CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 61 /822,795, filed May 13, 2013, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Therapeutic polypeptides are an important class of therapeutic biotechnology products, and therapeutic Fc containing polypeptides, such as IVIG, Fc-receptor fusions, and antibodies (including murine, chimeric, humanized and human antibodies and fragments thereof) account for the majority of therapeutic biologic products.

Alzheimer disease (AD) is an age-related disease involving neurodegeneration that results in progressive loss of cognitive function. Five medications are currently used to treat the cognitive manifestations of AD: four are acetylcholinesterase inhibitors (tacrine, rivastigmine, galantamine and donepezil) and the other (memantine) is an NMDA receptor antagonist.

SUMMARY OF THE INVENTION

The invention encompasses the discovery that Fc-containing polypeptides that include branched glycans and that are sialylated on the branched glycan (e.g., on an a 1 ,3 and/or a 1 ,6 arm in the Fc region's N-linked glycosylation site), with, e.g., a NeuAc-a 2,6-Gal or NeuAc-a 2,3-Gal terminal linkage, are useful in treating neurodegeneration, e.g., in the treatment of neurodegenerative diseases such as Alzheimer's Disease. The present disclosure provides, in part, methods for treating neurodegeneration or neurodegenerative diseases by administering compositions containing such Fc-containing polypeptides as well as methods for evaluating, identifying, and/or producing (e.g., manufacturing) such polypeptides for the treatment of neurodegeneration.

In the first aspect, the invention features a method for the treatment of neurodegeneration. The method includes administering (e.g., to a subject in need thereof) a preparation that includes polypeptides having an Fc region (e.g., an Fc region of IgA, IgD, IgE, IgG, or IgM) wherein at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to and including 100%) of the branched glycans (e.g., on the Fc region) have at least one galactose connected to a respective terminal sialic acid (i.e., are sialylated).

In certain embodiments, the branched glycans (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to and including 100%) are sialylated on an a 1 ,3 arm of the branched glycan (e.g., by way of a NeuAc-a 2,6-Gal and/or a NeuAc-a 2,3-Gal terminal linkage).

In some embodiments, the polypeptides are derived from IVIG (e.g., sialylated IgGs purified or enriched from IVIG ; modified (e.g., enzymatically sialylated) IVIG ; or Fc regions produced from IVIG). In other embodiments, the polypeptides are Fc regions derived from IVIG (e.g., papain digested and sialylated).

In certain embodiments, the preparation includes recombinant polypeptides having an Fc region

(e.g., an Fc region of IgA, IgD, IgE, IgG, or IgM) wherein at least 1 0% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to and including 100%) of the branched glycans (e.g., on the Fc region) have at least one galactose connected to a respective terminal sialic acid (i.e., are sialylated).

In certain embodiments of any of the foregoing methods, the polypeptides include an IgG Fab region that is or is not sialylated.

In other embodiments, the polypeptides are Fc regions (e.g., recombinant Fc regions) or Fc- region-containing (e.g., recombinant Fc-region-containing) polypeptides.

In further embodiments of any of the foregoing methods, the polypeptides are administered in a pharmaceutical formulation (e.g., including a pharmaceutically acceptable carrier or diluents).

In another aspect, the invention features a method of manufacturing a pharmaceutical product for the treatment of neurodegeneration. This method includes: providing a sample of a test preparation including polypeptides (e.g., polypeptides having an Fc region) ; determining the percent of branched glycans (e.g., on the Fc regions) of the polypeptides that possess a galactose moiety connected to a terminal sialic acid; and processing the test preparation into a pharmaceutical product for the treatment of neurodegeneration if the percent of branched glycans (e.g., on the Fc regions) of the polypeptides is greater than 10% (e.g., greater than 1 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to and including 1 00%), thereby manufacturing a pharmaceutical product for the treatment of neurodegeneration.

In a further embodiment, the invention features another method of manufacturing a

pharmaceutical product. This method includes: providing a sample of a test preparation including polypeptides (e.g., polypeptides having an Fc region) ; determining the percent of branched glycans (e.g., on the Fc regions) of the polypeptides that have at least one galactose connected to a terminal sialic acid (e.g., on an a 1 ,3 arm of the glycan, linked to the galactose via a NeuAc-a 2,6-Gal terminal linkage) ; and processing the test preparation into a pharmaceutical product if the percent of branched glycans (e.g., on the Fc regions) of the polypeptides is greater than 1 0% (e.g., greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to and including 100%), thereby manufacturing a pharmaceutical product.

In further embodiments, the processing step includes combining the test preparation with an excipient or buffer.

In other embodiments, the processing step includes: (a) combining (e.g., formulating) the preparation with a pharmaceutically acceptable excipient; and (b) packaging the combination with instructions for use in the treatment of one or more neurodegenerative diseases.

In still other embodiments, the processing step includes one or more of: formulating the test preparation; processing the test preparation into a drug product; combining the test preparation with a second component, e.g., an excipient or buffer; changing the concentration of the polypeptide in the preparation; lyophilizing the test preparation; combining a first and second aliquot of the polypeptide to provide a third, larger, aliquot; dividing the test preparation into smaller aliquots; disposing the test preparation into a container, e.g., a gas or liquid tight container; packaging the test preparation;

associating a container comprising the test preparation with a label (e.g., labeling) ; and shipping or moving the test preparation to a different location. In some embodiments of any of the foregoing methods, the polypeptides include a human IgG Fc region (e.g., lgG1 , lgG2, lgG3, or lgG4). In other embodiments of any of the foregoing methods, the polypeptides include human lgG1 , lgG2, lgG3 or lgG4, or a mixture thereof.

In a further aspect, the disclosure features a method of selecting a preparation useful for the treatment of neurodegeneration. This method includes the steps of providing a sample of a test preparation (e.g., derived from IVIG) including polypeptides (e.g., polypeptides having an IgG Fc region) ; acquiring an input value for the test preparation for one or more parameters listed in Table 1 ; acquiring one or more assessments made by comparing the input value for the test preparation with one or more target value, and selecting the test preparation as being useful for the treatment of neurodegeneration if the input values for at least one parameter in the test preparation listed in Table 1 meet the corresponding target value for the parameter.

In some embodiments, the test preparation is derived from IVIG. In certain embodiments, the test preparation is a modified IVIG preparation or IVIG fraction. In other embodiments, the test preparation includes an Fc region derived from IVIG.

In certain embodiments of any of the foregoing methods, neurodegeneration is related to a disease selected from the group consisting of age-related dementia, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), cerebellar ataxia, Creutzfedt-Jakob disease, Down's syndrome, frontotemporal lobar degenerations/dementia, Huntington's disease, inclusion body myositis, Lewy body dementia, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Charcot-Marie-Tooth syndrome, myasthenia gravis, Lambert-Eaton myasthenic syndrome, multifocal motor neuropathies, multiple sclerosis, multiple-system atrophy, Parkinson's disease, vascular dementia, Lennox-Gastaut syndrome, ataxia telangiectasia, neurodegenerative Lyme disease, acute disseminating

encephalomyelitis, acute idiopathic dysautonomia, adrenoleukodystrophy, demylelinative brain stem encephalitis, demyelinating neuropathy associated with monoclonal IgM, HTLV-1 -associated myelopathy, other paraneoplastic neurodegeneration, neuropathy or encephalopathies, lumbosacral or brachial plexitis, POEMS syndrome, post-infection cerebellar ataxia, presbycusis, spinocerebellar ataxia, other peripheral neuropathies (e.g., mononeuropathy, mononeuritis multiplex, polyneuropathy, autonomic neuropathy, and neuritis), and vascular dementia.

Table 1

All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

These and other aspects of the invention are described in more detail below and in the claims.

DESCRIPTION OF THE FIGURES

Figure 1 is a schematic illustration of a common core pentasaccharide (Man) ₃ (GlcNAc)(GlcNAc) of N-glycans.

Figure 2 is a schematic illustration of an IgG antibody molecule.

Figure 3A depicts an exemplary ST6 sialyltransferase amino acid sequence (SEQ ID NO:1 ). Figure 3B depicts an exemplary ST6 sialyltransferase amino acid sequence (SEQ ID NO:2). Figure 3C depicts an exemplary ST6 sialyltransferase amino acid sequence (SEQ ID NO:3).

Figure 4 is a schematic illustration of a reaction scheme for ST6 sialyltransferase (fucose: triangles, N-acetylglucosamine: squares, mannose: dark circles, galactose: light circles, sialic acid: diamonds).

Figure 5 is a graphic representation of relative abundance of glycans at various times during a sialylation reaction with ST6 sialyltransferase. Figure 6 is an image displaying the experimental design.

Figure 7 is an image displaying the clinical course of EAE.

Figures 8A and 8B are images displaying the increased frequency of T cells in the spleen following treatment.

Figure 9A-9D are images displaying the increased frequency of na ^' ive T cells and decreased frequency of memory T cells following treatment.

Figure 10A-10E are images displaying characterization of CNS populations using FACS analysis.

Figure 1 1 A and 1 1 B are images displaying the expression levels CCL2 (MCP-1 ) and Eotaxin following treatment.

DETAILED DESCRIPTION

Antibodies are glycosylated at conserved positions in the constant regions of their heavy chain. For example, IgG antibodies have a single N-linked glycosylation site at Asn297 of the C _H 2 domain. Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions. For human IgG, the core oligosaccharide normally consists of GlcNAc ₂ Man ₃ GlcNAc, with differing numbers of outer residues. Variation among individual IgGs can occur via attachment of galactose and/or galactose- sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).

The present disclosure relates to polypeptide preparations (e.g., Fc region-containing polypeptide preparations (e.g., IVIG, Fc or IgG antibodies)) having particular levels of branched glycans that are sialylated on an a1 ,3 arm , an a1 ,6 arm , or both, of the branched glycans in the Fc region (e.g., with a NeuAc-a2,6-Gal terminal linkage). The levels can be measured on an individual Fc region (e.g., the number of branched glycans that are sialylated on an a1 ,3 arm , an a1 ,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of polypeptides (e.g., the number or percentage of branched glycans that are sialylated on an a1 ,3 arm , an a1 ,6 arm , or both, of the branched glycans in the Fc region in a preparation of polypeptides).

The inventors have discovered that Fc region-containing polypeptides having branched glycans that are preferentially sialylated on an a 1 ,3 arm of the branched glycan in the Fc region (e.g., with a NeuAc-a 2,6-Gal terminal linkage) are useful for the treatment of neurodegeneration, e.g.,

neurodegenerative diseases. Described herein are polypeptides (e.g., antibodies or fusion proteins, such as Fc fusion proteins) having branched glycans sialylated on an a 1 ,3 arm of the branched glycan in the Fc region (e.g., with a NeuAc-a 2,6-Gal terminal linkage) and useful in the treatment of

neurodegeneration, e.g., neurodegenerative diseases. Methods of making and using such compositions are also described.

Preparations useful herein can be obtained from any source. In some instances, providing or obtaining a preparation (e.g., such as a biologic drug substance or a precursor thereof), e.g., that is or includes a polypeptide, can include providing a host cell, e.g., a mammalian host cell (e.g., a CHO cell) that is genetically engineered to express a polypeptide (e.g., a genetically engineered cell) ; culturing the host cell under conditions suitable to express the polypeptide (e.g., mRNA and/or protein) ; and, optionally, purifying the expressed polypeptide, e.g., in the form of a recombinant fusion protein) from the cultured cell, thereby producing a preparation. Definitions

As used herein, "acquire or acquiring (e.g., acquiring information)" means obtaining possession of a physical entity, or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., performing an assay or test on a sample or "analyzing a sample" as that term is defined herein) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). "Directly acquiring" a physical entity includes performing a process, e.g., analyzing a sample, that includes a physical change in a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. "Directly acquiring" a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent. Exemplary analytical methods are shown in Table 2.

As used herein, the term "antibody" refers to a polypeptide that includes at least one

immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as V _H ), and a light (L) chain variable region (abbreviated herein as V|_). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab, F(ab') ₂ , Fd, Fv, and dAb fragments) as well as complete antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin can be of types kappa or lambda.

In some instances, a preparation (e.g., such as a preparation of an Fc region-containing polypeptide) can be a sample from a proposed or test batch of a drug product. As used herein, a "batch" of a preparation refers to a single production run. Evaluation of different batches thus means evaluation of different production runs or batches. As used herein "sample(s)" refer to separately procured samples. For example, evaluation of separate samples could mean evaluation of different commercially available containers or vials of the same batch or from different batches. A batch can include a drug product.

As used herein, the term "constant region" refers to a polypeptide that corresponds to, or is derived from , one or more constant region immunoglobulin domains of an antibody. A constant region can include any or all of the following immunoglobulin domains: a C _H 1 domain, a hinge region, a C _H 2 domain, a C _H 3 domain (derived from an IgA, IgD, IgG, IgE, or IgM), and a C _H 4 domain (derived from an IgE or IgM).

As used herein, "IVIG" is a preparation of pooled, polyvalent IgG, including all four IgG subgroups, extracted from plasma of at least 1 ,000 human donors. IVIG is approved as a plasma protein replacement therapy for immune deficient patients. The level of IVIG Fc glycan sialylation varies between about 10-20% among IVIG preparations.

As used herein, the term "derived from IVIG" refers to polypeptides which result from

manipulation of IVIG. For example, polypeptides purified from IVIG (e.g., enriched for sialylated IgGs, modified IVIG (e.g., IVIG IgGs enzymatically sialylated), or Fc regions of IVIG (e.g., papain digested and sialylated) are derived from IVIG.

As used herein, "evaluating," e.g., in the evaluation/evaluating, identifying, and/or producing aspects disclosed herein, means reviewing, considering, determining, assessing, analyzing, measuring, and/or detecting the presence, absence, level, and/or ratio of one or more parameters in a preparation to provide information pertaining to the one or more parameters. In some instances, evaluating can include performing a process that involves a physical change in a sample or another substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. "Evaluating" can include performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent.

As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides," each "Fc polypeptide" including the constant region of an antibody excluding the first constant region

immunoglobulin domain. In some embodiments, an "Fc region" includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or the entire flexible hinge N-terminal to these domains. For IgG, "Fc polypeptide" comprises immunoglobulin domains Cgamma2 (Cy2) and Cgamma3 (Cy3) and the lower part of the hinge between Cgammal (Cy1 ) and Cy2. Although the boundaries of the Fc polypeptide may vary, the human IgG heavy chain Fc polypeptide is usually defined to comprise residues starting at T223 or C226 or P230, to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991 , N IH Publication 91 -3242, National Technical Information Services, Springfield, VA). For IgA, Fc polypeptide comprises immunoglobulin domains Calpha2 (Ca2) and Calpha3 (Ca3) and the lower part of the hinge between Calphal (Cert ) and Ca2. An Fc region can be synthetic, recombinant, or generated from natural sources such as IVIG.

An "Fc region-containing polypeptide" is a polypeptide that includes all or a substantial portion of an Fc region. Examples of an Fc region-containing polypeptide preparation include, e.g., a preparation of Fc fragments, a preparation of antibody molecules, a preparation of Fc-fusion proteins (e.g., an Fc- receptor fusion protein), and a preparation of pooled, polyvalent immunoglobulin molecules (e.g., IVIG). Such an Fc region-containing polypeptide may be recombinant (e.g., a recombinant Fc fragment preparation or a recombinant antibody preparation) or naturally derived (such as IVIG).

As used herein, the term "Fc region variant" refers to an analog of an Fc region that possesses one or more Fc-mediated activities described herein. This term includes Fc regions having one or more amino acid modifications (e.g., substitutions, additions, or deletions) relative to a wild-type or naturally existing Fc region. For example, variant Fc regions can possess at least about 50% homology, at least about 75% homology, at least about 80% homology, at least about 85%, homology, at least about 90% homology, at least about 95% homology, or more, with a naturally existing Fc region. For example, variant Fc regions can possess between 1 and 5 amino acid substitutions, e.g., 1 , 2, 3, 4 or 5 amino acid substitutions such as phenylalanine to alanine substitutions. Fc region variants also include Fc regions having one or more amino acid residues added to or deleted from the N- or C-terminus of a wild type Fc region.

As used herein, "glycan" is a sugar, which can be monomers or polymers of sugar residues, such as at least three sugars, and can be linear or branched. A "glycan" can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose,

phosphomannose, 6'sulfo N-acetylglucosamine, etc.). The term "glycan" includes homo and

heteropolymers of sugar residues. The term "glycan" also encompasses a glycan component of a glycoconjugate (e.g., of a polypeptide, glycolipid, proteoglycan, etc.). The term also encompasses free glycans, including glycans that have been cleaved or otherwise released from a glycoconjugate.

As used herein, the term "glycoprotein" refers to a protein that contains a peptide backbone covalently linked to one or more sugar moieties (i.e., glycans). The sugar moiety(ies) may be in the form of monosaccharides, disaccharides, oligosaccharides, and/or polysaccharides. The sugar moiety(ies) may comprise a single unbranched chain of sugar residues or may comprise one or more branched chains. Glycoproteins can contain O-linked sugar moieties and/or N-linked sugar moieties.

As used herein, the term "neurodegeneration" refers to the progressive loss of structure or function of neurons, including death of neurons. The term "neurodegenerative disease" refers to diseases in which neurodegeneration is, at least in part, a cause, symptom or phenotype. Exemplary neurodegenerative diseases include, but are not limited to, age-related dementia, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), cerebellar ataxia, Creutzfedt-Jakob disease, Down's syndrome, frontotemporal lobar degenerations/dementia, Huntington's disease, inclusion body myositis, Lewy body dementia, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Charcot-Marie- Tooth syndrome, myasthenia gravis, Lambert-Eaton myasthenic syndrome, multifocal motor

neuropathies, multiple sclerosis, multiple-system atrophy, Parkinson's disease, vascular dementia,

Lennox-Gastaut syndrome, ataxia telangiectasia, neurodegenerative Lyme disease, acute disseminating encephalomyelitis, acute idiopathic dysautonomia, adrenoleukodystrophy, demylelinative brain stem encephalitis, demyelinating neuropathy associated with monoclonal IgM, HTLV-1 -associated myelopathy, other paraneoplastic neurodegeneration, neuropathy or encephalopathies, lumbosacral or brachial plexitis, POEMS syndrome, post-infection cerebellar ataxia, presbycusis, spinocerebellar ataxia, other peripheral neuropathies (e.g., mononeuropathy, mononeuritis multiplex, polyneuropathy, autonomic neuropathy, and neuritis).

As used herein, an "N-glycosylation site of an Fc polypeptide" refers to an amino acid residue within an Fc polypeptide to which a glycan is N-linked. In some embodiments, an Fc region contains a dimer of Fc polypeptides, and the Fc region comprises two N-glycosylation sites, one on each Fc polypeptide.

As used herein "percent (%) of branched glycans" refers to the number of moles of glycan X relative to total moles of glycans present, wherein X represents the glycan of interest.

As used herein "percent (%) sequence identity" with respect to a sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. In some instances a product will include amino acid variants, e.g., species that differ at terminal residues, e.g., at one, two, three, or four N- terminal residues and/or one C-terminal residue. In instances of such cases the sequence identity which is compared is the identity between the primary amino acid sequences of the most abundant active species in each of the products being compared. In some instances sequence identity refers to the amino acid sequence encoded by a nucleic acid that can be used to make the product.

The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount (e.g., dose) effective in treating a patient, having a disorder or condition described herein. It is also to be understood herein that a "pharmaceutically effective amount" may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents.

As used herein, "polynucleotide" (or "nucleotide sequence" or "nucleic acid molecule") refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double- stranded, and represent the sense or anti- sense strand.

As used herein, "polypeptide" (or "amino acid sequence" or "protein") refers to a glycoprotein, oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. "Amino acid sequence" and like terms, such as "polypeptide" or "protein," are not meant to limit the indicated amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

"Predetermined level" as used herein, refers to a pre-specified particular level of one or more particular glycans, e.g., branched glycans having a sialic acid on an crt ,3 arm, and/or branched glycans having a sialic acid on an crt ,6 arm , and/or branched glycans having a sialic acid on an crt ,3 arm and on an crt ,6 arm. In some embodiments, a predetermined level is an absolute value or range. In some embodiments, a predetermined level is a relative value. In some embodiments, a predetermined level is the same as or different (e.g., higher or lower than) a level of one or more particular glycans (e.g., branched glycans having a sialic acid on an crt ,3 arm, and/or branched glycans having a sialic acid on an crt ,6 arm , and/or branched glycans having a sialic acid on an crt ,3 arm and on an crt ,6 arm) in a reference, e.g., a reference polypeptide product, or a reference document such as a specification, alert limit, or master batch record for a pharmaceutical product.

In some embodiments, a predetermined level is an absolute level or range of (e.g., number of moles of) one or more glycans (e.g., branched glycans having a sialic acid on an crt ,3 arm , and/or branched glycans having a sialic acid on an crt ,6 arm, and/or branched glycans having a sialic acid on an crt ,3 arm and on an crt ,6 arm) in a polypeptide preparation. In some embodiments, a predetermined level is a level or range of one or more glycans (e.g., branched glycans having a sialic acid on an crt ,3 arm , and/or branched glycans having a sialic acid on an crt ,6 arm , and/or branched glycans having a sialic acid on an crt ,3 arm and on an crt ,6 arm) in a polypeptide preparation relative to total level of glycans in the polypeptide preparation. In some embodiments, a predetermined level is a level or range of one or more glycans (e.g., branched glycans having a sialic acid on an crt ,3 arm, and/or branched glycans having a sialic acid on an crt ,6 arm , and/or branched glycans having a sialic acid on an crt ,3 arm and on an crt ,6 arm) in a polypeptide preparation relative to total level of sialylated glycans in the polypeptide preparation. In some embodiments, a predetermined level is expressed as a percent.

By "purified" (or "isolated") refers to a polynucleotide or a polypeptide that is removed or separated from other components present in its natural environment. For example, an isolated polypeptide is one that is separated from other components of a cell in which it was produced (e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA). An isolated polynucleotide is one that is separated from other nuclear components (e.g., histones) and/or from upstream or downstream nucleic acids. An isolated polynucleotide or polypeptide can be at least 60% free, or at least 75% free, or at least 90% free, or at least 95% free from other components present in natural environment of the indicated polynucleotide or polypeptide.

"Reference polypeptide", as used herein, refers to a polypeptide having substantially the same amino acid sequence as (e.g., having about 95-100% identical amino acids of) a polypeptide described herein, e.g., a polypeptide to which it is compared. In some embodiments, a reference polypeptide is a therapeutic polypeptide described herein, e.g., an FDA approved therapeutic polypeptide. As used herein, the term "ST6 sialyltransferase" refers to a polypeptide whose am ino acid sequence includes at least one characteristic sequence of and/or shows at least 1 00%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81 %, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71 % or 70% identity with a protein involved in transfer of a sialic acid to a term inal galactose of a glycan through an a2,6 linkage (e.g., ST6 Gal-I) . A wide variety of ST6 sialyltransferase sequences are known in the art, such as those described herein ; in some embodiments, an ST6 sialyltransferase shares at least one characteristic sequence of and/or shows the specified degree of overall sequence identity with one of the ST6 sialyltransferases set forth herein (each of which may be considered a "reference" ST6 sialyltransferase) . In some embodiments, an ST6 sialyltransferase as described herein shares at least one biological activity with a reference ST6 sialyltransferase as set forth herein. In some such embodiment, the shared biological activity relates to transfer of a sialic acid to a glycan .

The term "subject," as used herein, means any subject for whom diagnosis, prognosis, or therapy is desired. For example, a subject can be a mammal, e.g., a human or non-human primate (such as an ape, monkey, orangutan, or chimpanzee) , a dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow. In a preferred embodiment, the subject is a human.

As used herein, "target value" refers to a statistically significant change (e.g. a greater than 1 0%, 1 5%, 20%, 25%, 30%, 35%. 40%, 45%, 50% or more change) relative to an unmodified polypeptide having an IgG Fc region, e.g. , IVIG.

The term "treatment" or "treating," as used herein, refers to administering a therapy in an amount, manner, and/or mode effective to improve a condition, symptom , or parameter associated with a disorder or condition or to prevent or reduce progression of a disorder or condition to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject.

As used herein, the terms "coupled," "linked," "joined," "fused," and "fusion" are used

interchangeably. These terms refer to the joining together of two more elements or components by whatever means, including chemical conjugation or recombinant means.

The terms "overexpress," "overexpression," or "overexpressed" interchangeably refer to a polypeptide or polynucleotide that is transcribed or translated at a detectably greater level, such as in a cancer cell , in comparison to a control cell. The term includes expression due to transcription, post- transcriptional processing, translation, post-translational processing, cellular localization (e.g. , organelle, cytoplasm , nucleus, cell surface) , and RNA and protein stability, as compared to a control cell .

Overexpression can be detected using conventional techniques, e.g. , for detecting m RNA (i.e. , RT-PCR,

PCR, hybridization) or proteins (i.e. , ELISA, imm unohistochemical techniques) . Overexpression can be expression in an amount greater than about 1 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control cell. In certain instances, overexpression is 1 -fold, 2-fold, 3-fold, 4-fold, or more, higher level of transcription or translation compared to a control cell.

While the present disclosure provides exemplary units and methods for the evaluation, identification, and production methods disclosed herein , a person of ordinary skill in the art will appreciate that performance of the evaluation, identification, and production methods herein is not lim ited to use of those units and/or methods. For example, "percent of branched glycans" provided herein are generally described, as a value for a glycan or structure relative to total glycan or structure on a mol/mol basis. A person of skill in the art understands that although the use of other metrics or units (e.g., mass/mass, mole percent vs. weight percent) to measure a described parameter might give rise to different absolute values than those described herein, a test preparation meets a disclosed target value even if other units or metrics are used, as long as the test preparation meets the herein disclosed value when the herein disclosed units and metrics are used, e.g., allowing for the sensitivity (e.g., analytical variability) of the method being used to measure the value.

I. Polypeptides

Examples of an Fc region-containing polypeptide preparation include, e.g., a preparation of Fc fragments, a preparation of antibody molecules, a preparation of Fc-fusion proteins (e.g., an Fc-receptor fusion protein), and a preparation of pooled, polyvalent immunoglobulin molecules (e.g., IVIG). Fc region- containing polypeptides may be recombinant or naturally derived.

Naturally derived polypeptides that can be used in the methods of the invention include, for example intravenous immunoglobulin (IVIG) and polypeptides derived from IVIG (e.g., polypeptides purified from IVIG (e.g., enriched for sialylated IgGs), modified IVIG (e.g., IVIG IgGs enzymatically sialylated), or Fc regions of IVIG (e.g., papain digested and sialylated)).

Recombinant Fc region-containing polypeptides that can be used in the methods of the invention can be, for example expressed in and purified from CHO cells and sialylated using human ST6-Gal sialtransferase enzyme (expressed in and purified from E. coli cells) or expressed in and purified from CHO cells and sialylated using human ST6-Gal sialtransferase enzyme (expressed in and purified from CHO cells).

A. N-Linked Glycosylation

N-linked oligosaccharide chains are added to a protein in the lumen of the endoplasmic reticulum .

Specifically, an initial oligosaccharide (typically 14-sugar) is added to the amino group on the side chain of an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline. The structure of this initial oligosaccharide is common to most eukaryotes, and contains 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by specific glycosidase enzymes in the endoplasmic reticulum , resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the "a 1 ,3 arm ," and the second branch is referred to as the "a 1 ,6 arm," as denoted in Figure 1 .

N-glycans can be subdivided into three distinct groups called "high mannose type," "hybrid type," and "complex type," with a common pentasaccharide core (Man (a 1 ,6)-(Man(a 1 ,3))-Μ3η(β 1 ,4)- GlcpNAc^ 1 ,4)-GlcpNAc^ 1 ,N)-Asn) occurring in all three groups.

After initial processing in the endoplasmic reticulum, the polypeptide is transported to the Golgi where further processing may take place. If the glycan is transferred to the Golgi before it is completely trimmed to the core pentasaccharide structure, it results in a "high-mannose glycan."

Additionally or alternatively, one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form a "complex glycan." Galactose may be added to the N- acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with any of a sialic acid, a galactose or an N-acetylglucosamine residue.

Additionally, a fucose residue may be added to an N-acetylglucosamine residue of the core

oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.

"Hybrid glycans" comprise characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may comprise primarily or exclusively mannose residues, while another branch may comprise N-acetylglucosamine, sialic acid, galactose, and/or fucose sugars.

Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They bear a negative charge via a carboxylic acid group attached to the ring as well as other chemical decorations including N-acetyl and N-glycolyl groups. The two main types of sialyl residues found in polypeptides produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N- glycolylneuraminic acid (NeuGc). These usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing termini of both N- and O-linked glycans. The glycosidic linkage

configurations for these sialyl groups can be either a 2,3 or a 2,6.

Fc regions are are glycosylated at conserved, N-linked glycosylation sites. For example, each heavy chain of an IgG antibody has a single N-linked glycosylation site at Asn297 of the C _H 2 domain. IgA antibodies have N-linked glycosylation sites within the C _H 2 and C _H 3 domains, IgE antibodies have N- linked glycosylation sites within the C _H 3 domain, and IgM antibodies have N-linked glycosylation sites within the C _H 1 , C _H 2, C _H 3, and C _H 4 domains.

Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions. For example, IgG has a single N-linked biantennary carbohydrate at Asn297 of the C _H 2 domain in each Fc polypeptide of the Fc region, which also contains the binding sites for C1 q and FcyR. For human IgG, the core oligosaccharide normally consists of GlcNAc ₂ Man ₃ GlcNAc, with differing numbers of outer residues. Variation among individual IgG can occur via attachment of galactose and/or galactose- sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).

B. Antibodies

The basic structure of an IgG antibody is illustrated in Figure 2. As shown in Figure 2, an IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulphide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the antibody binding specificities found in each individual antibody. These are known as variable heavy (V _H ) and variable light (V _L ) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (C _H ) and constant light (C _L ) regions. As shown in Figure 2, for an IgG antibody, the light chain includes one variable region (V _L ) and one constant region (C _L ). An IgG heavy chain includes a variable region (V _H ), a first constant region (C _H 1 ), a hinge region, a second constant region (C _H 2), and a third constant region (C _H 3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (C _H 4).

Antibodies described herein can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above. Antibodies can be of any type (e.g., IgG,

IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.

The term "Fc fragment," as used herein, refers to one or more fragments of an Fc region that retains an Fc function and/or activity described herein, such as binding to an Fc receptor. Examples of such fragments include fragments that include an N-linked glycosylation site of an Fc region (e.g., an

Asn297 of an IgG heavy chain or homologous sites of other antibody isotypes), such as a CH2 domain.

The term "antigen binding fragment" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include a Fab fragment, a F(ab') ₂ fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature

341 :544-546), and an isolated complementarily determining region (CDR). These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies.

Reference Fc region-containing polypeptides described herein can be produced by any method known in the art for the synthesis of antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual,

(Cold Spring Harbor Laboratory Press, 2nd ed. 1 988) ; Brinkman et al., 1995, J. Immunol. Methods

182:41 -50; WO 92/22324; WO 98/46645).

Additional reference Fc region-containing polypeptides described herein are bispecific antibodies and multivalent antibodies, as described in, e.g., Segal et al., J. Immunol. Methods 248:1 -6 (2001 ) ; and Tutt et al., J. Immunol. 147: 60 (1991 ).

C. Polypeptide Conjugates

The disclosure includes polypeptides (or Fc regions or Fc fragments thereof containing one or more N-glycosylation sites) that are conjugated or fused to one or more heterologous moieties and that have different levels of sialylated glycans relative to a corresponding reference polypeptide.

Heterologous moieties include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. In some instances, a reference polypeptide is a fusion protein that comprises a peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or an antibody mimetic fused to an Fc region, such as a glycosylated Fc region. The fusion protein can include a linker region connecting the Fc region to the heterologous moiety (see, e.g., Hallewell et al. (1989), J. Biol. Chem . 264, 5260-5268; Alfthan et al. (1995), Protein Eng. 8, 725-731 ; Robinson & Sauer (1996)).

In some instances, a reference fusion protein includes an Fc region (or an Fc fragment containing one or more N-glycosylation sites thereof) conjugated to a heterologous polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids.

In some instances, a reference fusion protein can include an Fc region (or Fc fragment containing one or more N-glycosylation sites thereof) conjugated to marker sequences, such as a peptide to facilitate purification. A particular marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311 ). Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1 984, Cell 37:767) and the "Flag" tag.

In other instances, a reference polypeptide (or an Fc region or Fc fragment containing one or more N-glycosylation sites thereof) is conjugated to a diagnostic or detectable agent. Such fusion proteins can be useful for monitoring or prognosing the development or progression of disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by coupling the polypeptide to detectable substances including, but not limited to, various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin ; radioactive materials, such as but not limited to iodine ( ¹³¹ l, ¹²⁵ l, ¹²³ l), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ ln, ¹¹³ ln, ¹¹² ln, ¹¹¹ In), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵³ Gd, ¹⁵⁹ Gd, ¹⁴⁹ Pm , ¹⁴⁰ La, ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, and ¹¹⁷ Sn; positron emitting metals using various positron emission tomographies, non-radioactive paramagnetic metal ions, and molecules that are radiolabeled or conjugated to specific radioisotopes.

Techniques for conjugating therapeutic moieties to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss, Inc. 1985) ; Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987)).

D. Sialyltransferase Polypeptides

Methods and compositions described herein include the use of a sialyltransferase enzyme, e.g., an a 2,6 sialyltransferase (e.g., ST6 Gal-I). A number of ST6 sialyltransferases are known in the art and are commercially available (see, e.g., Takashima, Biosci. Biotechnol. Biochem . 72:1 155-1 167 (2008) ; Weinstein et al., J. Biol. Chem. 262:17735-17743 (1987)). ST6 Gal-I catalyzes the transfer of sialic acid from a sialic acid donor (e.g., cytidine 5'-monophospho-N-acetyl neuraminic acid) to a terminal galactose residue of glycans through an a 2,6 linkage. The sialic acid donor reaction product is cytidine 5'- monophosphate. Figures 3A-3C depict three exemplary ST6 sialyltransferase amino acid sequences (SEQ ID NOs:1 -3). In some embodiments, an ST6 sialyltransferase has or includes an amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, or in amino acid residues 95-416 of SEQ ID NO:3, or a characteristic sequence element thereof or therein. In some embodiments, an ST6 sialyltransferase has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81 %, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71 %, or 70% overall sequence identity with one or more of SEQ ID NO:1 , SEQ ID NO:2, or amino acid residues 95-416 of SEQ ID NO:3. Alternatively or additionally, in some embodiments, an ST6 sialyltransferase includes at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or 1 50 or more contiguous amino acid residues found in SEQ ID NO:1 , SEQ ID NO:2, or amino acid residues 95-416 of SEQ ID NO:3.

In some embodiments, an ST6 sialyltransferase differs from an amino acid sequence as set forth in SEQ ID NO:1 , SEQ ID NO:2, or in amino acid residues 95-416 of SEQ ID NO:3, or characteristic sequence elements thereof or therein, by one or more amino acid residues. For example, in some embodiments, the difference is a conservative or nonconservative substitution of one or more amino acid residues. Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of similar characteristics. Typical conservative substitutions are the following replacements: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine or vice versa; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.

In some embodiments, an ST6 sialyltransferase polypeptide includes a substituent group on one or more amino acid residues. Still other useful polypeptides are associated with (e.g., fused, linked, or coupled to) another moiety (e.g., a peptide or molecule). For example, an ST6 sialyltransferase polypeptides can be fused, linked, or coupled to an amino acid sequence (e.g., a leader sequence, a secretory sequence, a proprotein sequence, a second polypeptide, or a sequence that facilitates purification, enrichment, or stabilization of the polypeptide).

II. Methods for Producing Sialylated Polypeptides

The present disclosure relates to Fc region-containing polypeptide preparations (e.g., IVIG, Fc, or IgG antibodies) having higher levels of branched glycans that are sialylated on an a 1 ,3 or 1 ,6 arm of the branched glycans in the Fc region (e.g., with a NeuAc-a 2,6-Gal or NeuAc-a 2,3-Gal terminal linkage), relative to a corresponding reference polypeptide preparation. The higher levels can be measured on an individual Fc region (e.g., an increase in the number of branched glycans that are sialylated on an a 1 ,3 arm of the branched glycans in the Fc region), or the overall composition of a preparation of polypeptides can be different (e.g., a preparation of polypeptides can have a higher number or a higher percentage of branched glycans that are sialylated on an a 1 ,3 arm of the branched glycans in the Fc region) relative to a corresponding preparation of reference polypeptides).

In exemplary methods, Fc molecules were obtained or produced from various sources, glycan compositions were characterized, and activities were determined. The Fc molecules were tested for their ability to protect mice from neurodegeneration in a mouse EAE model.

ST6 Gal-I sialyltransferase catalyzes the transfer of sialic acid from a sialic acid donor (e.g., cytidine 5'-monophospho-N-acetyl neuraminic acid) to a terminal galactose residue of glycans through an a 2,6 linkage. The present disclosure exploits the discovery that ST6 sialyltransferase catalyzes the transfer of sialic acid to branched glycans (e.g., Fc branched glycans) comprising an a 1 ,3 arm and an a 1 ,6 arm in an ordered fashion. As shown in Figure 4, ST6 sialyltransferase transfers a sialic acid to an a 1 ,3 arm of a branched glycan, which can be followed by transfer of a second sialic acid to an a 1 ,6 arm (yielding a disialylated branched glycan), and can further be followed by removal of sialic acid from an a 1 ,3 arm (yielding a branched glycan having a sialic acid on an a 1 ,6 arm). Accordingly, by controlling and/or modulating activity (e.g., kinetics) of ST6 sialyltransferase, polypeptides having particular sialylation patterns can be produced.

Any parameter generally known to affect enzyme kinetics can be controlled and/or modulated to produce a polypeptide preparation having a predetermined level of sialic acid on an a 1 ,3 arm of a branched glycan, on an a 1 ,6 arm of a branched glycan, and/or on an a 1 ,3 arm and an a 1 ,6 arm of a branched glycan. For example, reaction time, ST6 sialyltransferase concentration and/or specific activity, branched glycan concentration, sialic acid donor concentration, sialic acid donor reaction product concentration, pH, buffer composition, and/or temperature can be controlled and/or modulated to produce a polypeptide preparation having a desired level of sialylation (e.g., a 1 ,3 arm and/or a 1 ,6 arm sialylation).

In some embodiments, to preferentially sialylate an crt ,3 arm of branched glycans (e.g., having an a 1 ,3 arm and an a 1 ,6 arm), branched glycans are contacted in vitro with an ST6 sialyltransferase under limited reaction conditions. Such limited reaction conditions are selected such that addition of a sialic acid to an a 1 ,3 arm is enhanced relative to addition of a sialic acid to an a 1 ,6 arm (e.g., rate of transfer of a sialic acid to an a 1 ,3 arm ("R _a ^{1 ,3} ") exceeds rate of transfer of a sialic acid to an a 1 ,6 arm ("R _a ^{1 ,6} "). In some embodiments, limited reaction conditions are further selected such that removal of a sialic acid from an crt ,6 arm is enhanced relative to addition of a sialic acid to an a 1 ,6 arm (e.g., rate of removal of a sialic acid from an a 1 ,6 arm ("R _r ^{1 ,6} ") exceeds rate of transfer of a sialic acid to an a 1 ,6 arm ("R _a ^{1 ,6} "). Limited reaction conditions can include, for example, reduced reaction time, reduced enzyme

concentration and/or activity, reduced amount of branched glycans, reduced level of sialic acid donor, and/or reduced temperature.

In some embodiments, to preferentially sialylate an crt ,6 arm of branched glycans (e.g., having an a 1 ,3 arm and an a 1 ,6 arm), branched glycans can be contacted in vitro with an ST6 sialyltransferase under extended reaction conditions. Such extended reaction conditions are selected such that addition of a sialic acid to an a 1 ,6 arm is enhanced relative to removal of a sialic acid from an a 1 ,6 arm (e.g., rate of transfer of a sialic acid to an a 1 ,6 arm ("R _a ^{1 ,6} ") exceeds rate of removal of a sialic acid from an a 1 ,6 arm ("R _r ^{1 ,6} ")). In some embodiments, extended reaction conditions are further selected such that, after initial conditions that enhance addition of sialic acid to an a 1 ,3 arm , conditions are extended such that removal of a sialic acid from an a 1 ,3 arm is eventually enhanced relative to addition of a sialic acid to an a 1 ,3 arm (e.g., rate of removal of a sialic acid from an a 1 ,3 arm ("R _r ^{1 ,3} ") exceeds rate of transfer of a sialic acid to an a 1 ,3 arm ("R _a ^{1 ,3} ")). Extended reaction conditions can include, for example, increased reaction time, increased enzyme concentration and/or activity, increased amount of branched glycans, increased level of sialic acid donor, and/or increased temperature.

In some embodiments, to preferentially sialylate both an a 1 ,3 arm and an a 1 ,6 arm of branched glycans (e.g., having an a 1 ,3 arm and an a 1 ,6 arm), branched glycans are contacted in vitro with an ST6 sialyltransferase under intermediate reaction conditions. Such intermediate reaction conditions are selected such that addition of a sialic acid to an a 1 ,3 arm is enhanced relative to removal of a sialic acid from an a 1 ,3 arm (e.g., rate of transfer of a sialic acid to an a 1 ,3 arm ("R _a ^{1 ,3} ") exceeds rate of removal of a sialic acid from an a 1 ,3 arm ("R _r ^{1 ,3} "). In some embodiments, intermediate reaction conditions are further selected such that addition of a sialic acid to an a 1 ,6 arm is enhanced relative to removal of a sialic acid from an a 1 ,6 arm (e.g., rate of addition of a sialic acid to an a 1 ,6 arm ("R _a ^{1 ,6} ") exceeds rate of removal of a sialic acid from an a 1 ,6 arm ("R _r ^{1 ,6} "). Intermediate reaction conditions can include, for example, intermediate reaction time, intermediate enzyme concentration and/or activity, intermediate amount of branched glycans, intermediate level of sialic acid donor, and/or intermediate temperature. In some embodiments, intermediate reaction conditions further include supplementing the sialic acid donor at least once during the reaction. In some embodiments, intermediate reaction conditions further include removing a sialic acid donor reaction product at least once during the reaction. In some embodiments, intermediate reaction conditions further include supplementing the sialic acid donor reaction product at least once during the reaction.

In some embodiments, a polypeptide, e.g., a glycosylated antibody, is sialylated after the polypeptide is produced. For example, a polypeptide can be recombinantly expressed in a host cell (as described herein) and purified using standard methods. The purified polypeptide is then contacted with an ST6 sialyltransferase (e.g., a recombinantly expressed and purified ST6 sialyltransferase) in the presence of reaction conditions as described herein. In certain embodiments, the conditions include contacting the purified polypeptide with an ST6 sialyltransferase in the presence of a sialic acid donor, e.g., cytidine 5'-monophospho-N-acetyl neuraminic acid, manganese, and/or other divalent metal ions. In some embodiments, IVIG is used in a sialylation method described herein.

In some embodiments, chemoenzymatic sialylation is used to sialylate polypeptides. Briefly, this method involves sialylation of a purified branched glycan, followed by incorporation of the sialylated branched glycan en bloc onto a polypeptide to produce a sialylated polypeptide.

A branched glycan can be synthesized de novo using standard techniques or can be obtained from a polypeptide preparation (e.g., a recombinant polypeptide, Fc, or IVIG) using an appropriate enzyme, such as an endoglycosidase (e.g., EndoH or EndoF). After sialylation of the branched glycan, the sialylated branched glycan can be conjugated to a polypeptide using an appropriate enzyme, such as a transglycosidase, to produce a sialylated polypeptide.

In one exemplary method, a purified branched N-glycan is obtained from a polypeptide (e.g., a polypeptide preparation, e.g., IVIG) using an endoglycosidase. The purified branched N-glycan is then chemically activated on the reducing end to form a chemically active intermediate. The branched N- glycan is then further processed, trimmed, and/or glycosylated using appropriate known glycosidases. The branched glycan is then sialylated using an ST6 sialylation as described herein. After engineering, the desired branched N-glycan is transferred onto a polypeptide using a transglycosidase (such as a transglycosidase in which glycosidic activity has been attenuated using genetically engineering).

In some embodiments, a branched glycan used in methods described herein is a galactosylated branched glycan (e.g., includes a terminal galactose residue). In some embodiments, a branched glycan is galactosylated before being sialylated using a method described herein. In some embodiments, a branched glycan is first contacted with a galactosyltransferase (e.g., a beta-1 ,3-galactosyltransferase) and subsequently contacted with an ST6 sialyltransferase as described herein. In some embodiments, a galactosylated glycan is purified before being contacted with an ST6 sialyltransferase. In some embodiments, a galactosylated glycan is not purified before being contacted with an ST6

sialyltransferase. In some embodiments, a branched glycan is contacted with a galactosyltransferase and an ST6 sialyltransferase in a single step. In some embodiments, a host cell is genetically engineered to express a polypeptide described herein and one or more sialyltransferase enzymes, e.g., an ST6 sialyltransferase. In some embodiments, the host cell is genetically engineered to further express a galactosyltransferase. The genetically engineered host cell can be cultured under conditions sufficient to produce a particular sialylated polypeptide. For example, to produce polypeptides preferentially sialylated on crt ,3 arms of branched glycans, a host cell can be genetically engineered to express a relatively low level of ST6

sialyltransferase, whereas to produce polypeptides preferentially sialylated on crt ,6 arms of branched glycans, a host cell can be genetically engineered to express a relatively high level of ST6

sialyltransferase. In some embodiments, to produce polypeptides preferentially sialylated on crt ,3 arms of branched glycans, a genetically engineered host cell can be cultured in a relatively low level of sialic acid donor, whereas to produce polypeptides preferentially sialylated on crt ,6 arms of branched glycans, a genetically engineered host cell can be cultured in a relatively high level of sialic acid donor.

Recombinant expression of a gene, such as a nucleic acid encoding a reference polypeptide and/or a sialtransferase described herein, can include construction of an expression vector containing a polynucleotide that encodes a reference polypeptide and/or a sialtransferase. Once a polynucleotide has been obtained, a vector for the production of the reference polypeptide can be produced by recombinant DNA technology using techniques known in the art. Known methods can be used to construct expression vectors containing polypeptide coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

An expression vector can be transferred to a host cell by conventional techniques, and the transfected cells can then cultured by conventional techniques to produce reference polypeptides.

A variety of host expression vector systems can be used (see, e.g., U .S. Pat. No. 5,807,715). Such host-expression systems can be used to produce polypeptides and, where desired, subsequently purified. Such host expression systems include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing polypeptide coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing polypeptide coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing polypeptide coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing polypeptide coding sequences; or mammalian cell systems (e.g., COS, CHO, BH K, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

For bacterial systems, a number of expression vectors can be used, including, but not limited to, the E. coli expression vector pUR278 (Ruther et al., 1 983, EMBO 12:1791 ) ; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101 -31 09; Van Heeke & Schuster, 1989, J. Biol. Chem . 24:5503-5509) ; and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). For expression in mammalian host cells, viral-based expression systems can be utilized (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1 :355-359). The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the polypeptide expressed. Such cells include, for example, established mammalian cell lines and insect cell lines, animal cells, fungal cells, and yeast cells.

Mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030 and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, host cells are engineered to stably express a polypeptide. Host cells can be transformed with DNA controlled by appropriate expression control elements known in the art, including promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and selectable markers. Methods commonly known in the art of recombinant DNA technology can be used to select a desired recombinant clone.

In some embodiments, a reference Fc region-containing polypeptide is recombinantly produced in cells as described herein, purified, and contacted with a sialtransferase enzyme in vitro to produce Fc region-containing polypeptides containing higher levels of glycans having higher levels of sialic acid on the a 1 ,3 arms of the branched glycans with a NeuAc-a 2,6-Gal terminal linkage, relative to the reference polypeptide. In some embodiments, a purified reference polypeptide is contacted with the sialtransferase in the presence of CMP-sialic acid, manganese, and/or other divalent metal ions.

A reference Fc region-containing polypeptide can be purified by any method known in the art for purification, for example, by chromatography (e.g., ion exchange, affinity, and sizing column

chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, a reference antibody can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column with chromatography columns, filtration, ultra filtration, salting-out and dialysis procedures (see Antibodies: A Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). Further, as described herein, a reference polypeptide can be fused to heterologous polypeptide sequences to facilitate purification.

In some embodiments, a polypeptide can be purified using a lectin column by methods known in the art (see, e.g., WO 02/30954). For example, a preparation of polypeptides can be enriched for polypeptides containing glycans having sialic acids in a 2,6 linkage as described in, e.g.,

WO2008/057634. Following enrichment of polypeptides containing glycans having sialic acids in a 2,6 linkage, the glycan composition of such polypeptides can be further characterized to identify polypeptides having sialic acids attached to the a 1 ,3 arm of a branched glycan. Preparations of polypeptides containing a predetermined level of glycans having sialic acids in a 2,6 linkage on the a 1 ,3 arm can be selected for use, e.g., for therapeutic use. Such compositions can have increased levels of antiinflammatory activity. In accordance with the present disclosure, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are described in the literature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1 989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. ; DNA Cloning: A Practical Approach, Volumes I and I I (D. N. Glover ed. 1985) ; Oligonucleotide Synthesis (M. J. Gait ed. 1984) ; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)) ; Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)) ; Animal Cell Culture (R. I. Freshney, ed. (1986)) ; Immobilized Cells and Enzymes (IRL Press, (1986)) ; B. Perbal, A Practical Guide To Molecular Cloning (1984) ; F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.

(1994).

Glycan compositions can be characterized using methods described in, e.g., Barb, Biochemistry 48:9705-9707 (2009) ; Anumula, J. Immunol. Methods 382:167-176 (2012) ; Gilar et al., Analytical Biochem. 417:80-88 (201 1 ) . Glycan Evaluation

Glycans of polypeptides can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are sialylated on an a1 ,3 arm and/or an a1 ,6 arm) can be characterized using methods described in, e.g., Barb, Biochemistry 48:9705- 9707 (2009) ; Anumula, J. Immunol. Methods 382:167-1 76 (2012) ; Gilar et al., Analytical Biochem.

417:80-88 (201 1 ) ; Wuhrer et al., J. Chromatogr. B. 849:1 1 5-128 (2007). In some embodiments, in addition to evaluation of sialylation of glycans, one or more parameters described in Table 2 are evaluated.

In some instances, glycan structure and composition as described herein are analyzed, for example, by one or more, enzymatic, chromatographic, mass spectrometry (MS), chromatographic followed by MS, electrophoretic methods, electrophoretic methods followed by MS, nuclear magnetic resonance (NMR) methods, and combinations thereof. Exemplary enzymatic methods include contacting a polypeptide preparation with one or more enzymes under conditions and for a time sufficient to release one or more glycan(s) (e.g., one or more exposed glycan(s)). In some instances, the one or more enzymes include(s) PNGase F. Exemplary chromatographic methods include, but are not limited to, Strong Anion Exchange chromatography using Pulsed Amperometric Detection (SAX-PAD), liquid chromatography (LC), high performance liquid chromatography (H PLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof. Exemplary mass spectrometry (MS) include, but are not limited to, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof. Exemplary electrophoretic methods include, but are not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, and combinations thereof. Exemplary nuclear magnetic resonance (NMR) include, but are not limited to, one-dimensional NMR (1 D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), and combinations thereof.

In some instances, techniques described herein may be combined with one or more other technologies for the detection, analysis, and or isolation of glycans or polypeptides. For example, in certain instances, glycans are analyzed in accordance with the present disclosure using one or more available methods (to give but a few examples, see Anumula, Anal. Biochem., 350(1 ) :1 , 2006; Klein et al., Anal. Biochem., 179:162, 1989; and/or Townsend, R.R. Carbohydrate Analysis" High Performance Liquid Chromatography and Capillary Electrophoresis., Ed. Z. El Rassi, pp 181 -209, 1995; WO2008/12821 6; WO2008/128220; WO2008/128218; WO2008/130926; WO2008/128225; WO2008/130924;

WO2008/128221 ; WO2008/128228; WO2008/128227; WO2008/128230; WO2008/128219;

WO2008/128222; WO2010/071817; WO2010/071824; WO2010/085251 ; WO201 1 /069056; and

WO201 1 /127322, each of which is incorporated herein by reference in its entirety). For example, in some instances, glycans are characterized using one or more of chromatographic methods, electrophoretic methods, nuclear magnetic resonance methods, and combinations thereof. In some instances, methods for evaluating one or more target protein specific parameters, e.g., in a polypeptide preparation, e.g., one or more of the parameters disclosed herein, can be performed by one or more of following methods.

In some instances, methods for evaluating one or more target protein specific parameters, e.g., in a polypeptide preparation, e.g., one or more of the parameters disclosed herein, can be performed by one or more of following methods.

Table 2: Exemplary methods of evaluating parameters:

ultracentrifugation 367:225-237 (2007)

References listed in Table 2 are hereby incorporated by reference in their entirety or, in the alternative, to the extent that they pertain to one or more of the methods disclosed in Table 2. Other methods for evaluating one or more parameters are disclosed in the examples.

III. Treatment of Neurodegeneration

The inventors have discovered that sialic acid-mediated biological activity of Fc-containing molecules is not only due to the level of sialylation, but due to particular branching arrangements. Accordingly, Fc region-containing polypeptides described herein (e.g., Fc region-containing polypeptides containing glycans containing sialic acid on a 1 ,3 arms of branched glycans with a NeuAc-a 2,6-Gal terminal linkage ) have increased activity relative to a reference polypeptide.

In some embodiments, Fc region-containing polypeptides having sialic acids in both the a 1 ,3 and a 1 ,6 arms of branched glycans may be useful in the treatment of neurodegeneration.

IV. Pharmaceutical Compositions and Administration

A polypeptide of the present disclosure, e.g., an Fc region-containing polypeptide comprising branched glycans that are sialylated on an a 1 ,3 arm of the branched glycan in the Fc region, e.g., with a NeuAc-a 2,6-Gal terminal linkage, can be incorporated into a pharmaceutical composition and can be useful in the treatment of neurodegeneration. Such a pharmaceutical composition is useful as an improved composition for the prevention and/or treatment of diseases relative to the corresponding reference polypeptide. Pharmaceutical compositions comprising a polypeptide can be formulated by methods known to those skilled in the art. The pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, the pharmaceutical composition can be formulated by suitably combining the sulfated polypeptide with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided.

The sterile composition for injection can be formulated in accordance with conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D- mannitol, and sodium chloride may be used as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80™, HCO-50 and the like.

Non-limiting examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampoule.

Route of administration can be parenteral, for example, administration by injection, transnasal administration, transpulmonary administration, or transcutaneous administration. Administration can be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.

A suitable means of administration can be selected based on the age and condition of the patient. A single dose of the pharmaceutical composition containing a modified polypeptide can be selected from a range of 0.001 to 1000 mg/kg of body weight. On the other hand, a dose can be selected in the range of 0.001 to 100000 mg/body weight, but the present disclosure is not limited to such ranges. The dose and method of administration varies depending on the weight, age, condition, and the like of the patient, and can be suitably selected as needed by those skilled in the art.

EXAMPLES

Example 1 - Galactosylation and sialylation of IVIG

The sialylation of IVIG by the sialyltransferase ST6 was analyzed. IVIG was first galactosylated and then sialylated. The reactions were performed sequentially. There was no purification between galactosylation and sialylation reactions. The relative abundance of glycoforms was analyzed following the sialylation reactions.

A. Galactosylation

A reaction was set up that contained the following components at the concentrations indicated:

The reaction was incubated for 72 hours at 37 Ό.

B. Sialylation

To an aliquot of the galactosylation reaction were added CMP-NANA, MOPS buffer and ST6Gal1 The final volume was adjusted so that the final concentration of components in the reaction was as indicated.

e react on was ncu ate at 37 . quots were extracte at the times indicated in Figure 2 and frozen at -20 °C for later analyses.

C. Results

As shown in Figure 5, the predominant glycoform changed over time from G2F to A1 F (1 ,3) to A2F to A1 F (1 ,6). The results are summarized in the reaction scheme depicted in Figure 4. As shown in Figure 4, the product glycoform can change between G2F, A1 F (1 ,3), A2F, and A1 F (1 ,6) during the course of a reaction due to competing addition (forward reaction) and removal (back reaction) steps.

The sialyltransferase ST6 can add sialic acid to either branch of a substrate's biantennary N- glycan. However, these results demonstrate that addition to each branch happens at different rates, resulting in different end products depending on the reaction conditions. Addition of sialic acid to the a 1 ,3 branch is much faster than addition to the crt ,6 branch.

These data also demonstrate that sialyltransferase ST6 can also catalyze the removal of sialic acids from N-glycans. The removal of sialic acid from the a 1 ,3 branch is much faster than removal from the a 1 ,6 branch. This can surprisingly lead to the production of Fc glycans substantially or primarily monosialylated on the a 1 ,6 branch by modulating reaction conditions.

This Example demonstrates that reaction conditions can be controlled to produce a glycoprotein product having a predetermined sialylation levels. Such conditions can include time, ST6

sialyltransferase concentration, substrate concentration, donor sugar nucleotide concentration, product nucleotide concentration, pH, buffer composition, and/or temperature.

Example 2 - Treatment of neurodegeneration with sialylated IVIG

To test the efficacy and mechanisms of action of these agents in Alzheimer's disease (AD) and other neurodegenerative diseases, IVIG a 2,6 sialylated on the N-linked biantennary glycan at ASN-297 in the Fc region was administered to mice with experimental autoimmune encephalitis.

The specific sialylated preparations of IVIG tested in these studies include:

1 ) Monosialylated (s1 ) IVIG is a preparation where 63-79 percent of the Fc glycans are sialylated exclusively on the a 1 ,3 arm , fucosylated, and not bisected. Less than 1 5% of the Fc glycans are sialylated exclusively on the a 1 ,6 arm , fucosylated, and not bisected. Less than 15% of the Fc glycans are sialylated on both the a 1 ,3 and a 1 ,6 arms, fucosylated, and not bisected. Bisected species and afucosylated species, all of which are minor components, were likewise sialylated predominately on the a 1 ,3 arm only.

2) Disialylated (s2) IVIG is a preparation where 87 percent of the Fc glycans are sialylated on both the a 1 ,3 and a 1 ,6 arms, fucosylated, and not bisected. Less than 6% of the Fc glycans are sialylated on the a 1 ,3 arm , fucosylated, and not bisected. Fc glycans sialylated on the a 1 ,6 arm , fucosylated, and not bisected were not detected. Bisected species and afucosylated species, all of which are minor components, were likewise sialylated predominately on both arms.

Experimental autoimmune encephalomyelitis (EAE) is the most commonly used model of CNS autoimmunity, demyelination, cell trafficking, and tolerance induction.

EAE is mediated by myelin-specific CD4 ⁺ T cells but is also characterized by a critical involvement of the innate immune compartment, especially monocytes, macrophages, dendritic cells and microglia. EAE induces inflammatory infiltration of the CNS through the blood-brain barrier. Also, damage in the CNS in EAE is driven by adaptive immune cells (T cells) and microglia, CNS-specific innate immune cells.

EAE was induced in C57BL/6 mice by subcutaneous immunization with the myelin

oligodendrocyte protein (MOG) ₃ 5-55 peptide in a complete Freund's adjuvant (CFA) emulsion, followed by administration of Pertussis Toxin (PTX). Typically, EAE developed 8-10 days after immunization and was characterized by an ascending paralysis, starting with the loss of tail tone.

Mice were injected subcutaneously at two sites in the back with the MOG35-55/CFA Emulsion. One site of injection was in the area of upper back, approximately 1 cm caudal of the neck line. The second site was in the area of lower back, approximately 2 cm cranial of the base of the tail. The injection volume was 0.1 m L at each site.

Within 2 hours of the injection of emulsion, and then again 24 hours after immunization, 0.1 ml (143 ng) PTX was administered intraperitoneally (i.p.).

Mice were scored on a scale from 0 to 5, as follow, by a person unaware of both treatment and of previous scores for each mouse.

0 No signs of disease

1 Limp tail. When the mouse was picked up by the tail, instead of being erect, the whole tail draped over finger.

2 Limp tail and weakness of hind legs. When mouse was picked up by tail, legs are not spread apart, but held closer together. When the mouse was observed walking, it had a clearly apparent wobbly walk.

3 Limp tail and complete paralysis of hind legs (most common) OR Limp tail with paralysis of one front and one hind leg OR ALL of:

• Severe head tilting,

· Walking only along the edges of the cage,

• Pushing against the cage wall,

• Spinning when picked up by the tail.

4 Limp tail, complete hind leg and partial front leg paralysis. Mouse was minimally moving around the cage but appears alert and feeding. Usually, euthanasia was recommended after the mouse scores level 4 for 2 days. When the mouse was euthanized because of severe paralysis, score of 5 was entered for that mouse for the rest of the experiment.

5 Moribund or dead.

Mice were dosed i.p. Q3D, starting at the day of immunization (Figure6). Tissues were harvested at 3 days (one injection), 8 days (3 injections) and at the end point of the experiment (d21 ; 7 injections). 8 C57BI/6 females were used per condition (per time point and per treatment).

These experiments aim to compare the efficacy of IVIG and sialylated IVIG (s1 and s2) at modulating clinical EAE scores as well as cellular and molecular readouts. Readouts include clinical scores, FACS analysis, Luminex, shotgun proteomics and quantitative RT-PCR. Tissues analyzed include Blood (FACS), serum (Luminex), Spleen (FACS/Proteomics/ qRT-PCR), Lymph nodes

(Proteomics), Bone marrow (FACS/ qRT-PCR/Proteomics) and Spinal cord (FACS).

Methods

FACS

Tissues were processed and filtered through a 70 Dm strainer. Red blood cells (spleen/blood) were lysed for 10 min. at 4°C. Samples were then resuspended in FACS buffer and plated in a V-bottom 96 well plate Fc receptors were blocked for 10 min in FBS and stained for 20 min in various antibody cocktails (Table 3) at 4°C. Samples were resuspended in 1 % PFA. Data was acquired using a FACS Verse (Becton Dickinson) and analyzed using the FlowJo software (TreeStar).

Table 3: Antibody cocktails

Luminex

Millipore's M ILLIPLEX™ Mouse Cytokine / Chemokine kit was used to quantify the following 32 mouse cytokines and chemokines from plasma:

Eotaxin, G-CSF, GM-CSF, IFN D , IL-1□, IL-1□, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL- 12(p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1 , M-CSF, M IG, M IP-1□, M IP-1□, M IP-2, RANTES, TNFD , and VEGF.

Characterization of the clinical efficacy of IVIG and sl-IVIG in EAE

Both IVIG and sIVIG treatments appeared to have postponed EAE onset in a dose-dependent fashion (Figure 7). Neither agent showed a statistically significant decrease in maximal EAE severity (MMS). For delay of disease onset, IVIG and s1 -IVIG appeared to be equally effective at 0.5 g/kg, but s1 -IVIG at 0.05 g/kg was as effective as IVIG at a dose of 0.5 g/kg. However, no statistically significant differences regarding EAE onset or EAE severity were observed between the vehicle treated group and either the IVIG or s1 -IVIG treated groups at 0.005 g/kg (Table 4). These results demonstrate the increased potency of s1 -IVIG which was effective in delaying disease onset at 0.05 g/kg while IVIG was not effective at this dose, but did reach this level of efficacy at the 0.5 g/kg dose.

Table 4: Statistical analysis of clinical EAE. Comparison of Incidence, Median Day of onset and Mean

Maximal clinical score (MMS).

Modulation of T cell differentiation by IVIG, sl-IVIG or S2-IVIG

We found that CD3 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ regulatory T cells were increased in the spleen following

IVIG, s1 - and S2-IVIG treatment after only one injection (d3). This increased frequency is maintained and potentiated at day 8 post-immunization (Figures 8A and 8B). No difference was observed between the IVIG, s1 - and S2-IVIG groups.

EAE is dependent on T cell activation and differentiation. The frequency of CD44 ^" CD62L ⁺ CD4 ⁺ and CD8 ⁺ na ^' ive T cells is reduced following EAE induction (Figures 9A-9D). However, the frequency of both CD4 ⁺ and CD8 ⁺ na ^' ive T cells was significantly elevated following s1 - and S2-IVIG compared to vehicle and IVIG treatment. Conversely, the frequency of both CD4 ⁺ and CD8 ⁺ CD44 ⁺ CD62L ^" effector memory cells was significantly more reduced in s1 -IVIG and s2-IVIG-treated groups.

This result suggests that s1 - and S2-IVIG are significantly more potent than IVIG at preventing T cell activation. These results could be beneficial in AD considering the alteration of the memory cell compartment associated with the disease.

Modulation of myeloid cell differentiation and activation by IVIG, sl-IVIG or S2-IVIG

Microglia are often found near damaged tissue in AD patients. Studies have shown that microglia play opposite roles in disease pathogenesis by eliminating beta-amyloid aggregates via phagocytosis but also by killing nearby neurons through the release of neurotoxic factors. Microglia can be subdivided in two different subsets: resident microglia and parenchymal microglia (Figure 10A). Parenchymal microglia originate from the extravasation of peripheral monocytes through the blood brain barrier and are influenced by chemotactic gradients.

Expression of CCR2, a chemokinereceptor on monocytes and parenchymal microglia, has been demonstrated to play a positive role in the clearance of Αβ plaques in a mouse model of AD by promoting the infiltration of monocytes into the CNS. In our model, we observed a reduction of the frequency of parenchymal microglia in the CNS, following treatment with s1 - and S2-IVIG (Figure 1 0B). Treatment with IVIG and sialylated IVIG was associated with a higher frequency of CCR2 positive cells in the CNS (Figure 10C), but also in periphery (bone marrow (Figure 10D) and spleen (Figure 10E). These results suggest an increased ability for sialylated IVIG to positively regulate microglial mechanisms beneficial for the removal of

neurodegenerative plaque as well as to decrease the percentage of detrimental inflammatory microglia. .

Modulation of chemokine expression by IVIG and sl-IVIG

Similar to many other diseases which involve neurodegeneration, such as amyotrophic lateral sclerosis (ALS) and Huntington disease (HD), the blood brain barrier is intact in AD. Chronic

inflammatory responses are believed to be caused by resident CNS cells. An impressive number of chemokines and receptors have been found in resident CNS cells, including MCP-1 and eotaxin, which both are CCR2 ligands.

MCP-1 is involved in monocyte trafficking. In AD, it has been found in mature, but not in immature, senile plaques and in reactive microglia of brain tissues from patients, suggesting that it may contribute to the maturation of senile plaques. Serum levels have been shown to be increased in patients transitioning from mild cognitive impairment (MCI) to AD

Concentrations of eotaxin were previously shown to be elevated in serum from AD patients and increased levels of eotaxin may be associated with aging. Also, a role for eotaxin was recently demonstrated in neurogenesis. Therefore, inhibition of both MCP-1 and eotaxin may decrease plaques and preserve neurons in aging.

Levels of MCP-1 were significantly reduced in the s1 -IVIG-treated group, compared to vehicle (Figure 1 1 A), suggesting a role for sialylation in regulating the expression of this chemokine.

Levels of eotaxin were reduced in both IVIG and s1 -IVIG 0.5 g/kg groups (Figure 1 1 B). This result suggests a role for IVIG in regulating the expression of the cytokine, but not necessarily for sialylation.

Relevance to neurodegeneration for sialylated IVIG

• Reduction of CNS infiltration: reduction of CNS/PNS inflammation.

· Increased expression of CCR2: increased ability for plaque clearance/lesion resorption.

• Increased regulatory T cell differentiation: regulation of inflammation/T cell activation.

• Decreased memory T cell differentiation (and conversely increased na ^' ive phenotype) : reduction of inflammation and migration to CNS/PNS.

• Reduction of MCP-1 expression: decreased migration of monocytes to the CNS/PNS.

Decreased secretion of pro-inflammatory cytokines (IL-1□).

• Reduction of Eotaxin levels: improved neurogenesis.

While the methods have been described in conjunction with various instances and examples, it is not intended that the methods be limited to such instances or examples. On the contrary, the methods encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

What is claimed is: