was observed for the stressed samples, which was reflected in an increase in the main peak (about 0.5% to 1%). In the determination of polysorbate content by general fluorescence micelle assay (FMA), no relevant changes were observed after stress conditions.

[0372] Conclusion

[0373] Overall, polysorbate protects the interleukin - 12 metal - binding polypeptide fusion agent from instability that can be triggered by oscillatory stress. A higher concentration of polysorbate (0.04%) does not seem to be beneficial for the IL - 12 fusion polypeptide compared to a lower concentration (0.02%). When polysorbate is present in the composition, no real difference was observed between the two stress durations (2 days and 5 days).

[0374] Example 4: Influence of other composition components on stability

[0375] This example demonstrates that methionine has a positive effect on the stability of the interleukin-12 metal-binding polypeptide fusion agent, particularly on preventing the formation of high molecular weight species (HMWS). This example demonstrates that the evaluated IL-12 fusion polypeptide compositions can be stored at 2 - 8°C. This example also presents exemplary IL-12 fusion polypeptide compositions used according to the present invention.

[0376] Evaluated compositions

[0377] The addition of methionine and sucrose was tested against trehalose in compositions containing polysorbate 20 or 80 (0.02% (w / v)), see the compositions in Table 11. Surfactant evaluations were performed using compositions containing 2 mg / ml interleukin-12 metal-binding polypeptide fusion agent, 20 mM Tris buffer (pH 7.0, 7.3 or 7.6). For all candidate compositions, the buffer substances NaCl (50 mM) and L-methionine (10 mM) were kept constant. Six different compositions (F1 - F6) were tested.

[0378] Table 11. Components of evaluated compositions

[0379]

[0380]

[0381] The compositions as described in Table 11 were prepared by buffer exchange to achieve the target buffer concentration and pH value. The protein concentration, osmotic pressure, and pH value of each composition were determined. All composition solutions were filtered using a 0.22 μm polyvinylidene fluoride (PVDF) membrane filter.

[0382] Primary packaging materials were prepared according to standard procedures, and each composition was manually (observing aseptic technique) transferred into 2R / 13 mm Type I glass vials with a target fill volume of 1.0 mL, stoppered with a 13 mm bromobutyl rubber stopper (injection stopper), and sealed with a 13 mm aluminum flip-off seal. Samples of all compositions were labeled and stored at 5 ± 3°C until dispensed for stability evaluation.

[0383] Test samples of each composition were subjected to shaking stress at a target speed of 200 rpm in a reciprocating (horizontal) shaker at room temperature for approximately 5 days. Additionally, test samples of each composition were subjected to five freeze / thaw cycles from -65°C or below to room temperature in the vertical position. The vials were maintained at the 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 assignments.

[0384] Table 12. Vial Dispensing

[0385]

[0386]

[0387] At initial and subsequent time points, test samples of each composition are withdrawn and evaluated as follows:

[0388] · Visible particles at all stability time points.

[0389] · Solution clarity and turbidity (nephelometry) at all stability time points.

[0390] · Color measurement using a colorimeter at all stability time points.

[0391] · Subvisible particles according to the light obscuration (LO) low volume method at all stability time points.

[0392] · pH measurement at all stability time points.

[0393] · Osmotic pressure determined from freezing point depression at all stability time points.

[0394] · Protein content according to SoloVPE at all stability time points.

[0395] · Purity according to size exclusion - HPLC at all stability time points.

[0396] · Purity according to anion exchange chromatography (AEX) at all stability time points, except for FT (freeze / thaw) and agitation.

[0397] · Polysorbate 20 / 80 content determination according to fluorescence micellar assay (HPLC) at all stability time points.

[0398] · Purity according to reverse phase - HPLC (CR - HPLC) (reduced and non - reduced conditions) at all stability time points.

[0399] In addition, at baseline for each composition (stored at - 65 °C), 200 μl aliquots are taken for alum binding assay, 100 μl aliquots are taken for potency assay, and 100 μl aliquots are taken for ELISA.

[0400] The composition after compounding

[0401] The pH value, protein concentration, and osmotic pressure of each composition were determined after compounding and filtration. The results are shown in Table 13. For completeness, the protein concentration measured by UV spectrophotometer (A280) at the starting time point of the stability study was also included.

[0402] · The pH value and protein concentration of each composition (F1 - F5) were close to the desired values

[0403] · As expected for each of the compositions, the osmotic pressure results showed a wide range

[0404] · All compositions were colorless and free of visible particles after compounding and filtration.

[0405] Table 13. Characterization results of the compositions after compounding.

[0406]

[0407] a Values obtained from the T0 results

[0408] Freeze - thaw and oscillation studies

[0409] Compared with the initial non - stressed samples, all compositions subjected to 5 freeze - thaw cycles (-65 °C to room temperature) or oscillation stress at ambient temperature did not show any relevant changes in any of the analytical methods, indicating that the compositions effectively stabilized the IL - 12 polypeptide fusion agent against freeze - thaw and oscillation stresses. After 5 days of oscillation stress, only F1 and F5 seemed to have slightly higher particle counts.

[0410] Short - term stability study (thermal stress): stored at -20 °C, 5 °C, 25 °C, 40 °C

[0411] No visible particles were observed in any of the compositions, the turbidity was stable (0 to 1 NTU), and the color was in the range from colorless to B9. The protein concentration and pH value remained stable over time.

[0412] The number of sub - visible particles was generally low. Sub - visible particles ≥25 μm were detected in very few samples (and only 1 or 2 particles).

[0413] For all compositions analyzed using the SEC method, no significant differences in the percentage of the main peak over time were observed at temperatures up to 25 °C. After 6 weeks at 40 °C, some LMWS (≤0.1%) appeared and the HMWS showed an increase of about 0.3% compared to T0 of F1, and up to 0.5% for F4 and F5.

[0414] In RP-HPLC, reducing conditions appear to be more indicative of stability than non-reducing conditions. Some signs of degradation were observed at 40 °C, especially for F5 (without L-methionine) (5% loss of the main peak after 6 weeks), indicating that methionine stabilizes the fusion polypeptide.

[0415] In the polysorbate content determination, only minor variability was observed for the samples containing PS20. Samples containing PS80 (F2 and F5) showed some degree of degradation at 40 °C.

[0416] The T12W time point was withdrawn for the designated composition (F1) to judge the expected storage conditions (refrigeration vs. freezing at -20 °C).

[0417] Long-term (12-week time point) stability study of the designated composition (F1)

[0418] Analysis of only one additional time point was performed for the designated composition (F1) to support the determination of storage conditions.

[0419] After 12 weeks, no visible particles were observed at any storage temperature. No differences were observed in turbidity (remaining at 1 NTU), color, pH, and protein concentration. Less than or equal to 25 subvisible particles with a diameter greater than or equal to 10 μm / mL were observed in the composition, and less than or equal to 3 subvisible particles with a diameter greater than or equal to 10 μm / mL were observed in the composition.

[0420] By SEC, only the 40 °C samples showed some degree of main peak reduction (1%), which was reflected in an increase in HMWS (an increase of about 1.0 - 1.2% compared to T0 and the frozen analysis reference) and the appearance of 0.1% LWMS.

[0421] Non-reducing RP-HPLC of the composition showed no relevant changes. Under reducing conditions, a main peak reduction (about 10 - 13%) was observed upon storage at 40 °C, which was reflected in a corresponding increase in peak A (about 10 - 11%).

[0422] No decrease in polysorbate content was observed under any storage conditions.

[0423] Overall, no differences were observed between the 5 °C and -20 °C storage conditions for all purity methods; the potential differences in SVP counts may be related to the method variability inherent in the particle counting method.

[0424] In summary, the tested interleukin-12 metal-binding polypeptide fusion agent composition can be stored at 2 - 8 °C.

[0425] Sample analysis by the method developed through anion exchange chromatography (AEX)

[0426] The method uses a phosphatase kit for digestion during sample preparation and then analyzes by HPLC. FT and oscillatory stress are not analyzed by AEX. Composition samples for short-term stability studies are frozen after withdrawal, and F1 is analyzed together before and after a 12-week withdrawal time, in sequence according to time points, and includes frozen analysis controls. AEX runs in the form of repeated injections, and some variability is observed.

[0427] During the implementation run, the profiles obtained in DPS look similar to the profiles known from assay development, with two highly similar peaks separated by a valley. Results are reported for a main peak and a second (earlier) peak labeled as acidic peak 1. This profile is generally maintained during formulation studies, with only minor differences between compositions. Samples stored at -20 °C, 5 °C, and 25 °C produce similar profiles, possibly showing a slight shift in peak height towards the earlier peak (e.g., at 25 °C for 6 weeks). In contrast, in all compositions, samples stored at 40 °C start to show a loss of peak resolution at 3 weeks (still two peaks, but with a less distinct valley), and at 6 weeks, the signal can no longer be integrated into two separate peaks; the distinction between the "main peak" and the "acidic" peak is not possible because in some samples, the highest signal occurs at the retention time between peak assignments.

[0428] It should be noted that for the F1 analyzed, at 12 weeks, the 25 °C sample shows a certain loss of clarity of the valley between the two peaks. The 40 °C sample shows a profile change towards the front peak shoulder and a sharper peak, with a retention time similar to that assigned as the main peak during method development.

[0429] Conclusion

[0430] Temperature can affect the composition, as observed particularly in the reduction of the main peak on RP-HPLC at 40 °C. Inclusion of methionine improves the stability of the interleukin-12 metal-binding polypeptide fusion agent, especially for the production of HMWS. All pH values within the detection range are acceptable. Polysorbate 20 is proven to be more suitable for the IL-12 fusion polypeptide than polysorbate 80 (especially at 40 °C). The provided compositions are proven to be stable even for freeze-thaw and oscillatory stress, indicating suitable performance for handling during manufacturing.

[0431] Example 5: Aluminum hydroxide (alum) retention assay

[0432] This example presents an exemplary pharmaceutical composition comprising a fusion polypeptide metal hydroxide complex. This example demonstrates that such pharmaceutical compositions are stable and that aluminum hydroxide complexation does not affect fusion polypeptide stability.

[0433] The binding and retention of the IL-12 fusion polypeptide to aluminum hydroxide were tested in vitro. The 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 number alu-vac-250) to a final concentration of 250 μg / mL IL-12 fusion polypeptide and 2.5 mg / mL aluminum hydroxide, or the composition buffer alone as a control to a final volume of 40 μL.

[0434] The IL-12 fusion polypeptide / alum mixture was resuspended thoroughly by pipetting and incubated at room temperature for 30 minutes. Then the IL-12 fusion polypeptide / alum mixture or the IL-12 fusion polypeptide control alone was diluted 25-fold in an 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 the sample was removed and centrifuged at 18,000 x 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 R&D Systems MAB219 as the capture reagent and Biolegend antibody 508802 as the detection reagent. All dilutions were prepared in TBS + 1% BSA + 0.1% Tween-20. At the highest concentration of 0.5 ng / mL and 2-fold dilutions, a standard curve was obtained using the test reagent, and if all polypeptides were released, the supernatant samples were diluted to a theoretical concentration of 0.25 ng / mL.

[0435] 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, pH 7.3.

[0436] All alum samples: 10 mg / mL (pure)

[0437] A-H described below were eluted in TBS / PBS containing 1 mM phosphate and 40% human serum. PBS: 1x PBS (pH 7.4; 11.8 mM PO4), via the CSH protocol; 20 mM TBS (pH 7.4). Pooled human serum sex; BioVT, catalog number HUMA NSERM-0001255, batch number: HMN749277. Time points: Incubated at 37 °C for 2 h and 24 h. B1 and B2 are two different preparations of purified IL-12 fusion polypeptide.

[0438] Experimental conditions, proteins, and concentrations:

[0439] · A: 10 μg / mL AK346B (TBS); 30 μL protein + 10 μL TBS - no alum control

[0440] · B: 10 μg / mL ANK101_B1 (F1); 30 μL protein + 10 μL F1 buffer - no alum control

[0441] · C: 10 μg / mL ANK101_B2 (F1); 30 μL protein + 10 μL TBS - no alum control

[0442] · D: 10 μg / mL AK346B (TBS); 30 μL protein + 10 μL alum - (10:1 alum:protein ratio)

[0443] · E: 10 μg / mL ANK101_B1 (F1); 30 μL protein + 10 μL alum - (10:1 alum:protein ratio)

[0444] · F: 10 μg / mL ANK101_B2 (F1); 30 μL protein + 10 μL alum - (10:1 alum:protein ratio)

[0445] · G: 10 μg / mL ANK101_B1 (TBS); 30 μL protein + 10 μL alum - (10:1 alum:protein ratio)

[0446] · H: 10 μg / mL ANK101_B2 (TBS); 30 μL protein + 10 μL alum - (10:1 alum:protein ratio)

[0447] The IL-12 fusion polypeptide in the formulation buffer forms a complex with aluminum hydroxide, and the complex remains stable after incubation in 1 mM phosphate and 40% human serum. Only about 4% of the protein is released at 2 hours, and about 14% is released at 24 hours ( Figure 5, condition E / F), which is highly similar to the release of the IL-12 fusion polypeptide when formulated in TBS prior to complexation with aluminum hydroxide( Figure 5 , condition G / H).

[0448] Example 6: IL-12 signal transduction activity assay

[0449] This example demonstrates that the IL-12 fusion polypeptide retains its biological activity when formulated as a pharmaceutical composition according to the present disclosure.

[0450] The IL12 signal transduction activity in vitro was evaluated using the Promega IL12 Biotest Kit (JA2601) according to the manufacturer's instructions. The IL12 biotest uses human cells that have been engineered to express the IL12 receptor and a luciferase reporter under the control of an IL12-inducible promoter. The Promega IL12 reporter cells are supplied in a frozen ready-to-use format that does not require cell culture.

[0451] The IL-12 fusion polypeptide formulated in TBS or the optimized 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 diluted in the assay medium to produce a titration series with a maximum concentration of 3 μg / mL and 3-fold dilutions. For samples mixed with alum, the fusion polypeptide at a final concentration of 250 μg / mL was mixed with a 10-fold mass excess of aluminum hydroxide (as determined by the metal mass in the formulation buffer) and incubated for 30 minutes at room temperature with shaking, then diluted in the assay medium as described above. 25 μL of each sample in the titration series was transferred to a 96-well plate and mixed with 50 μL of the Promega cell suspension to give a final maximum fusion polypeptide concentration of 1 μg / mL. The plate was then incubated overnight at 37 °C in 5% CO2 for 6 hours. 75 μL of the Bio-Glo reagent was added to the sample wells, incubated for 10 minutes, and luminescence was measured.

[0452] The IL12 fusion polypeptide in the optimized IL12 fusion polypeptide composition induced efficient signal transduction both alone and after Alhydrogel complexation in the Promega IL12 reporter assay( Figure 6 ). The EC50 values were highly similar to the IL12 fusion polypeptide in TBS, indicating that formulation does not affect biological activity.

[0453] Example 7: Influence of other composition components on the stability of IL-12 fusion polypeptide

[0454] This example demonstrates that the IL-12 fusion polypeptide exhibits low oxidation and low deamidation when formulated into a composition containing Tris buffer, sucrose, salt, L-methionine, and a surfactant at a pH of about 7 - 7.5. Specifically, this example demonstrates that L-methionine in the composition prevents oxidation of methionine and tryptophan in the IL-12 fusion polypeptide and the low pH (about 7 - 7.5) prevents deamidation of asparagine and glutamine.

[0455] Prepare compositions F1, F2, F4, and F5 as described in Example 4. Incubate the compositions at 40 °C for 6 weeks.

[0456] The oxidation (%) of methionine and tryptophan is shown in Table 14 below.

[0457] Table 14.

[0458] · Oxidation of methionine and tryptophan [%]

[0459]

[0460] Composition F5 (without methionine) exhibited approximately 27% oxidized material, whereas F1, F2, and F4 (all containing methionine) exhibited approximately 18 - 20% oxidized material. This indicates that L-methionine can reduce or prevent oxidation of methionine and tryptophan in compositions F1, F2, and F4.

[0461] The deamination (%) of asparagine (N) and glutamine (Q) is shown in Table 15 below.

[0462] Table 15

[0463] · Deamidation of asparagine and glutamine [%]

[0464] Q56 Q65 Q144 N162 N218 Q220 Q254 F1 T0 control 0.3 n.d. 0.2 n.d. 0.1 0.2 0.9 F1 T6w 40°C 0.3 0.2 0.3 9.0 0.1 0.2 0.4 F2 T6w 40°C 0.3 0.2 0.3 9.9 <0.1 0.2 0.9 F4 T6w 40°C 0.3 0.2 0.3 11.1 0.1 0.1 1.0 F5 T6w 40°C 0.4 0.2 0.3 9.3 0.1 0.3 n.d.

[0465] N323 N349 Q352 Q434 N441 Q456 Q467 N472 F1 T0 control 0.2 0.1 0.4 0.1 n.d. <0.1 n.d. 3.8 F1 T6w 40°C 1.0 0.2 0.4 0.1 0.1 n.d. n.d. 18.4 F2 T6w 40°C 0.9 0.1 0.4 0.1 0.1 n.d. 0.5 16.2 F4 T6w 40°C 1.6 0.2 0.4 0.1 n.d. 0.2 <0.1 24.1 F5 T6w 40°C 1.1 0.1 0.3 0.2 n.d. n.d. <0.1 18.4

[0466] n.d.: No change detected.

[0467] No change was detected at other positions.

[0468] Table 15 shows the effect of pH on deamidation. For N162, N323, and N472, the composition with the highest pH (pH 7.6) (F4) exhibited a significantly higher level of deamidation compared to the compositions with lower pH values (F1, F2, and F5 with a pH of about 7.3).

[0469] Example 8: Formulation development of IL-12 fusion polypeptide alone or in combination with aluminum hydroxide

[0470] This example demonstrates that the IL-12 fusion polypeptide drug product can be complexed with aluminum hydroxide (alum). This example also demonstrates that the IL-12 fusion polypeptide complexed with alum does not significantly affect the potency of the IL-12 fusion polypeptide or the viability of PBMCs. The preparation of the complexed IL-12 fusion polypeptide using a syringe does not cause loss of the IL-12 fusion polypeptide.

[0471] Exemplary IL-12 fusion polypeptide drug product

[0472] The IL-12 fusion polypeptide drug product is prepared to be obtained at 1.5 mg / vial and is manufactured directly from the fully formulated drug substance. The IL-12 fusion polypeptide drug product is a sterile preparation contained in a disposable vial, and each vial nominally contains 1.5 mg.

[0473] At the drug substance stage, the IL-12 fusion polypeptide drug substance components (nominal concentration 2 mg / mL) are fully formulated at a target pH of 7.3 in 20 mM Tris, 50 mM sodium chloride, 150 mM sucrose, 0.02% polysorbate 20 (w / v), 10 mM L-methionine. The IL-12 fusion polypeptide drug substance is the only active ingredient in the drug product (1.5 mg of IL-12 fusion polypeptide drug product per vial).

[0474] The qualitative and quantitative composition of the IL-12 fusion polypeptide drug product is the same as that of the IL-12 fusion polypeptide drug substance. As shown in the stability studies of both the drug substance and the drug product, there is no incompatibility between the excipients and the active substance.

[0475] The IL-12 fusion polypeptide drug product is developed for intratumoral administration in clinical trials. The IL-12 fusion polypeptide drug product consists of 1.5 mg of IL-12 fusion polypeptide per vial in a glass vial.

[0476] Early formulation development studies (including agitation, freeze / thaw, and storage stability studies) have confirmed the suitability of this formulation and dosage form. The buffer and pH value are selected to provide a stable solution for the protein while maintaining the pH value during the storage of the drug substance and the drug product. Polysorbate 20 has been added to reduce the likelihood of agitation- and / or freeze / thaw-induced aggregation. Sucrose has been added to adjust the osmotic pressure of the product. The formulation is designed to be robust to freeze / thaw cycles.

[0477] Administration components and simulated use

[0478] Simulated administration studies were conducted to evaluate the initial steps in dose preparation, including the compatibility of the product with several components and contact materials expected to be used during dose preparation for clinical drug administration via the intratumoral route.

[0479] The compatibility of the diluted IL-12 fusion polypeptide drug product with the product-specific diluent was studied in type 1 glass vials (6R) to determine the stability of the diluted drug product and to determine the materials for preparing the representative clinical dose. The IL-12 fusion polypeptide drug product (batch 101) was in liquid form 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, with a nominal concentration of 2 mg / mL (a representative batch with a nominal fill volume was 1.0 mL in a 2R glass vial [type I], including overfill). The product-specific diluent was in liquid form 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 (a representative batch with a nominal fill volume was 6 mL in a 6R glass vial [type I]). The IL-12 fusion polypeptide drug product and the product-specific diluent were transferred to sealed empty sterile 6R glass vials using commercially available silanized syringes (1 mL or 2 mL) and needles (21 gauge).

[0480] The IL-12 fusion polypeptide drug product was diluted to a target concentration of 0.25 mg / mL with the product-specific diluent solution in 6R vials. The diluted drug product (0.25 mg / mL) was prepared in triplicate (n = 3). Physicochemical analysis data supported the physicochemical stability of the diluted IL-12 fusion polypeptide drug product solution in 6R sterile sealed vials when exposed to ambient storage conditions (ambient temperature and exposure to light) for up to 4 hours.

[0481] Throughout the study, no significant changes were observed in the physicochemical analysis tests (clarity, color), purity according to size exclusion-high performance liquid chromatography, and activity, indicating good compatibility with the selected materials. The time 0 samples tested had few visible particles (1 fibrous particle in 1 of 3 replicates). For the 4-hour time point (T4h), the reported results were that the samples had few visible particles (1 fiber-like particle each in 2 of 3 replicates). In further studies by particle characterization, these visible particles were reported to be non-protein, mainly cellulose fibers and some oleamide particles. Therefore, the visible particles are inherent to the dose preparation. Considering the expected administration volume of less than 100 mL, the subvisible particles fully comply with the pharmacopoeial requirements <USP 787> for subvisible particle counts of ≥25 μm (≤600 per container) and ≥10 μm (≤6,000 per container).

[0482] All samples had generally high recoveries at T4h (all above 99%), indicating good compatibility with the contact materials.

[0483] The cell-based assay results of the IL-12 fusion polypeptide drug product at T0 and T4 showed 102% activity (T0) and 103% activity (T4).

[0484] These results indicate that under ambient conditions, the IL-12 fusion polypeptide drug product is stable when in contact with glass vials in the product-specific diluent at the target concentration (0.25 mg / mL) for up to 4 hours.

[0485] At the time of dose preparation, the potency and strength of the IL-12 fusion polypeptide complexed with

[0486] aluminum hydroxide (alum) serves as the chemical basis.

[0487] Use in-use compatibility studies to examine the compatibility of the IL-12 fusion polypeptide drug product complexed with diluent, sterile empty vials (SEV), and commonly available ancillary components (e.g., syringes, needles) in drug preparation that simulates the use of the IL-12 fusion polypeptide drug product or active pharmaceutical ingredient in a clinical pharmacy. A bracketing design is used to cover the expected dose range including intermediate doses. The dose preparation is described as follows:

[0488] High dose: 0.25 mg / mL IL-12 fusion polypeptide complexed with 2.5 mg of (dose group 6)

[0489] 1) Add 3.75 mL of the diluent drug product to a 6R SEV.

[0490] 2) Add 0.75 mL of the IL-12 fusion polypeptide drug product / active pharmaceutical ingredient (2 mg / mL) to the 6R vial containing the diluent and gently agitate to ensure mixing.

[0491] 3) Vigorously shake the vial to ensure homogeneity.

[0492] 4) Add 1.5 mL of (10 mg / mL) to the diluted solution of the IL-12 fusion polypeptide drug product and gently agitate to ensure mixing.

[0493] a. Concentration of the IL-12 fusion polypeptide drug product / active pharmaceutical ingredient = 0.250 mg / mL

[0494] b. Concentration of = 2.5 mg / mL

[0495] The 6R vial contains a total volume of 6 mL of IL-12 fusion polypeptide

[0496] 5) Incubate the mixed preparation at room temperature for 30 minutes or 6 hours.

[0497] Medium dose: 0.02 mg / mL of IL-12 fusion polypeptide and 0.2 mg of Complex (Dose Group 3)

[0498] Starting with the 0.250 mg / mL IL-12-fusion polypeptide / 2.5 mg / mL (Dose Group 6) solution prepared by the initial mixing method described above, perform the following dilution protocol:

[0499] 1) Gently mix the vial prepared for Dose Group 6.

[0500] 2) Add 5.52 mL of diluent drug product to the 6R SEV.

[0501] 3) Add 0.48 mL of Dose Group 6 to the 6R vial containing the diluent and gently agitate to ensure mixing.

[0502] a. Concentration of IL-12 fusion polypeptide drug product / bulk drug = 0.02 mg / mL.

[0503] b. The concentration of = 0.2 mg / mL

[0504] 4) Incubate the mixed preparation at room temperature for 30 minutes or 6 hours.

[0505] Low dose: 0.002 mg / mL IL-12-ABP and 0.02 mg of Complex (Dose Group 1).

[0506] Starting with the 0.02 mg / mL IL-12 fusion polypeptide / 0.2 mg / mL (Dose Group 3) solution prepared by the mixing method described above, perform the following dilution protocol:

[0507] 1) Gently mix the vial prepared for Dose Group 3.

[0508] 2) Add 5.4 mL of diluent drug product to the 6R SEV.

[0509] 3) Add 0.6 mL of Dose Group 3 to the 6R vial containing the diluent and gently agitate to ensure mixing.

[0510] a. Concentration of IL-12 fusion polypeptide drug product / bulk drug = 0.002 mg / mL.

[0511] b. Concentration = 0.02 mg / mL.

[0512] 4) Incubate the mixed preparation at room temperature for 30 minutes or 6 hours.

[0513] Interleukin-12 (IL-12) signals through a heterodimeric complex of IL-12Rβ1 and IL-12Rβ2 expressed on T cells and natural killer (NK) cells to induce interferon γ (IFNγ) expression through phosphorylation and activation of STAT4. The potencies of free IL-12-ABP protein and complexed IL-12 fusion polypeptide to induce IFNγ expression in activated primary human peripheral blood mononuclear cells (PBMC) from healthy donors were evaluated in comparison to a human IL-12 control protein lacking an alum-binding protein. For PBMC, cells were treated with titrated human IL-12 (control), IL-12 fusion polypeptide, or IL-12 fusion polypeptide complexed with alum in the presence of 100 ng / mL soluble α-cluster of differentiation (CD) 3 antibody (clone OKT3). After 3 days, the IFNγ concentration in the supernatant was measured by time-resolved fluorescence energy transfer (TR-FRET) assay. The concentration of the α-CD3 antibody was chosen to suboptimally activate immune cells and increase their responsiveness to IL-12 agents while inducing minimal IFNγ by itself.

[0514] In this study, human PBMC were isolated from 2 healthy donors and seeded at 5 x 10 5 cells / well in round-bottom 96-well plates. PBMC were stimulated with anti-CD3 (100 ng / mL) in the presence of the IL-12 fusion polypeptide drug substance (Good Manufacturing Practice batch 1205114). The IL-12 fusion polypeptide was prepared via syringe (for all steps or only for extraction from the dose vial) or by using all pipettes (study ATXFTE-06). Appropriate controls included a negative control (unstimulated PBMC), a positive control (soluble CD3 [5 μg / mL] + soluble CD28 [2 μg / mL]), and a diluent (formulation buffer). After 72 hours of incubation, the cell culture supernatant was harvested and stored at -80 °C until completion of the cytokine analysis for IFNγ by TR-FRET. Cell viability was determined using 2.0 Cell Viability Assay. The study was performed using PBMC from 2 donors. Each experimental condition was tested in triplicate, and each immunoassay reading was repeated once (Table 16).

[0515] Table 16: Overview of the PBMC Assay Group Study

[0516]

[0517]

[0518] CD = Cluster of Differentiation; IFNγ = Interferon γ; NA = Not applicable; PBMC = Peripheral blood mononuclear cells; TR-FRET = Time-resolved fluorescence energy transfer

[0519] Use 2.0 Cell viability assay determines the number of viable cells in a culture by quantifying the amount of adenosine triphosphate (ATP) present. This is used as an indicator of the presence of metabolically active cells.

[0520] Results

[0521] In all 2 donors, stimulation with the positive control (anti-CD3 [5 μg / mL] + anti-CD28 [2 μg / mL]) increased the ATP level to above the unstimulated condition (Figures 7 and 8). In the presence of the IL-12 fusion polypeptide complexed with alum, in all dose preparation groups, for most protein concentrations, the ATP level was consistent with the vehicle control, and a decrease in the ATP level was observed at the highest dose of the IL-12 fusion polypeptide complexed with alum. The test compound preparation and culture methods did not seem to have a consistent effect on PBMC viability.

[0522] Sub-optimal stimulation of PBMC was induced by stimulation with 100 ng / mL anti-CD3, generating moderate IFNγ. The IL-12 fusion polypeptide complexed with alum exhibited enhanced IFNγ production in a dose-dependent manner in all dose preparation groups (Figures 9 and 10). There were limited differences in IFNγ production between the two donors between the syringe and pipette preparation groups. The incubation method (static versus rotation) did not seem to affect IFNγ production. Increasing the incubation time only moderately decreased the potency of the IL-12 fusion polypeptide complexed with alum under the rotation condition. However, at all 3 doses, the half-maximal effective concentration (EC50) was still consistent with the non-rotation condition.

[0523] Overview

[0524] IL-12 is an important T cell and NK cell stimulant and plays an important role in driving the differentiation of T cells towards a pro-inflammatory phenotype by inducing the production of IFNγ. To study the effects of the preparation and administration methods of the IL-12 fusion polypeptide complexed with alum, the EC of IFNγ produced from activated primary human PBMC was determined 50Values. The effect of the preparation method on PBMC viability was also determined. In this study (preparation with or without a syringe and with a rotating device), three preparation methods (syringe, syringe only when removing from the vial, and without a syringe [pipette]) and three incubation times (6 hours static, 6 hours with rotation, and 30 minutes with rotation) were compared. The preparation conditions with or without a syringe were compared in three different dose groups (high, medium, or low).

[0525] At the end of the incubation time, in the absence of rotation, a high level of complex sedimentation was noted. Each condition was thoroughly inverted back and forth to ensure homogeneity.

[0526] In two donors, the cell viability of PBMC (measured by ATP) remained consistent with a moderate reduction in cell viability at the highest dose concentration (donor 2), which was similar to the observations of previous studies. This reduction in cell viability was not always affected by the formulation dose of the IL-12 fusion polypeptide complexed with alum. Overall, the preparation of the IL-12 fusion polypeptide complexed with alum by syringe did not seem to have a consistent effect on PBMC viability.

[0527] In two donors, the IL-12 fusion polypeptide complexed with alum enhanced IFNγ production in a dose-dependent manner, with the maximum response higher than the positive control (anti-CD3 + anti-CD28). There were limited differences in IFNγ production between the syringe and pipette preparation groups in all dose preparation groups. The method of dose preparation did not seem to affect the maximum response of the cells, and the responses at all doses were similar whether prepared by syringe or pipette. Only the EC 50 value was calculated for donor 2 because donor 1 did not reach the maximum response. Increasing the incubation time decreased the potency of the IL-12 fusion polypeptide complexed with alum under the rotation-only condition. However, at all three doses, the EC 50 remained consistent with the non-rotation condition. The potencies in each dose group in donor 2 remained consistent, within the range of 0.0541 ng / mL to 0.129 ng / mL, consistent with the historical IL-12 fusion polypeptide complexed with alum.

[0528] Overall, the data indicate that the dose preparation method (syringe / no syringe), incubation method (static / rotation), and length of incubation (30 minutes vs. 6 hours) have no significant effect on the potency of the IL-12 fusion polypeptide complexed with alum or PBMC viability.

[0529] Micro BCA assay of the complexed IL-12 fusion polypeptide.

[0530] The content (protein concentration) of the composite IL-12 fusion polypeptide was examined using in-use compatibility studies, which simulated drug preparation in a clinical pharmacy. The studies used the IL-12 fusion polypeptide bulk drug substance (batch P4130826), (batch 152 - 001 - 001), diluent (batch 152 - 002 - 001), and SEV, as well as commonly available ancillary components (such as syringes, needles). A bracketing design was used to cover the expected dose range including intermediate doses. The micro BCA assay (Micro BCA Protein Assay Kit, Thermo Scientific) was used to measure the amount of IL-12 fusion polypeptide at 3 different doses.

[0531] Samples for dose group 6 (250 μg / mL), dose group 3 (20 μg / mL), and dose group 1 (2 μg / mL) were prepared. A group of dose controls was also prepared, where all preparations were done by pipette.

[0532] The micro BCA assay was used to quantify the IL-12 fusion polypeptide that bound to during the in-use bracketing study. The study included the highest potential dose for patients, as well as intermediate and lowest doses. Improvements in sample handling enabled more accurate quantification of high and medium doses of the protein, while low doses remained a challenge. The measured concentration of the dosing formulation (using the same materials and techniques as those described in pharmaceutical handbooks) showed no significant change relative to the dosing control (where each group was prepared by pipette). It appears that using a syringe during preparation does not cause significant protein loss. The precision was good in all cases, indicating that liquid handling by syringe is accurate enough to ensure reproducible dose dispensing at all volumes.

[0533] Example 9: Stability data of bulk IL-12 fusion polypeptide drug substance

[0534] This example confirmed the high stability of the IL-12 fusion polypeptide bulk drug substance reference standard batch and the IL-12 fusion polypeptide bulk drug substance GMO batch.

[0535] Two batches of IL-12 fusion polypeptide bulk drug substance were tested:

[0536] · 500 L scale bioreactor batch (analytical reference standard batch) (P4130826ARS); and

[0537] · 1000 L scale bioreactor batch (Good Manufacturing Practice (GMP) batch) (1205114).

[0538] An overview of the IL-12 bulk drug substance stability is shown in Table 17.

[0539] Table 17

[0540]

[0541] The stability protocol for the reference standard batch of IL-12 fusion polypeptide is shown in Table 18.

[0542] Table 18. Stability protocol for the IL-12 fusion polypeptide drug substance batch P4130826 1 .

[0543]

[0544] 1 Residual host cell deoxyribonucleic acid (DNA) and host cell protein (HCP) were not tested in the stability protocol; bioburden was only tested at the T = 0 time point; malachite green was only tested at T = 0; anion exchange high performance liquid chromatography (AEX-HPLC) was for reference only; endotoxin was only tested at the T = 0 time point

[0545] X = clarity, color, protein concentration (content, pH, reducing reverse phase HPLC (RP-HPLC), size exclusion HPLC (SE HPLC), reducing capillary SDS, anion exchange HPLC (AEX-HPLC), activity

[0546] B = Remove six containers from storage (two at the start of the aliquoting process, two in the middle, and two at the end) and transfer to QC Microbiology for dedicated bioburden testing

[0547] B* = Remove three bags from storage (one at the start of the aliquoting process, one in the middle, and one at the end) and transfer to QC Microbiology for dedicated bioburden testing

[0548] BR = Release testing for batches that will report the bioburden and osmotic pressure at the T = 0 time point

[0549] HDPE = high density polyethylene; LDPE = low density polyethylene; NA = not applicable; NT = not tested

[0550] The stability protocol for the IL-12 fusion polypeptide GMO batch is shown in Table 19.

[0551] Table 19. Stability protocol for the IL-12 fusion polypeptide drug substance batch 1205114 1

[0552]

[0553] 1 ​Residual host cell DNA and HCP were not tested in the stability protocol; bioburden was tested only at the T = 0 time point; malachite green was tested only at T = 0; AEX-HPLC testing is for reference only; endotoxin was tested only at the T = 0 time point

[0554] X = clarity, color, protein concentration (content, pH, reducing reverse-phase HPLC (RP-HPLC), size exclusion HPLC (SE HPLC), reducing capillary SDS, anion exchange HPLC (AEX-HPLC), activity

[0555] B* = Remove three bags from storage (one at the start of the aliquoting process, one in the middle, and one at the end) and transfer to QC Microbiology dedicated for bioburden testing

[0556] BR = The batch release test at the T = 0 time point will be reported, except for bioburden and osmolality

[0557] LDPE = low density polyethylene; NA = not applicable; NT = not tested

[0558] Analytical method

[0559] An exemplary stability testing method is described in this section. The AEX-HPLC method is used to separate the dephosphorylated IL-12 fusion polypeptide drug substance and its charged variants. The AEX-HPLC column is used to quantify the charged variants present in the LI-12 fusion polypeptide drug substance. The sample is dephosphorylated using phosphatase and injected onto the column. The charged variants are separated based on the differences in the surface charges of different molecular species. Acidic molecules with negative charges elute later than basic molecules with positive charges. For the IL-12 fusion polypeptide drug substance, the charged species can be determined by detecting the elution peaks by fluorescence detection using an excitation wavelength of 280 nm and an emission wavelength of 320 nm based on the differences in their surface charges. The relative quantification of acidic and basic substances in the IL-12 fusion polypeptide drug substance is achieved by relative area % evaluation.

[0560] Overview

[0561] To date, stability data for 1 month for the GMP batches of the IL-12 fusion polypeptide bulk drug substance under each storage condition have been collected, and the stability for 9 months for the analytical reference standard batches of the IL-12 fusion polypeptide bulk drug substance under each storage condition has been collected. All stability storage conditions (-70 °C, +5 °C, and +25 °C) comply with the stability study specifications. All samples tested to date comply with the current GMP release specifications. Compared to the start of the study, the percentage of the main peak in the samples of the analytical reference standard batches of the bulk drug substance stored at +5 °C showed a slight but significant decrease by reductive capillary electrophoresis sodium dodecyl sulfate (CE SDS) analysis at nine months, however this was within the expected intermediate precision CV percentage range for the main peak (CV < 1% for reductive CE SDS).

[0562] For the IL-12 fusion polypeptide from the analytical reference standard batches, comparison of samples stored at -70 °C for nine months in low-density polyethylene (LDPE) bags and high-density polyethylene (HDPE) bottles showed consistent results, confirming that the container type has no significant effect on the product according to any test method.

[0563] The decrease in the percentage of the main peak of the analytical reference standard batches of the bulk drug substance was only confirmed by CE SDS during the stress condition at 25 °C and at the 6-month time point (analytical reference standard batches).

[0564] The similar slight decrease in the GMP batches at the 1-month time point under all conditions as confirmed by AEX-HPLC may be attributed to assay variability, which will be investigated during the planned assay validation.

[0565] Example 10: Stability data of IL-12 fusion polypeptide drug product

[0566] This example demonstrates the high stability of the IL-12 fusion polypeptide drug product (reference standard batches and GMP batches).

[0567] An overview of the batches of the IL-12 fusion polypeptide drug product for which stability studies were conducted is provided in Table 20. The stability indicating tests and related conditions are listed in Tables 21 and 22 below.

[0568] Table 20: Overview of the IL12-fusion polypeptide stability study

[0569]

[0570]

[0571] Table 21: Stability protocol for batch 101 of the IL-12 fusion polypeptide drug product

[0572]

[0573] BR = Batch Release; INV = Inverted, vial stored head down; NT = Not Tested; Opt. = Optional; RH = Relative Humidity; Up = Upright, vial stored head up; X = Color, Clarity, pH, Protein Concentration (Content), Size Exclusion - High Performance Liquid Chromatography (SE HPLC), Capillary Electrophoresis Sodium Dodecyl Sulfate (CE SDS), Reverse Phase High Performance Liquid Chromatography (RP HPLC), Anion Exchange High Performance Liquid Chromatography (AEX HPLC), Surfactant Content, Activity, Visible and Sub - visible Particles

[0574] O = Only Bioburden; Note: Batch release testing includes Color, Clarity, pH, Osmolality, Extractable Volume, Visible and Sub - visible Particles, Protein Concentration (Content), Size Exclusion HPLC, Activity, Endotoxin, Bioburden, Identity according to Reductive Reverse Phase HPLC, Purity according to Reductive Reverse Phase HPLC, Reductive Capillary Electrophoresis SDS, Anion Exchange HPLC; Extractable Volume, Osmolality, Identity, Endotoxin are only tested at BR; Surfactant Content is tested starting from 1M under all conditions; Bioburden is tested at BR and then annually at 5°C ± 3°C (Up) and - 20°C ± 5°C (Up).

[0575] Table 22: Stability Protocol for IL - 12 Fusion Polypeptide Drug Product Batch 101

[0576]

[0577]

[0578] BR = Batch Release; INV = Inverted, vial stored head down; NT = Not Tested; Opt. = Optional; RH = Relative Humidity; UP = Upright, vial stored head up; O = Color, Clear Clarity, pH, Content, SE HPLC, CE SDS, RP HPLC, Surfactant Content, Activity, Visible and Non - Sub - visible Particles; X = Color, Clear Clarity, pH, Content, SE HPLC, CE SDS, RP HPLC, Surfactant Content, Activity, Visible and Non - Sub - visible Particles, CCIT, Osmolality, Malachite Green, LC MS (Full Mass). 1 LC MS (Full Mass) was not used to test the conditions of 1M - 20°C ± 5°C (Up), 5°C ± 3°C (INV), and 25°C ± 2°C / 60% RH ± 5% RH (INV). 2The malachite green test was not used under the conditions of 1M - 20°C ± 5°C (upward). Note: Batch release tests include color, clarity, pH, osmotic pressure, extractable volume, visible and sub-visible particles, protein concentration (content), SE HPLC, activity, endotoxin, sterility, identity according to reductive RP-HPLC, purity according to reductive RP-HPLC, reductive CE-SDS, surfactant content, AEX HPLC; extractable volume, identity, endotoxin, sterility are only tested at BR; for stability, only the container closure integrity (CCIT) is tested at 5°C ± 3°C (INV), starting at 12M and annually thereafter. Osmotic pressure is tested at 5°C ± 3°C (INV) and 25°C ± 2°C / 60% RH ± 5% RH (INV), both starting at the 3M time point. Malachite green, AEX HPLC, and LC MS (intact mass) are used for characterization purposes

[0579] Exemplary analytical methods

[0580] The AEX-HPLC method is used to separate the dephosphorylated IL-12 fusion polypeptide drug product and its charged variants. The AEX-HPLC column is used to quantify the charged variants present in the IL-12 fusion polypeptide drug substance. The sample is dephosphorylated using phosphatase and injected onto the column. The charged variants are separated based on the differences in the surface charges of different molecular species. Acidic molecules with negative charges elute later than basic molecules with positive charges. For the IL-12 fusion polypeptide drug substance, the charged species can be determined by detecting the elution peaks by fluorescence detection using an excitation wavelength of 280 nm and an emission wavelength of 320 nm based on the differences in their surface charges. The relative quantification of acidic and basic substances in the IL-12 fusion polypeptide drug product is achieved by relative area% evaluation

[0581] The container closure integrity (CCI) test is performed using a helium leak test. A helium leak physical container closure integrity method based on quantitative mass spectrometry is used to test the microbiological tightness of the container closure system. The container closure system is placed in an airtight flange connected to a mass spectrometer, and a vacuum pump creates a pressure difference between the inside of the mass spectrometer and the inside of the container closure system, where helium is continuously applied to the container closure system. The mass spectrometry instrument quantifies the helium flow rate (mbar L / s) leaking through potential leaks in the container closure system

[0582] Conclusion

[0583] At the 9-month time point and at the expected storage temperature of 5°C ± 3°C, the non-GMP batches of the IL-12 fusion polypeptide drug product remained stable and met all release specifications. In upright and inverted vials, there was minimal increase in subvisible particles ≥ 2 μm. Similar profiles were obtained when the IL-12 fusion polypeptide drug product was stored frozen at -20°C ± 5°C. At the accelerated temperature of 25°C ± 2°C / 60% ± 5% relative humidity (RH) (inverted) and at the 6-month time point, when tested by reducing reverse-phase high performance liquid chromatography (RP-HPLC) and reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS), the IL-12 fusion polypeptide drug product showed a slight decrease in the main peak compared to the previous time points. The molecule showed little (if any) aggregation (size exclusion - high performance liquid chromatography [SE-HPLC]) or tendency to form subvisible particles. Only at the stress condition of 40°C ± 2°C / 75% ± 5% RH (inverted) and at the 3-month time point did the IL-12 fusion polypeptide drug product show a significant decrease in the main peak (RP-HPLC, CE-SDS). Under the same conditions, the IL-12 fusion polypeptide drug product also showed a slight decrease in monomer % as determined by SE-HPLC. The GMP batches of the IL-12 fusion polypeptide drug product met all specifications at batch release (0-month time point) and were similar to the non-GMP batches of the IL-12 fusion polypeptide drug product (0-month time point). It is expected that the GMP batches of the IL-12 fusion polypeptide drug product will exhibit a stability profile similar to that of the non-GMP batches of the IL-12 fusion polypeptide drug product.

[0584] Equivalent schemes

[0585] Those skilled in the art will recognize or be able to determine many equivalent schemes of the specific embodiments of the invention described herein using only routine experimentation. The scope of the invention is not intended to be limited to the above specification, but rather as set forth in the following claims.

Claims

1. A composition, the composition comprising i) a phosphorylated form of a fusion polypeptide, the fusion polypeptide comprising: a) an immunomodulatory polypeptide, the immunomodulatory polypeptide comprising an interleukin-12 immunostimulatory moiety; and b) a metal hydroxide-binding polypeptide, the amino acid sequence of which includes a plurality of phosphorylation sites such that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms; ii) Tris buffer; iii) salt; iv) sucrose; v) L-methionine; and vi) a surfactant, wherein the pH value of the composition ranges from about 6.5 to about 8.

2. The composition according to any one of the preceding claims, wherein the fusion polypeptide forms a complex with the metal hydroxide when exposed to the metal hydroxide.

3. The composition according to any one of the preceding claims, wherein the metal hydroxide is aluminum hydroxide.

4. The composition according to any one of the preceding claims, wherein the pH value ranges from about 6.5 to about 7.

8.

5. The composition according to any one of the preceding claims, wherein the pH value ranges from about 7 to about 7.

6.

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

4.

7. The composition according to any one of the preceding claims, wherein the concentration of the fusion polypeptide ranges from about 0.1 g / L to about 15 g / L.

8. The composition according to any one of the preceding claims, wherein the concentration of the fusion polypeptide ranges from about 0.5 g / L to about 5 g / L.

9. The composition according to any one of the preceding claims, wherein the concentration of the fusion polypeptide ranges from about 1 g / L to about 3 g / L.

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

11. The composition according to any one of the preceding claims, wherein the concentration of the Tris buffer ranges from about 1 mM to about 50 mM.

12. The composition according to any one of the preceding claims, wherein the concentration of the Tris buffer ranges from about 10 mM to about 25 mM.

13. The composition according to any one of the preceding claims, wherein the concentration of the salt ranges from about 1 mM to about 750 mM.

14. The composition according to any one of the preceding claims, wherein the concentration of the salt ranges from about 10 mM to about 100 mM.

15. The composition according to any one of the preceding claims, wherein the salt is NaCl or Na2SO4.

16. The composition according to any one of the preceding claims, wherein the concentration of L-methionine ranges from about 1 mM to about 20 mM.

17. The composition according to any one of the preceding claims, wherein the concentration of L-methionine ranges from about 5 mM to about 15 mM.

18. The composition according to any one of the preceding claims, wherein the concentration of L-methionine is 10 mM.

19. The composition according to any one of the preceding claims, wherein the surfactant is polysorbate.

20. The composition according to any one of the preceding claims, wherein the surfactant is polysorbate 20 or polysorbate 80.

21. The composition according to any one of the preceding claims, wherein the surfactant is polysorbate 20.

22. The composition according to any one of the preceding claims, wherein the concentration of the polysorbate is in the range of about 0.005% w / v to about 0.1% w / v.

23. The composition according to any one of the preceding claims, wherein the concentration of the polysorbate is in the range of about 0.01% w / v to about 0.05% w / v.

24. The composition according to any one of the preceding claims, wherein the concentration of the polysorbate is about 0.02% w / v.

25. The composition according to any one of the preceding claims, wherein the concentration of sucrose is in the range of about 100 mM to about 200 mM.

26. The composition according to any one of the preceding claims, wherein the concentration of sucrose is 150 mM.

27. The composition according to any one of the preceding claims, wherein the composition comprises 2 mg / mL fusion polypeptide, 20 mM Tris buffer, 50 mM NaCl, 10 mM L-methionine, 0.02% polysorbate 20, and 150 mM sucrose, and wherein the pH value of the composition is in the range of 6 to 8.

28. The composition according to any one of the preceding claims, wherein the composition has been stored at a temperature of at most -50 °C, such as at most -55 °C, such as at most -60 °C, such as at most -65 °C.

29. The composition according to any one of the preceding claims, wherein the composition has been stored for about 1 day to about 500 days.

30. The composition according to any one of the preceding claims, wherein the pH value of the composition after storage is similar to the pH value of the composition before storage.

31. The composition according to any one of the preceding claims, wherein the concentration of the fusion polypeptide in the composition after storage is similar to the concentration of the fusion polypeptide in the composition before storage.

32. The composition according to any one of the preceding claims, wherein the osmotic pressure of the composition after storage is similar to the osmotic pressure of the composition before storage.

33. The composition according to any one of the preceding claims, wherein the color of the composition after storage is similar to the color of the composition before storage.

34. The composition according to any one of the preceding claims, wherein the amount of visible particles in the composition is similar to the amount of visible particles in the composition before storage.

35. The composition according to any one of the preceding claims, wherein the formulation is a liquid composition.

36. The composition according to any one of the preceding claims, wherein the formulation is a solid composition.

37. The composition according to any one of the preceding claims, wherein the composition is in a dry form.

38. The composition according to any one of the preceding claims, wherein the formulation is a powder.

39. The composition according to any one of the preceding claims, wherein the composition is frozen.

40. The composition according to any one of the preceding claims, wherein the composition is in a vial.

41. The composition according to any one of the preceding claims, wherein the vial is light - resistant.

42. A pharmaceutical preparation, the pharmaceutical preparation comprising i) a fusion polypeptide metal hydroxide complex, the fusion polypeptide metal hydroxide complex comprising: (a) a phosphorylated form of a fusion polypeptide, the fusion polypeptide comprising: ai) an immunomodulatory polypeptide, the immunomodulatory polypeptide comprising an interleukin - 12 immune agonist moiety; and aii) a metal hydroxide - binding polypeptide wherein the amino acid sequence of the fusion polypeptide metal hydroxide complex includes a plurality of phosphorylation sites such that it can exist in phosphorylated and non - phosphorylated forms; and (b) a metal hydroxide ii) Tris buffer; iii) a salt; iv) sucrose; v) L - methionine; and vi) a surfactant, wherein the pH value of the composition is in the range of about 6.5 to about 8.

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

44. The pharmaceutical preparation according to claims 41 - 42, wherein the metal hydroxide is aluminum hydroxide.

45. The pharmaceutical preparation according to 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.

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

47. The pharmaceutical preparation according to claims 41 - 45, wherein the concentration of the aluminum hydroxide is 2.5 mg / mL.

48. The pharmaceutical preparation according to claims 41 - 46, wherein the pH value is in the range of about 6.8 to about 7.

8.

49. The pharmaceutical preparation according to claims 41 - 47, wherein the pH value is in the range of about 7 to about 7.

6.

50. The pharmaceutical preparation according to claims 41 - 48, wherein the pH value is about 7.

3.

51. The pharmaceutical preparation according to 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.

52. The pharmaceutical preparation according to 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.

53. The pharmaceutical preparation according to 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.

54. The pharmaceutical preparation according to claims 41 - 52, wherein the concentration of the fusion polypeptide is 0.25 mg / mL.

55. The pharmaceutical preparation according to claims 41 - 53, wherein the concentration of the Tris buffer is in the range of about 1 mM to about 50 mM.

56. The pharmaceutical preparation according to claims 41 - 54, wherein the concentration of the Tris buffer is in the range of about 10 mM to about 40 mM.

57. The pharmaceutical preparation according to claims 41 - 55, wherein the concentration of the Tris buffer is in the range of about 15 mM to about 20 mM.

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

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

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

61. The pharmaceutical preparation according to claims 41 - 59, wherein the salt is NaCl or Na2SO4.

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

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

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

65. The pharmaceutical preparation according to claims 41 - 63, wherein the surfactant is polysorbate.

66. The pharmaceutical preparation according to claims 41 - 64, wherein the surfactant is polysorbate 20 or polysorbate 80.

67. The pharmaceutical preparation according to claims 41 - 65, wherein the surfactant is polysorbate 20.

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

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

70. The pharmaceutical preparation according to claims 41 - 68, wherein the concentration of polysorbate is about 0.015% w / v.

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

72. The pharmaceutical preparation according to claims 41 - 70, wherein the concentration of sucrose is 113 mM.

73. The pharmaceutical preparation according to claims 41 - 71, wherein the preparation is a liquid composition.

74. The pharmaceutical preparation according to claims 41 - 72, wherein the preparation is a solid composition.

75. The pharmaceutical preparation according to claims 41 - 73, wherein the preparation is a powder.

76. The pharmaceutical preparation according to 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 aluminum hydroxide, and wherein the pH value of the composition ranges from 6 to 8.

77. The pharmaceutical preparation according to claims 41 - 75, wherein the preparation is a liquid preparation.

78. The pharmaceutical preparation according to claims 41 - 76, wherein the preparation is formulated for parenteral delivery.

79. The pharmaceutical preparation according to claims 41 - 77, wherein the preparation is formulated for intratumoral injection.

80. The pharmaceutical preparation according to claims 41 - 79, wherein the preparation is in a vial.

81. A method of treating a subject, the method comprising administering the pharmaceutical composition according to claim 41.

82. The method according to claim 80, wherein the subject has cancer.

83. The method according to claim 81, wherein the cancer is associated with a tumor.

84. The method according to claim 80, wherein the subject is a human.

85. The method according to claim 80, wherein the composition is administered by parenteral administration.

86. The method according to claim 82, wherein the composition is administered by intratumoral injection.

87. The method according to claim 83, wherein the composition is administered by peritumoral injection.

88. The method according to claim 80, wherein the composition is administered in combination with a second therapy.

89. The method according to claim 87, wherein the second therapy is a checkpoint inhibitor.

90. A method of manufacturing the composition according to claim 1, the method comprising combining the phosphorylated form of the fusion polypeptide with Tris buffer, salt, sucrose, L - methionine, and a surfactant.

91. A method of manufacturing the pharmaceutical preparation according to claim 41, the method comprising: i) contacting the composition according to claim 1 with a metal hydroxide.

92. The method according to claim 90, wherein the contacting is carried out for 1 minute to 60 minutes.

93. The method according to claim 91, wherein the contacting is carried out at room temperature.

94. A method of characterizing the composition according to claim 1, the method being carried out by evaluating the degree of phosphorylation of the fusion polypeptide.

95. The method according to claim 93, wherein the characterization includes evaluating the purity of the fusion polypeptide in the composition.

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