Methods and compositions for modulating GM-CSF bioactivity

Abstract

Compositions and methods for modifying granulocyte-macrophage colony-stimulating factor (GM-CSF) as described. Non-naturally occurring cell engineered for production of GM-CSF with an altered glycosylation pattern comprising an expression vector having the CSF2 gene, and one or more knockout genes selected from the group consisting of MGAT5, ST3GAL3, ST3GAL4, ST3GAL6, B3GNT2, SPPL3, MGAT4A, and MGAT4B. In some embodiments, the cell additionally comprises a knock-in gene consisting of HST6GAL1.

Description

Attorney Docket No. 24978-1010METHODS AND COMPOSITIONS FOR MODULATING GM-CSF BIOACTIVITYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 685,158, filed August 20, 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present invention relates to modified proteins and cells used for the production of recombinant proteins.BACKGROUND

[0003] Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is an important cytokine that stimulates the proliferation of neutrophils, eosinophils, and monocytes. It can be used to treat cancer and neutropenia1,2. Much like any biologic it is critical to understand how to enhance its production, not only in quantity, but also in quality. Indeed, GM-CSF is glycosylated in mammalian cells with two N-linked glycosylation sites3 4and four O-linked glycosylation sites3’6. Recombinant GM-CSF can be produced using various expression systems such as bacteria, yeast, and mammalian cells7. Clinical studies indicate that E. co / / -derived GM-CSF elicits higher adverse reactions, attributed to the absence of glycans8 l0, underscoring the role of glycans in reducing immunogenicity.

[0004] Bioactivity studies have shown that increased glycosylation decreases GM-CSF activity4,11. However, conflicting reports exist regarding the role of sialic acid. While one study suggests that sialic acid removal does not affect GM-CSF activity12, another indicates that sialic acid enhances its activity13. In addition to sialic acid, the meta-analysis of human transcriptomics data revealed that MGAT5, involved in glycan branching, is co-expressed with high GM-CSF-producing cells, suggesting a potential role in both production yield and bioactivity enhancement.Atorney Docket No. 24978-1010SUMMARY OF THE INVENTION

[0005] The disclosure provides non-naturally occurring GM-CSF proteins and cells for expression of recombinant proteins and other research. The invention provides that glycan microheterogeneity, both at the level of branching and changes in sialylation indeed impacts GM-CSF activity. The invention establishes that the role of MGAT5 in maintaining GM-CSF bioactivity.

[0006] The invention provides a modified GM-CSF comprising glycoengineered GM- CSF having changes to glycan branching and sialylation to modulate pharmaceutical activity, as described herein.

[0007] Specifically, the invention provides a non-naturally occurring cell engineered for production of granulocyte-macrophage colony-stimulating factor (GM-CSF) with an altered glycosylation pattern comprising an expression vector having the CSF2 gene, and one or more knockout genes selected from the group consisting of MGAT5, ST3GAL3, ST3GAL4, ST3GAL6, B3GNT2, SPPL3, MGAT4A, and MGAT4B. In some embodiments, the cell additionally comprises a knock-in gene consisting of HST6GAL1.

[0008] Also provided are methods of producing a GM-CSF protein with a modified glycosylation pattern comprising preparing the non-naturally occurring cell described above and isolating the GM-CSF protein.

[0009] The invention provides a non-naturally occurring cell comprising genes engineered for glycosylation modification of GM-CSF, as described herein.

[0010] The invention provides a method of treating a subject comprising administering to a subject in need an effective amount of a modified GM-CSF, as described herein. In embodiments, the subject is immunosuppressed.

[0011] The invention provides a method of modifying a GM-CSF, as described herein.-2-53562250.5Atorney Docket No. 24978-1010BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows a flowchart of the study. First, an HPC4-tagged GM-CSF expression vector was generated, and was used to transiently transfect glycoengineered CHO cell lines to produce various GM-CSF glycovariants. After confirming the successful production of multiple glycoforms using Western blot, the variants were purified using HPC4 affinity chromatography. After verifying product purity, the glycans were profiled, and GM-CSF bioactivity was assessed using a TF-1 assay.

[0013] Figure 2 shows an overview of TF-1 assay to determine GM-CSF bioactivity. The assay began with 24-hour GM-CSF starvation of TF-1 cells, followed by treatment with GM- CSF glycovariants across a serial dilution. After a 72-hour incubation, cell proliferation was measured.

[0014] Figure 3 shows a western blot image of harvests from CHO-S and glycoengineered cell lines (Marker, GM-CSF-A: GM-CSF-B: CL107, GM-CSF-C: CL187, GM- CSF-D: CL419, GM-CSF-E: CL5957, GM-CSF-F: CL5967).

[0015] Figure 4 shows a SDS-PAGE image for purified GM-CSF products (M:Marker, EGM-CSF-A, 2:GM-CSF-B, 3:GM-CSF-C, 4: GM-CSF-D, 5:GM-CSF-E and 6: GM-CSF-F).

[0016] Figure 5: Terminal sialic acid abundance results regarding their linkage in total peak area percentage (TPA%) for GM-CSF-A, C, D, E, and F are depicted. Glycan structures are shown above the graph. Terminal sialic acids are represented as NANA(Neu5Ac), and all galactose (Gal) and GalNAc can potentially be occupied by sialic acid. Only one sialic acid is shown since these occupancies were not specifically analyzed. (ND: not determined).

[0017] Figure 6: TF-1 assays were conducted to measure bioactivity, and dose-response curves were generated for GM-CSF-A, B, C, D, E, and F, along with E. coli-produced GM-CSF as a control greyscale curve. All GM-CSF variants were assayed at increasing concentrations, and data are depicted as log-transformed concentration (ng / mL). The results of these assays are depicted in panels A-F, respectively.-3-53562250.5Atorney Docket No. 24978-1010

[0018] Figure 7: EC50 (Panel A) and top response (Panel B) values for erh-GM-CSF as E. coll and GM-CSF-A, C and D, respectively.

[0019] Figure 8: Illustration of tetra-antennary N-glycans capped with sialic acids (NeuAc) alone (left) and featured with poly-N-acetyllactosamine extension the P6 antenna and capped with sialic acids (NeuAc)(right). Lines indicate the reported functions of glycosyltransferase genes examined in this study (Yang et al. 2015). Transparent glycan moieties indicate their absence in cells with deletions of the genes listed in the grey box. The (+ hST6GALl) represents the knock-in of that gene, resulting in the incorporation of a novel glycan moiety typically absent from CHO-produced proteins.

[0020] Figure 9: Log-transformed EC50 values (base- 10) calculated from non-linear regression analysis using a three-parameter equation across GM-CSF products. The data represent the mean log-transformed EC50 values from three biological replicates, with error bars indicating standard deviation. The statistical significance of GM-CSF bioactivity responses was assessed by comparing WT GM-CSF using a t-test. Significance levels are indicated on the graph as follows: p < 0.01 (**), p < 0.001 (***).

[0021] Figure 10: MGAT5 expression is coexpressed in high GM-CSF producing T lymphocytes. Panel A shows GM-CSF expression in GM-CSF+ and GM-CSF- T cell populations shown as boxplots with the median lines indicated. Panel B shows scatter plot of MGAT5 and GM-CSF expression levels, with Pearson’s correlation used for statistical analysisDETAILED DESCRIPTION

[0022] The present invention provides compositions, methods, and systems comprising a non-naturally occurring GM-CSF protein.

[0023] The invention provides a modified GM-CSF comprising glycoengineered GM- CSF having changes to glycan branching and sialylation to modulate pharmaceutical activity, as described herein.

[0024] The invention provides a non-naturally occurring cell comprising genes engineered for glycosylation modification of GM-CSF, as described herein.-4-53562250.5Atorney Docket No. 24978-1010

[0025] The invention provides a method of treating a subject comprising administering to a subject in need an effective amount of a modified GM-CSF, as described herein. In embodiments, the subject is immunosuppressed.

[0026] The invention provides a method of modifying a GM-CSF, as described herein. Specifically, the invention provides a non-naturally occurring cell engineered for production of granulocyte-macrophage colony-stimulating factor (GM-CSF) with an altered glycosylation pattern comprising a tagged CSF2 gene, and one or more knockout genes selected from the group consisting of MG AT 5, ST3GAL3, ST3GAL4, ST3GAL6, B3GNT2, SPPL3, MGAT4A, and MGAT4B. In some embodiments, the cell additionally comprises a knock-in gene consisting ofHST6GALl.

[0027] Also provided are methods of producing a GM-CSF protein with a modified glycosylation pattern comprising preparing the non-naturally occurring cell described above and isolating the GM-CSF protein.

[0028] In some embodiments, the cell is a CHO cell, a CHO-S cell, or a human cell.

[0029] Also provided are methods of preparing a cell culture of the non-naturally occurring cell comprising: inactivation of one or more knockout genes selected from the group consisting of MGAT5, ST3GAL3, ST3GAL4, ST3GAL6, B3GNT2, SPPL3, MGAT4A, and MGAT4B; transfection of a CHO-S cell with a plasmid vector; and cultivation of the resulting cell. In some embodiments, the method additionally comprises purification with an anti-protein C affinity column.

[0030] In some embodiments, the invention provides a protein having modified glycan branching or sialylation. In some embodiments, the protein displays an activity which is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the wild-type activity. In some embodiments, the protein displays an activity which is at least 100%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, or at least 190% of the wild-type activity.-5-53562250.5Atorney Docket No. 24978-1010

[0031] In some embodiments, genes are transfected into the cell. In some embodiments, the genes are transfected into the cell on one or more vectors. In some embodiments, the vector or vectors which re-introduce the genes are each independently selected from a plasmid, a viral vector, and a cosmid. In some embodiments, each of the genes are transfected into the cell on plasmids. In embodiments, the invention provides that the genes are transfected into the cell on separate plasmids. In some embodiments, the genes are transfected into the cell on the same plasmid.

[0032] In some embodiments, one or more of the vectors which are transfected into the cell to reintroduce the knocked out genes (e. ., Gs and Aspg) further comprise the gene encoding the heterologous protein of interest. In some embodiments, the gene encoding the heterologous protein of interest is controlled by a different promoter than that which controls the gene.

[0033] The cells and systems provided herein are useful for the expression of heterologous proteins of interest. The heterologous protein of interest can be of a wide variety of types and used in a variety of applications. For example, in some embodiments, the heterologous protein of interest is an active pharmaceutical ingredient, a reagent in an industrial process, or an ingredient in a diagnostic reagent. Examples of heterologous proteins of interest compatible with the systems provided herein include antibodies and antigen binding fragments thereof, antibody fusions, Fc domain fusions, cytokines, cytokine receptors, signaling proteins, enzymes, viral proteins, cancer antigens, peptide hormones, fusion proteins of any of these, and variants of any of these. In some embodiments, the heterologous protein of interest is a mammalian protein or a derivative thereof. In some embodiments, the heterologous protein of interest is a human protein or a derivative thereof.

[0034] In some embodiments, the heterologous protein of interest has at least one CHO sialic acid residue. In some embodiments, the protein has no human sialic acid residues. In some embodiments, the glycan branching is tetra-antennary. In some embodiments, the glycan branching is bi-antennary. In some embodiments, the protein is deglycosylated. Also provided are methods of treating a subject comprising administering to a subject in need an effective amount of a modified GM-CSF protein prepared with the methods described above. In some cases, the subject is immunosuppressed. In some cases, the subject is being treated for cancer.-6-53562250.5Atorney Docket No. 24978-1010

[0035] Also provided herein are selection systems for heterologous protein expression in the cells provide herein. In some embodiments, the selection system comprises a cell as provided herein (e.g., a non-naturally occurring mammalian cell) comprises one or more vectors (e.g., plasmids) encoding a protein of interest and a selection medium.

[0036] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0037] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0038] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, 2nded. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Remington, The Science and Practice of Pharmacy, 20thed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22thed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0039] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a protein, a pharmaceutical composition, and / or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other-7-53562250.5Atorney Docket No. 24978-1010 elements (or components or steps) not expressly listed or inherent to the protein, pharmaceutical composition and / or method.

[0040] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0041] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a fusion protein, pharmaceutical composition, and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0042] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0043] The term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B and / or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0044] It is understood that aspects and embodiments of the invention described herein include “consisting” and / or “consisting essentially of’ aspects and embodiments.-8-53562250.5Atorney Docket No. 24978-1010

[0045] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and / or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0046] As used herein, the term “tagged” refers to proteins or genes which have had “tag” sequences appended to either end of the protein sequence to aid in purification or identification. For example, an HPC4 tag comprises amino acids which are appended to the beginning or end of a protein sequence to create binding with anti-protein C antibodies, typically for the purposes of purification with anti-protein C affinity chromatography.

[0047] As used herein, the term “specific productivity” refers to the amount of an indicated protein produced by an average cell in a culture over a given time. Specific productivity herein is typically given in units of pg / cell / day (unless otherwise specified) and is sometimes denoted qP. Unless otherwise specified, specific productivity values provided herein are calculated over an entire culture duration (e.g., time from transfer of cell culture to new media until final measurement).-9-53562250.5Atorney Docket No. 24978-1010

[0048] As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and / or non-human mammals.

[0049] As used herein the term “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which demethylation compound(s), is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0050] In an aspect, the disclosure provides a pharmaceutical composition comprising the engineered protein or cell of the disclosure and one or more pharmaceutically acceptable excipients or diluents.

[0051] As used herein the term “pharmaceutical composition” refers to pharmaceutically acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers.-10-53562250.5Atorney Docket No. 24978-1010

[0052] As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and / or non-human mammals.

[0053] As used herein the term “pharmaceutically acceptable diluent or excipient” or “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which an cell of the disclosure, is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable diluent or excipient” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0054] Formulations of a pharmaceutical composition suitable for administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable diluents or excipients, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.-11-53562250.5Atorney Docket No. 24978-1010Formulations may also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents or sterile, pyrogen-free, water. Exemplary administration forms may include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.

[0055] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, and / or aromatic substances and the like which do not deleteriously interact with the formulation. In some embodiments, the pharmaceutical composition comprises said cells in combination with other therapeutically active agents. In some embodiments, the disease cell phenotype is that of a malignant cell.

[0056] The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances, the combination partners show a cooperative, e.g., synergistic effect. The terms “co-admini strati on” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the-12-53562250.5Atorney Docket No. 24978-1010 active ingredients, e g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0057] The present invention provides engineered cells derived from a renewable source.

[0058] In an aspect, the disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering the engineered protein or cell of the disclosure or the pharmaceutical composition of the disclosure to the subject. In some embodiments, the disease or disorder is a malignancy.

[0059] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0060] In some embodiments, administering comprises administering a therapeutically effective amount to a subject.

[0061] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a cell to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some-13-53562250.5Atorney Docket No. 24978-1010 manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0062] As used herein, the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0063] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0064] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can-14-53562250.5Atorney Docket No. 24978-1010 encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. In some embodiments, the engineered cell or pharmaceutical composition comprising said engineered cell of the disclosure is administered in a prophylactically effective amount.

[0065] The proteins, cells or pharmaceutical compositions of the disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired. The proteins, cells or pharmaceutical compositions are typically suitable for parenteral administration, wherein administration includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intranasal, intratracheal, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, intraocular, intradermal, intrasynovial injection or infusions, intra-tumoral; and kidney dialytic infusion techniques. In some embodiments, the cells, or pharmaceutical compositions of the present disclosure comprise intravenous administration. In some embodiments, the cells, or pharmaceutical compositions of the present disclosure comprise intra-tumoral administration. In some embodiments, the cells, or pharmaceutical compositions are administered to a patient in a similar fashion to previous clinical work with cell-based therapies using unmodified cells.

[0066] In some embodiments, the engineered cell or pharmaceutical composition comprising said cells of the disclosure are administered in combination with a combination partner. The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where the engineered cell, or pharmaceutical composition comprising said engineered cell of the disclosure, and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some-15-53562250.5Atorney Docket No. 24978-1010 circumstances the combination partners show a cooperative, e.g., synergistic effect. The terms “co- administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0067] “Polymerase chain reaction” (PCR) generally refers to a process that uses multiple cycles of nucleic acid denaturation, annealing of primer pairs to opposite strands (forward and reverse), and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. There are many permutations of PCR known to those of ordinary skill in the art.

[0068] A “sequence” of a nucleic acid refers to the order and identity of nucleotides in the nucleic acid. A sequence is typically read in the 5' to 3' direction. The terms “identical” or percent “identity” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection.-16-53562250.5Atorney Docket No. 24978-1010

[0069] In some embodiments, the Cas protein or the variant thereof is a Cas9 protein or a variant thereof. Isolated Cas9-crRNA complex from the S. thermophilus CRISPR-Cas system as well as complex assembled in vitro from separate components demonstrate that it binds to both synthetic oligodeoxynucleotide and plasmid DNA bearing a nucleotide sequence complementary to the crRNA. It has been shown that Cas9 has two nuclease domains — RuvC- and HNH-active sites / nuclease domains, and these two nuclease domains are responsible for the cleavage of opposite DNA strands. In some embodiments, the Cas9 protein is derived from Cas9 protein of . thermophilus CRISPR-Cas system. In some embodiments, the Cas9 protein is a multi-domain protein having about 1,409 amino acids residues.

[0070] It should be appreciated that any CRISPR-Cas systems capable of disrupting the double stranded nucleic acid and creating a loop structure can be used in the present methods. For example, the Cas proteins provided herein may include, but not limited to, the Cas proteins described in Haft et al., PLoS Comput BioL, 2005, 1(6): e60, and Zhang et al., NucL Acids Res., 2013, 10.1093 / nar / gktl262. Some of these CRISPR-Cas systems require that a specific sequence be present for these CRISPR-Cas systems to recognize and bind to the target sequence. For instance, Cas9 may require the presence of a 5'-NGG protospacer-adjacent motif (PAM). Thus, in some embodiments, a PAM sequence or a sequence complementary to a PAM sequence is engineered into the target nucleic acid for initiating the binding of the CRISPR-Cas systems to the target nucleic acid.

[0071] As used herein, the term “guide polynucleotide” (e.g., a guide RNA), refers to a polynucleotide sequence that can form a complex with an endonuclease ( .g., Cas protein such as Cas9) and enables the endonuclease to recognize and optionally cleave a target site on a polynucleotide such as DNA. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence). Optionally, the guide polynucleotide can comprise at least one nucleotide, phosphodiester bond, or linkage modification such as, but not limited, to locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), 5-methyl dC, 2,6-Diaminopurine, 2'-Fluoro A, 2'-Fluoro U, 2’-O-Methyl RNA, Phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or 5' to 3' covalent-17-53562250.5Atorney Docket No. 24978-1010 linkage resulting in circularization. In some embodiments, the guide polynucleotide does not solely comprise ribonucleic acids (RNAs). In other embodiments, the guide polynucleotide does solely comprise ribonucleic acids (RNAs). A guide polynucleotide that solely comprises ribonucleic acids is also referred to as a “guide RNA”.

[0072] In general, a guide polynucleotide is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide polynucleotide and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0073] In some embodiments, the cell of the instant invention is an adherent cell line. In some embodiments, gene knockout and transfection with the genes encoding the protein occur with the cell or cells in an adherent format. In some embodiments, the cells are then adapted to a suspension culture. In some embodiments, the cell is in a suspension culture. Adaptation to a suspension culture can be performed using a wide variety of methods known in the art, including clonal isolation and transfer to suspension growth and culture conditions. Alternatively, transfection and / or gene knockout can also be performed in a suspension format. Once in a suspension culture format, the cells can be cultured using a wide variety of methods, including without limitation shake flask formats, batch bioreactors, and the like. In suspension culture, cells can be grown to a desired volume (e.g., at least IL, at least 5L, at least 10L, at least 50L, at least 100L, or larger volumes for industrial scale production) and grown for a desired amount of time to achieve optimal expression and characteristics of the heterologous protein of interest. In addition to large volume suspension culture formats, large scale production of heterologous proteins of interest using the cells provided herein can also be accomplished using adherent cell-18-53562250.5Atorney Docket No. 24978-1010 formats, such as through use of fixed bed bioreactors, roller bottle formats, and the like. Cell cultures can be supplemented in a variety of methods to enhance protein production and cell survival, such as perfusion technologies to continuously provide fresh media to the culture.

[0074] In some embodiments, the cell includes one or more genes of the cell which have been modified. In some embodiments, one or more genes of the cell have been knocked-out or otherwise made inoperative. In some embodiments, knock-out genes are prepared by introducing one or more mutations into the gene such that the protein encoded by the gene is either not expressed or expressed in an inactive form. Non-limiting examples of gene modifications which can accomplish the knock-out include frameshift modifications (e. ., insertions or deletions of nucleotides which change the open reading frame of the gene), point mutations (e.g., substitution of one or more nucleotides such that the gene encodes a protein which contains a substituted amino acid which renders it inactive), deletion or modification of the start codon, insertion or deletion of one or more codons encoding amino acids of the encoded protein, and the like. In some embodiments, the modification includes deletion (e.g., excision) of the entire gene from the genome of the organism. In some embodiments, generating a knock-out of a given gene involves modification of both alleles of the gene.

[0075] Knock-out versions of genes as provided herein can be prepared using a variety of methods and / or gene editing systems well known in the art. Exemplary methods and gene editing systems are described in Gaj et al., Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023754 doi: 10.1101 / cshperspect.a023754. A gene editing system as provided herein is a system which comprises all the necessary components in order to effectuate the desired alterations into a target gene. In some embodiments, the gene editing system comprises exogenous functionalities which work with endogenous systems (e.g, host cell proteins implicated in homology directed repair) to effectuate the desired alteration to the desired allele.

[0076] In some embodiments, the gene editing system comprises a guide nucleic acid. In some embodiments, the gene editing system comprises a guide RNA. In some embodiments, the guide RNA is configured to recruit the endonuclease enzyme (e.g., CRISPR-Cas9 and derivatives thereof) in order to effectuate a cut in the desired gene.-19-53562250.5Atorney Docket No. 24978-1010

[0077] In some embodiments, the gene editing system comprises an endonuclease. In some embodiments, the endonuclease is configured to perform a cut in the target gene. In some embodiments, the endonuclease is configured to perform a cut in the target gene when in complex with the guide RNA. In some embodiments, the endonuclease is selected from a meganuclease, a Transcription Activator Like Effector Nucleases (TALEN), a Zinc-Finger Nucleases (ZFN), and a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- associated system (Cas), and derivatives thereof. In some embodiments, the endonuclease is a meganuclease or a derivative thereof. In some embodiments, the endonuclease is a TALEN or a derivative thereof. In some embodiments, the endonuclease is a ZFN or a derivative thereof. In some embodiments, the endonuclease is a Cas or a derivative thereof. In some embodiments, the endonuclease is a Cas9 or a derivative thereof. In some embodiments, the gene editing system comprises a guide RNA and an endonuclease enzyme.

[0078] In some embodiments, the gene editing system comprises an exogenous nucleic acid (e.g., DNA) repair template configured to introduce a desired modification into the targeted gene. In some embodiments, the nucleic acid repair template comprises homology arms which overlap with the target gene and flank the target modification site.

[0079] In some embodiments, the gene editing system or a portion thereof is encoded in a vector. In some embodiments, the vector is a plasmid, a viral vector, a cosmid, or an artificial chromosome. In some embodiments, the vector is a plasmid, a viral vector, or a cosmid. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. Examples of viral vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, and the like. In some embodiments, all exogenous components of the gene editing system are encoded on vectors. In some embodiments, all components of the gene editing system are encoded on a single vector.EXAMPLES

[0080] Also provided are methods of treating a subject comprising administering to a subject in need an effective amount of a modified GM-CSF protein prepared with the method-20-53562250.5Atorney Docket No. 24978-1010 above. This example investigated the effects of glycosylation, glycan branching, and sialic acid variations on GM-CSF bioactivity. The workflow is summarized in Figure 1.Material and MethodsPlasmid design and cloning

[0081] An expression vector harboring GM-CSF with an HPC4 tag was generated with NEBuilder HiFi DNA assembly (NEB #M5520). The plasmid sequence was confirmed using Sanger sequencing (Eurofins) and extracted with gigaprep kit (Macherey -Nagel, Cat#740548).Cell cultivation

[0082] CHO-S cells (Gibco, Cat#AT 11364-01) were obtained and glycoengineered as previously described14Both wild-type and glycoengineered cell lines were used for GM-CSF production. The glycoengineered cell lines used here were generated with one or more knockouts or knock-ins of glycosyltransferase genes, responsible for variations in N-linked glycan branching or sialylation (Table 1).

[0083] For cultivations, CD CHO medium (Gibco, Cat#l 0743029) supplemented with 8mM L- Glutamine (Gibco, Cat#A2916801) was used. Cell were maintained in 125 mL Polycarbonate Erlenmeyer shake flasks with vent caps (Coming, Cat #431143) with 30 mL working volume and 6-well flat bottom non-treated well plates (Coming, Cat#351146) with 3 mL working volume at 37°C with 5% CO2 on an orbital shaker platform at 120 rpm with 25 mm amplitude. NucleoCounter NC-250 was used to monitor viable cells.

[0084] The expected glycan structures resulting from the deletion or insertion of glycosyltransferases are illustrated in Figure 8. However, the function of signal peptide peptidase like 3 (Sppl3) is not shown, as it does not add or remove specific glycan bonds. Rather, Sppl3 decreases glycosyltransferase abundance by cleaving them in the Golgi, which can, in turn, increase the glycan content of proteins. The a2,3 linkage is referred to as CHO-like sialylation because CHO cells can attach sialic acids through this linkage. In contrast, St6gall, which is responsible for attaching sialic acids with an a2,6 linkage, is found to be silenced in CHO cells and the dominant linkage in humans.-21-53562250.5Attomey Docket No. 24978-1010

[0085] Specifically, these cell lines demonstrate reduced glycan branching, no CHO sialylation, only CHO sialylation, and no CHO sialylation with human sialic acid as biantennary glycan structure and the same formation as tetraantennary structure. The same cultivation methods were used for CHO cell maintenance. Table 1: Glycoengineered cell lines detailed information with corresponding phenotype.-22-53562250.5Atorney Docket No. 24978-1010

[0086] To test bioactivity, the TF-1 cell line was used (ICLC, Cat#HTL05001) since it is a cell line derived from the bone marrow tissue of an erythroleukemia patient and is dependent on GM-CSF for cell maintenance. TF-1 cells were cultured in RPMI-1640 medium (Sigma- Aldrich, Cat#R5886-500ML) containing 2mM L-Glutamine (Gibco, Cat#A2916801), 20% FBS (Gibco, Cat#A5256801) and 5 ng / mL E.coli produced GM-CSF (Peprotech, Cat#300-03). Cells were maintained in T25, T75, and T125 untreated flasks with vent caps (Coming, Cat#431463, 431464U, #431465). Flasks were incubated at 37°C with 5% CO2 without shaking. Cells were counted with AO / PI staining solution, prepared by a 1 : 1 mixture of acridine orange (Nexcelom, Cat#CS2-0106) and propidium iodide (Nexcelom, Cat#CSl-0109). For counting, 10 pL dye and 10 pL cells were mixed and incubated for 30 seconds, and 10 pL solution was loaded on the cell counter (Denovix Celldrop FL). Viable cell densities were measured using an AO / PI assay.Transfection of GM-CSF and batch culture

[0087] One day before transfection, cells were seeded as 1.4 xlO6cells / mL in 500 mL CD-CHO medium in 2L shake flasks (Corning, Cat#431255). The next day, transfection mixes were prepared with a final concentration of 1 pg / mL plasmid DNA in OptiPRO SFM media (Gibco) and 3.75 pg / mL LPEI MAX (Polysciences, Cat#24765-1). Cells were incubated at 37°C with 5% CO2 on an orbital shaker platform at 120 rpm as explained previously. On Day 1, in addition to the 0.2% anti-clumping reagent, cells were also fed with 1% Tryptone N1 (Organotechnie, Cat#19553) and 0.6 mM Valproic acid sodium salt solution (Sigma, Cat#P4543- 10g). The cultures were incubated at 32°C, while keeping other conditions the same. On Day 4, the supernatants were harvested through a two-step centrifugation process - first at 300 g for 5 minutes, and then the resulting supernatant was centrifuged at 1000 g for 10 minutes. The harvested materials were then stored at -70°C.Purification of GM-CSF products

[0088] Supernatants from a 500 mL cell culture batch, grown in 2L shake flasks (Corning, Cat#431255), were thawed overnight at 4°C and fully clarified by centrifugation at 4500 g for 30 minutes. Samples were concentrated fivefold by consecutive centrifugation using AMICON® Ultra Centrifugal Filters with a 3 kDa molecular weight cutoff (MWCO) (Sigma Aldrich, Cat#UFC900308) at 4500 g for 8 minutes at 4°C.-23-53562250.5Attomey Docket No. 24978-1010

[0089] Purification was conducted on an AKTA pure system (GE Healthcare) with affinity chromatography using an HPC4 (anti-protein C) tag antibody matrix (Roche, Cat#l 1815024001). The matrix was packed in a 2mL HiScale(Cytiva) column, and the purification method relies on the interaction between the HPC4 antibody and the Protein C-tag. This reaction is calcium-dependent, requiring the addition of ImM CaCk to the harvests. Buffers were prepared according to the manufacturer’s instructions. The running method is explained in Table 2. The column was regenerated to discard all non-bound proteins, and the proteins were eluted as 1 mL fractions into a 96-deep well plate (Coming, Cat#CLS3960).Table 2: Log-transformed EC50 values (base- 10) calculated from non-linear regression analysis using a three-parameter equation for GM-CSF variants. Data represent individual values from triplicate experiments.

[0090] After purification, products were concentrated for bioactivity and glycan analysis by sequential centrifugation using AMICON® Ultra Centrifugal Filters with the same cutoff (Sigma Aldrich, Cat#UFC8003) at 4500 g for 8 minutes at 4°C. The goal was to achieve a concentration range of 0.2-0.5 mg / mL for glycan analysis.Verification of GM-CSF production with Western blot-24-53562250.5Atorney Docket No. 24978-1010

[0091] The same procedure explained in section 3.2.7 was followed. GM-CSF production was detected by using Protein C-Tag antibody (HPC4) (GenScript, Cat#A00637-40) as the primary antibody with 1 : 1000 dilution in blocking solution and Goat Anti -Rabbit IgG antibody (Abeam, Cat#ab6721) as a secondary antibody, which is prepared with 1 :3000 dilution.Determination of purity of purified products with SDS-PAGE

[0092] Products were prepared in 2 pg amounts, and the procedures explained in the first part of the Western blot were followed to run the SDS-PAGE electrophoresis.TF1 assay

[0093] TF-1 cells were subjected to starvation by passaging them in media that did not contain GM-CSF and then incubated for 24 hours. The next day, cells were washed by centrifuging at 300 g for 10 minutes and then counted. The cells were resuspended in fresh media without GM-CSF, and 50,000 cells (0.1 mL per well) were seeded into each well of a 96- well plate (Corning Costar, Cat#3595). Recombinant E. co / z-produced GM-CSF and the glycovariant products were diluted to concentrations of 100, 25, 6.25, 1.56, 0.39, 0.10, 0.024, and 0.002 ng / mL through serial dilution in GM-CSF-free culture media. The plate setup was designed with triplicates for each condition, with a total volume of 200 pL per well. GM-CSF- free media was added to blank wells, and E. co / z-produced GM-CSF and glycoengineered product dilutions were added to the wells for GM-CSF treatment. Plates were incubated for 72 hours. Then, viable cell proliferation was monitored using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Cat#3582), which contains MTS tetrazolium inner salt. MTS solution is reduced by mitochondrial enzymes, generating formazan salt with color change. After 3 hours of incubation, the plate was read at 490 nm (MTS) signal and 630 nm background by a plate reader (BMG LabTech). Bioactivity assay steps are demonstrated in Figure 2.Glycan analysis

[0094] Glycan analysis was outsourced to Glyxera. First, A-glycan moieties were released with the PNGase enzyme, and A-glycome was further treated with two neuraminidases with different specificities: Sialidase A(a 2-3, 6, 8, 9) and Sialidase S(a 2-3). Sialic acid-25-53562250.5Atorney Docket No. 24978-1010 structures were analyzed with multiplexed capillary gel electrophoresis (CGE) with laser- induced fluorescence detection (xCGE-LIF) based Standard GlycoprofilingPLUS15.Calculations

[0095] Nonlinear regression analysis was conducted to generate a dose-response curve model with a 3-parameter logistic equation using GraphPad Prism 10. EC50 values, which represent the concentration of the agonist that gives a response halfway between the maximum and minimum response levels, were calculated for each GM-CSF glycovariant using the following equation, where X represents the logarithms of GM-CSF concentrations, Y represents absorbance values, and Maximum and Minimum represent the top and bottom absorbance values:Results

[0096] The transcriptomics analysis of GM-CSF+ and GM-CSF- human T cells was investigated. The study fractionated GM-CSF+ and GM-CSF- populations using a cytokine secretion assay (Emming et al. 2020) (Figure 10, panel A). Interestingly, MGAT5 (baseMean: 14183.86, log2 fold-change: 0.83, p-value: 1.05xl06, Benjamini-Hochberg corrected p-value: 2.50xl0'5) significant upregulated in the differential expression analysis and positively correlated with the increased GM-CSF expression (Figure 10, panel B).

[0097] In this study, MGAT5 (GnT-V, gene ID:4249) was selected, whose function matches the glycan structure of GM-CSF. MGAT5 (Gene ID: 4249) catalyzes the addition of a GlcNAc residue to the al, 6 mannose through a pi,6-linkage, which is important for forming tetra-antennary glycan structures (Dennis et al. 2014). This enzyme is present in CHO cells, as CHO-derived GM-CSF exhibits bi-, tri-, and tetra-antennary glycans (Forno et al. 2004).Identification of GM-CSF products re¬53562250.5Attomey Docket No. 24978-1010

[0098] GM-CSF derived from CHO-S and glycoengineered cell lines was assessed using Anti-HPC4 Western blots, showing the samples had heterogeneous glycans spanning from 20 to 40 kDa. Wild-type (WT) and Mgat5 knock-out (KO) were observed as smear bands between 35- 40 kDa. GM-CSF lacking CHO-like sialylation exhibited glycoforms spanning from 20 to 38 kDa, while GM-CSF with CHO-like sialylation predominantly appeared around 38 kDa with a smear band at 35 kDa. GM-CSF with human sialic acids in bi-antennary form, with less branching, had more uniform glycoforms between 30-36 kDa, whereas the tetra-antennary variant showed more branched glycans, resulting in glycoforms between 30-40 kDa (Figure 3).Purity check for GM-CSF products

[0099] Six products, referred to as GM-CSF-IDs, were purified and analyzed via SDS-PAGE for purity assessment (Table 3, Figure 4). These bands corresponded to those observed in the Western blot of the harvest samples (Figure 3). Except for GM-CSF-F, all products exhibited a band around 70 kDa, likely due to the upconcentration process. These additional bands were considered negligible due to the dilutions used for bioactivity assays. The products were concentrated to the concentrations outlined in Table 3, nearly in a 0.1 mL volume. GM-CSF-F, produced by host cells generating tetraantennary structures, showed more smear band formations than GM-CSF-E, which has bi-antennary glycoforms. Following purity confirmation, all samples were selected for further glycan analysis, except GM-CSF-B, due to insufficient quantity.Table 3: Purified GM-CSF products and concentrations-27-53562250.5Atorney Docket No. 24978-1010Confirmation of glycan structure

[0100] Glycan analysis confirmed that glycoengineered cell lines producing GM-CSF-D and GM-CSF-E exhibited expected glycan branching patterns based on their host cell line genotypes, as depicted in Table 4 (see host cell lines in Table 3). GM-CSF-C predominantly displayed tri-antennary and tetra-antennary glycans, with tri-antennary glycans being the most abundant. Interestingly, only 2.4% of GM-CSF-F glycans were identified as tetra-antennary, while 20.3% of the total peak area (TP A) corresponded to unannotated peaks. It is anticipated that these peaks may result from N-acetyllactosamine (LacNAc) extensions that can be generated due to B3gnt2 expression from GM-CSF-producing host cells (Table 1). Since such glycans with LacNAc extensions are not in the Glyxera database (glyXtoolCE, glyXera GmbH, Germany), further analysis would be needed to confirm the presence of these moieties.

[0101] As determined by glycan analysis, GM-CSF-E exhibited more homogeneous glycan branching, consisting only of mono- and bi-antennary structures (Table 4). It also showed more uniform band formation than other GM-CSF glycoforms in SDS-PAGE analysis (Figure 4). In contrast, other samples, including GM-CSF WT, GM-CSF-C, GM-CSF-D, and GM-CSF- F, displayed a broader range of glycan branching, including mono-, bi-, tri-, and tetra-antennary structures. These glycovariants were detected as more pronounced smeared bands, which is common when multiple glycoforms of a protein are present (Table 4, Figure 4).Table 4: Glycoprofiling results of GM-CSF products with glycan branching information. The total glycoforms, categorized by their corresponding branching structures, are presented as percentages of the total peak area (TP A). The glycoforms include mono-antennary, bi-antennary,-28-53562250.5Attomey Docket No. 24978-1010 tri-antennary, and tetra-antennary structures, with their respective distributions shown for each GM-CSF product.

[0102] Glycan analysis with neuraminidase treatment also verified that the GM-CSF glycoforms exhibited the expected terminal sialic acid composition, as terminal sialic acids predominantly attached via a2,3 linkages in GM-CSF-A and GM-CSF-D and a2,6 linkages in GM-CSF-E and -F (produced from ST6GAL1 expressing cells) in line with the host cell line glycosylation (Figure 5, Table 1). GM-CSF produced in WT CHO-S cells featured terminal sialic acids, with 41.7% linked via a2,3 linkage and 7.4% via a2,6 linkage. While inadequate amounts of GM-CSF-B were obtained to allow for glycoprofiling, its genotype indicates it should have sialylation patterns that mirror that of WT CHO-S cells, albeit with no tetra- antennary glycans. GM-CSF-C exhibited greatly reduced amounts of sialylation, with only 7.9%-29-53562250.5Atorney Docket No. 24978-1010 of glycans harboring terminal sialic acid linked via a2,3 linkage. In addition, GM-CSF-D showed 66.7% a2,3-linked terminal sialic acids. Meanwhile, GM-CSF produced from human ST6GAL1 inserted host cells (CL5957 and CL5967, respectively) displayed only a2,6-linked terminal sialic acids at 85.1% and 55.5%, respectively, as expected. These findings confirmed the sialic acid composition of the products and expected glycan branching, allowing testing their biological activity to proceed.Comparison of bioactivity among glycoengineered GM-CSF

[0103] The biological impact of glycoengineered GM-CSF on TF-1 cell proliferation was assessed by comparing their activity to E. co / z-produced GM-CSF (erh-GM-CSF) as a control. GM-CSF-A produced from CHO WT cells showed a proliferation rate similar to E. coli- produced GM-CSF. Although erh-GM-CSF had a lower ECso value than GM-CSF-A, this difference may be influenced by error bars in the high concentration readings of GM-CSF-A (Table 3, Figure 6A). The ECso values and top responses within confidence intervals were comparable between the two proteins (Figures 7A-B).

[0104] Notably, GM-CSF-B showed no detectable activity (Figure 6B). GM-CSF-C and D exhibited similar activity, although their maximum responses were lower (Figure 6C-D and Figure 7B). In addition, GM-CSF-C and D products had higher ECso values than erh-GM-CSF within the 95% confidence interval but with high variability (Figure 7A-B). GM-CSF-E and F displayed reduced bioactivity compared to erh-GM-CSF, as their maximum biological responses could not be identified even with a maximum concentration of 100 ng / mL (Figure 6E-F).

[0105] Notably, GM-CSF derived from the Mgat5 KO cell line (GM-CSF-B) and the human 5T6GAL7-inserted cell lines (CL5957 and CL5967, corresponding to GM-CSF-E and GM-CSF -F, respectively) exhibited significantly reduced proliferation rates as 20x, 16x, and 17x higher ECso values, respectively, compared to E. COII-^Q NQA GM-CSF (Figure 6 Panels B, E, F; Figure 9; Table 5). GM-CSF-C and D (produced from CL187 and CL419 host cell lines) showed comparable activity to E. coli-derived GM-CSF, with similar ECso values (Figure 6 Panels C-D; Figure 9; Table 5). However, GM-CSF-D exhibited variability in ECso values, potentially due to differences in biological responses among triplicates (Figure 6D, Table 2). Furthermore, GM--30-53562250.5Atorney Docket No. 24978-1010CSF-B, GM-CSF-E, and GM-CSF-F displayed significantly reduced bioactivity compared to GM-CSF derived from the WT cell line (GM-CSF-A) (Figure 9).Table 5: Mean of loglO-transformed EC50 values for each GM-CSF glycoform across three biological triplicates (Table 2) calculated by non-linear regression analysis using a three- parameter equation.

[0106] GM-CSF is a promising cytokine for treating immunosuppression patients h It contains two N- and four O- glycosylation sites3 6. Glycosylation is a critical quality attribute for glycoproteins, impacting on their characteristics such as bioactivity and immunogenicity16. GM- CSF glycans reduce immunogenicity by masking epitopes8. However, the influence of branched glycans and sialic acids on bioactivity has not been clearly defined,2 13. In this study, a panel of GM-CSF glycoforms were tested to rigorously evaluate the impact of sialic acid and glycan branching glycosylation on the protein's biological activity.

[0107] Notably, the study revealed that glycan branching is crucial for the bioactivity of glycosylated GM-CSF. GM-CSF was not active when it was produced by MGAT5 deleted cell line. Moreover, glycan branching did not influence GM-CSF bioactivity in the presence of human sialic acids.

[0108] Additionally, it was found that sialic acids exhibited different effects on bioactivity depending on their linkage to the A-glycan core. Human sialic acids appeared to reduce GM-CSF bioactivity. This aligns with a previous study that reported that a highly glycosylated GM-CSF variant from human T cells showed reduced bioactivity, even though the products (35-40 kDa) were heavier than theirs (28-32 kDa)4In contrast, CHO-specific sialic acids did not significantly affect GM-CSF bioactivity, as the removal or increase of a2,3 linked sialic acid abundance did not change the activity. This is consistent with the initial study, which did not report any difference in bioactivity between desialylated CHO-derived GM-CSF and-31-53562250.5Atorney Docket No. 24978-1010 wild-type GM-CSF12. These results contradict those of Hashimoto et al., as no enhanced bioactivity with sialic acid on GM-CSF was observed.

[0109] The sialic acid content of the products was verified, which matched with the host cell phenotypes. Glycan results showed that GM-CSF-A harbored 7.4% a2,6 linked terminal sialic acids, which was unexpected since ST6GAL1, encoding an enzyme responsible for a2,6- linked sialic acid addition to glycan structures, is reported to be silenced in CHO cells17-19. This may have resulted from the activation of St6gall and 2 genes or a technical error. GM-CSF-C exhibited 7.9% a.2,3 linked terminal sialic acid, which should not have existed since St3gal3, St3gal4, and St3gal6 genes were deleted in the host cell line (CL 187) to disrupt CHO sialylation. Some of these genes20may have been reactivated, adding sialic acid moieties to glycan groups.

[0110] Glycoengineered cell lines with B3gnt2 and Sppl3 knockouts enhanced these products’ sialic acid abundance since these genes are associated with competing sialic acid attachment and reducing glycosyltransferase prevalence in cells, respectively. B3GNT2 competes with sialyltransferases for the same substrate on tetra-antennary glycans for adding poly-N-acetyllactosamine chains21, and SPPL3 cleaves glycosyltransferases and decreases their abundance in cells22. These genes were deleted from GM-CSF-D and GM-CSF-E host cell lines. As expected, GM-CSF-D had more sialic acid and A. Additionally, around 30% more terminal sialic acid was found in GM-CSF-E compared to GM-CSF-F, which have biantennary and tetraantennary glycan structures, respectively. However, it was not possible to conduct a detailed glycan analysis for the GM-CSF-B produced by the Mgat5 KO cell line due to insufficient protein quantities.

[0111] GM-CSF glycans have been shown to decrease its immunogenicity8,10and determine the cytokine activity4,n. Human-derived GM-CSF variants with fewer glycosylation levels (16-18 kDa and 23-25 kDa) had similar bioactivity with erh-GM-CSF, whereas the highly glycosylated variant exhibited lower activity4Moonen el al. also stated that deglycosylation improved the CHO-produced GM-CSF activity. Regarding the impact of sialic acid, there is no consensus on the effect of sialic acids in glycome on GM-CSF bioactivity12,13. These results confirmed that CHO sialic acids do not affect the activity.-32-53562250.5Attorney Docket No. 24978-1010

[0112] Six GM-CSF glycoforms were produced, and five were characterized, confirming the glycan structures and sialic acid compositions. The results highlight the essential role of MGAT5 in maintaining GM-CSF bioactivity and provide insights into the differential effects of human and CHO-specific sialic acids on GM-CSF functionality.Table 6: HPC4 affinity chromatography running method.Bibliography

[0113] 1. Armitage, J. O. Emerging applications of recombinant human granulocyte-macrophage colony-stimulating factor. Blood 92, 4491-4508 (1998).

[0114] 2. Lazarus, H. M., Ragsdale, C. E., Gale, R. P. & Lyman, G. H.Sargramostim (rhu GM-CSF) as Cancer Therapy (Systematic Review) and An Immunomodulator. A Drug Before Its Time? Front. Immunol. 12, 706186 (2021).

[0115] 3. Wong, G. G. et al. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 228, 810-815 (1985).-33-53562250.5Atorney Docket No. 24978-1010

[0116] 4 Cebon, J. et al. Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity. J. Biol. Chem. 265, 4483-4491 (1990).

[0117] 5. Kaushansky, K., O’Hara, P. J., Hart, C. E., Forstrom, J. W. & Hagen, F. S.Role of carbohydrate in the function of human granulocyte-macrophage colony-stimulating factor. Biochemistry 26, 4861-4867 (1987).

[0118] 6. Kaushansky, K., Lopez, J. A. & Brown, C. B. Role of carbohydrate modification in the production and secretion of human granulocyte macrophage colonystimulating factor in genetically engineered and normal mesenchymal cells. Biochemistry 31, 1881-1886 (1992).

[0119] 7. Hussein, A. M. et al. Effects of granulocyte-macrophage colony stimulating factor produced in Chinese hamster ovary cells (regramostim), Escherichia coli (molgramostim) and yeast (sargramostim) on priming peripheral blood progenitor cells for use with autologous bone marrow after high-dose chemotherapy. Eur. J. Haematol. 55, 348-356 (1995).

[0120] 8. Gribben, J. G. et al. Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF. Lancet 335, 434-437 (1990).

[0121] 9. Dorr, R. T. Clinical properties of yeast-derived versus Escherichia coli- derived granulocyte-macrophage colony-stimulating factor. Clin. Ther. 15, 19-29; discussion 18 (1993).

[0122] 10. Rosier, B. et al. Mimicking Behcet’s disease: GM-CSF gain of function mutation in a family suffering from a Behcet's disease-like disorder marked by extreme pathergy. Clin. Exp. Immunol. 204, 189-198 (2021).

[0123] 11. Okamoto, M. et al. Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor. Arch. Biochem. Biophys. 286, 562-568 (1991).-34-53562250.5Atorney Docket No. 24978-1010

[0124] 12. Moonen, P., Mermod, J. J., Ernst, J. F., Hirschi, M. & DeLamarter, J. F.Increased biological activity of deglycosylated recombinant human granulocyte / macrophage colony-stimulating factor produced by yeast or animal cells. Proc. Natl. Acad. Set. U. S. A. 84, 4428-4431 (1987).

[0125] 13. Hashimoto, A. et al. Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments longterm bioactivity with delayed clearance in vitro. Cytokine 68, 118-126 (2014).

[0126] 14. Voldborg, B. G., Koi, S., Hansen, A. H. & Kildegaard, H. F.Glycosylation of proteins. US Patent (2023).

[0127] 15. Pralow, A., Cajic, S., Alagesan, K., Kolarich, D. & Rapp, E. State-of-the-Art Glycomics Technologies in Glycobiotechnology. Adv. Biochem. Eng. Biotechnol. 175, 379- 411 (2021).

[0128] 16. Walsh, G. & Jefferis, R. Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 24, 1241-1252 (2006).

[0129] 17. Lewis, N. E. et al. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nat. Biotechnol. 31, 759-765 (2013).

[0130] 18. Takeuchi, M. et al. Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J. Biol. Chem. 263, 3657-3663 (1988).

[0131] 19. Xu, X. et al. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat. Biotechnol. 29, 735-741 (2011).

[0132] 20. Chung, C.-Y. et al. Assessment of the coordinated role of ST3GAL3,ST3GAL4 and ST3GAL6 on the a2,3 sialylation linkage of mammalian glycoproteins. Biochem. Biophys. Res. Commun. 463, 211-215 (2015).-35-53562250.5Attomey Docket No. 24978-1010

[0133] 21. Zhuang, X. et al. Functional genomics identifies N-acetyllactosamine extension of complex N-glycans as a mechanism to evade lysis by natural killer cells. Cell Rep. 43, 114105 (2024).

[0134] 22. Voss, M. et al. Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation. EMBO J. 33, 2890-2905 (2014).-36-53562250.5

Claims

Attorney Docket No. 24978-1010We claim:

1. A non-naturally occurring cell engineered for production of granulocyte-macrophage colony-stimulating factor (GM-CSF) with an altered glycosylation pattern comprising a tagged CSF2 gene, and one or more knockout genes selected from the group consisting of MGAT5, ST3GAL3, ST3GAL4, ST3GAL6, B3GNT2, SPPL3, MGAT4A, and MGAT4B.

2. The cell of claim 1, wherein the cell additionally comprises a knock-in gene consisting of HST6GAL1.

3. The cell of claim 1, wherein the cell is a CHO cell.

4. The cell of claim 3, wherein the cell is a CHO-S cell.

5. The cell of claim 1, wherein the cell is a human cell.

6. The cell of claim 1, wherein the vector comprises one or more plasmids.

7. A modified GM-CSF protein produced by the cell of claim 1.

8. The protein of claim 7, wherein the protein has an activity which is at least 40% of the wild type GM-CSF activity.

9. The protein of claim 7, wherein the protein has at least one CHO sialic acid residue.

10. The protein of claim 7, wherein the protein has no human sialic acid residues.

11. The protein of claim 7, wherein the glycan branching is tetra-antennary.

12. The protein of claim 7, wherein the glycan branching is bi-antennary.

13. The protein of claim 7, wherein the protein is deglycosylated.

14. A method of preparing a cell culture of the non-naturally occurring cell of claim 1 comprising:Attomey Docket No. 24978-1010 inactivation of one or more knockout genes selected from the group consisting of MGAT5, ST3GAL3, ST3GAL4, ST3GAL6, B3GNT2, SPPL3, MGAT4A, and MGAT4B; transfection of a CHO-S cell with a plasmid vector; and cultivation of the resulting cell.

15. A method of producing a GM-CSF protein with a modified glycosylation pattern comprising preparing the non-naturally occurring cell of claim 1 and isolating the GM- CSF protein.

16. The method of claim 15, wherein the method additionally comprises purification with an anti-protein C affinity column.

17. A method of treating a subject comprising administering to a subject in need an effective amount of a modified GM-CSF protein prepared with the method of claim 15.

18. The method of claim 17, wherein the subject is immunosuppressed.

19. The method of claim 17, wherein the subject is being treated for cancer.-38-53562250.5

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