TW202426480A - Vectors and cells for producing alpha 2 macroglobulin and soluble klotho - Google Patents

Abstract

The present disclosure relates generally to polynucleotides, vectors, and cells for producing alpha 2 macroglobulin (A2M), methods of making A2M, and compositions comprising A2M. The present disclosure relates generally to polynucleotides, vectors, and cells for producing soluble klotho (sKL), methods of making sKL, and compositions comprising sKL. The present disclosure also relates to methods of making compositions comprising A2M and skL and compositions comprising A2M and sKL.

Description

Vectors and cells for the production of α2-macroglobulin and soluble Klotho

The present invention generally relates to polynucleotides, vectors and cells for producing alpha 2 macroglobulin (A2M), methods of making A2M and compositions comprising A2M. The present invention generally relates to polynucleotides, vectors and cells for producing soluble klotho (sKL), methods of making sKL and compositions comprising sKL. The present invention also relates to methods of making compositions comprising A2M and skL and compositions comprising A2M and sKL.

α2 macroglobulin (A2M) acts as a broad spectrum protease binding protein that serves as a major component of the innate immune system and has a dual role as a pan-protease inhibitor and scavenger protein. Recently, many growth factors, interleukins, and hormones have been reported to bind to A2M via different mechanisms. It is a highly abundant protein that can interact with a range of molecules, giving it the ability to affect a variety of biological processes, such as interleukin clearance and bacterial opsonization. There is evidence that A2M may play an important role in the neuroinflammatory response to the pathogenesis of Alzheimer's disease (AD) (Varma et al., Mol Psychiatry. (2017) 22:1; Wu et al., J Immunol. (1998) 161(8):4356-65).

Klossot, also known as Klossot-α, is a founding member of the Klossot family within the glycosidase-1 superfamily (Nabeshima Y, Imura H. Sci. Aging Knowl. (2008) 28:455-464; Kuro-o, M. et al., Nature. (1997) 390 (6655):45-51). Klossot is expressed in areas associated with calcium regulation, primarily in the distal tubules of the kidney, but also in the cerebral plexuses (which produce cerebrospinal fluid) and parathyroid glands (Nabeshima Y, Imura H. Sci. Aging Knowl. (2008) 28:455-464). The phenotype of Krossow-deficient mice is similar to premature aging, including arteriosclerosis, osteoporosis, skin atrophy, infertility, emphysema and premature death (Kuro-o, M. et al., Nature. (1997) 390 (6655): 45-51).

Klosso exists primarily in two forms: full-length transmembrane Klosso and soluble Klosso, the latter formed by proteolytic cleavage of membrane Klosso. The role of soluble Klosso in disease and repair is not fully understood. Emerging evidence suggests that soluble Klosso exhibits humoral activity, as Klosso-null mice have functional deficiencies in organs that do not express Klosso. Other reports suggest that soluble Klosso may serve as an on-demand co-receptor for fibroblast growth factor 23 (FGF-23), or that it may act independently of FGF23 and interact with monosialogangliosides in lipid rafts of the plasma membrane, thereby altering its lipid organization and modulating biological pathways that regulate oxidative stress, apoptosis, stem cell renewal, fibrogenesis, and angiogenesis. Soluble Krost reduces oxidative stress by promoting forkhead transcription factor activation and nuclear translocation, leading to upregulation of manganese superoxide dismutase (MnSoD). It also inhibits fibrosis by binding to the type II transforming growth factor-β1 (TGF-β) receptor, preventing TGF-β binding and inhibiting SMAD 2/3 signaling (Sunil B et al., Scientific Reports (2020) 10:12368; Kurosu, H. et al., Science (2005) 309:1829-1833).

The present invention provides A2M protein and sKL protein and preparation methods thereof, polynucleotides (e.g., codon-optimized polynucleotides), vectors and cells (e.g., CHO cells, CHOK1 cells, CHOK1 GenS cells). The present invention also provides compositions (e.g., pharmaceutical compositions) comprising A2M protein and sKL protein and a pharmaceutically acceptable carrier.

Provided herein are polynucleotides (e.g., codon-optimized polynucleotides) comprising a nucleic acid encoding an A2M protein, wherein the nucleic acid sequence encoding the A2M protein is operably linked to a promoter (e.g., a heterologous promoter). In some cases, the polynucleotide is codon-optimized. In some cases, the A2M protein comprises the sequence set forth in any one of SEQ ID NOs: 2, 4, and 6. In some cases, the promoter is a heterologous promoter. In some cases, the heterologous promoter is a cytomegalovirus (CMV) promoter.

Also provided herein is a vector comprising a polynucleotide (eg, a codon-optimized polynucleotide) encoding an A2M protein described herein. In some cases, the vector comprises a glutamine synthetase gene.

Also provided herein is a cell (e.g., a CHO cell, a CHOK1 cell, a CHOK1 GenS cell) comprising a polynucleotide described herein (e.g., a codon-optimized polynucleotide) encoding an A2M protein. In some cases, the cell is a mammalian cell. In some cases, the cell is a Chinese hamster ovary (CHO) cell. In some cases, the cell is a CHO K1 cell. In some cases, the cell is a CHO K1 GenS cell. In some cases, the cell stably expresses the A2M protein.

Also provided herein is a cell comprising a vector described herein, the vector comprising a polynucleotide encoding an A2M protein (e.g., a codon-optimized polynucleotide). In some cases, the cell is a mammalian cell. In some cases, the cell is a CHO cell. In some cases, the cell is a CHO K1 cell. In some cases, the cell is a CHO K1 GenS cell. In some cases, the cell stably expresses A2M.

Also provided herein is a method of producing an A2M protein, the method comprising (a) culturing cells (e.g., CHO cells, CHOK1 cells, CHOK1 GenS cells) comprising a polynucleotide (e.g., a codon-optimized polynucleotide) or a vector described herein encoding an A2M protein, and (b) isolating the A2M protein. In some cases, the method further comprises formulating the isolated A2M protein into a sterile pharmaceutical composition comprising the isolated A2M and a pharmaceutically acceptable carrier. In some cases, the isolated A2M is in a dimeric form. In some cases, the concentration of the isolated A2M protein is at least 5,000 mg/L, at least 6,000 mg/L, at least 7,000 mg/L, at least 8,000 mg/L, or at least 9,000 mg/L.

Also provided herein is a polynucleotide (e.g., a codon-optimized polynucleotide) comprising a nucleic acid sequence encoding a soluble Klossol (sKL) protein, wherein the nucleic acid sequence encoding the sKL protein is operably linked to a promoter (e.g., a heterologous promoter). In some cases, the polynucleotide is codon-optimized. In some cases, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some cases, the promoter is a heterologous promoter. In some cases, the heterologous promoter is a CMV promoter.

Also provided herein is a vector (e.g., a PiggyBac vector) comprising a polynucleotide (e.g., a codon-optimized polynucleotide) encoding an sKL protein described herein. In some cases, the vector comprises a glutamine synthetase gene. In some cases, the vector is a PiggyBac vector. In some cases, the vector comprises, in 5' to 3' order, a 5' PiggyBac transposon-specific inverted terminal repeat (ITR) sequence, a nucleic acid sequence (e.g., a codon-optimized polynucleotide) encoding an sKL protein described herein, and a 3' PiggyBac transposon-specific ITR.

Also provided herein is a cell comprising a polynucleotide described herein (e.g., a codon-optimized polynucleotide) encoding an sKL protein. In some cases, the cell is a mammalian cell. In some cases, the cell is a CHO cell. In some cases, the cell is a CHO K1 cell. In some cases, the cell is a CHO K1 GenS cell. In some cases, the cell stably expresses the sKL protein.

Also provided herein is a cell comprising a vector described herein (e.g., a PiggyBac vector) comprising a polynucleotide encoding an sKL protein. In some cases, the cell is a mammalian cell. In some cases, the cell is a CHO cell. In some cases, the cell is a CHO K1 cell. In some cases, the cell is a CHO K1 GenS cell. In some cases, the cell stably expresses the sKL protein.

Also provided herein is a method of producing an sKL protein, the method comprising (a) culturing a cell comprising a polynucleotide (e.g., a codon-optimized polynucleotide) or a vector (e.g., a piggyBac vector) described herein encoding an sKL protein, and (b) isolating the sKL protein. In some cases, the method further comprises formulating the isolated sKL protein into a sterile pharmaceutical composition comprising the isolated sKL and a pharmaceutically acceptable carrier. In some cases, the concentration of the isolated sKL protein is at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, or at least 1,1000 mg/L.

Also provided herein is a method for producing an sKL protein, the method comprising: (a) transfecting a cell with a first vector and a second vector, (b) culturing the cell, and (c) isolating the sKL protein, wherein the first vector comprises, in 5' to 3' order, a 5' PiggyBac transposon-specific ITR sequence, a nucleic acid sequence encoding an sKL protein described herein (e.g., a codon-optimized nucleic acid sequence), and a 3' nucleic acid sequence PiggyBac transposon-specific ITR, and wherein the second vector comprises a nucleic acid sequence encoding a PiggyBac transposase.

Cross-references to Related Applications

This application claims priority to U.S. Provisional Application No. 63/416,765 filed on October 17, 2022, the contents of which are incorporated herein by reference in their entirety. Sequence Listing

This application contains a sequence listing that has been submitted electronically as an XML file named 51771-0009WO1_SL_ST26.xml. The size of the XML file created on October 13, 2023 is 39,746 bytes. The material in the XML file is incorporated herein by reference in its entirety.

The present invention generally relates to polynucleotides (e.g., codon-optimized polynucleotides), vectors and cells (e.g., host cells, such as CHO cells, CHOK1 cells, CHOK1 GenS cells) for producing α2 macroglobulin (A2M) proteins, methods for preparing A2M proteins, and compositions (e.g., pharmaceutical compositions) comprising A2M proteins. The present invention generally also relates to polynucleotides (e.g., codon-optimized polynucleotides), vectors (e.g., PiggyBac vectors) and cells (e.g., host cells, such as CHO cells, CHOK1 cells, CHOK1 GenS cells) for producing soluble Klossol (sKL) proteins, methods for preparing sKL proteins, and compositions (e.g., pharmaceutical compositions) comprising sKL proteins. The present invention also relates to methods for preparing compositions (e.g., pharmaceutical compositions) comprising A2M protein and sKL protein, compositions comprising A2M protein and sKL protein, and reagents used in the preparation methods.

The present invention provides A2M protein, methods for preparing A2M protein, including polynucleotides (e.g., codon-optimized polynucleotides), vectors and cells (e.g., CHO cells, CHO K1 cells, CHO K1 GenS cells), and compositions (e.g., pharmaceutical compositions comprising A2M protein). In some cases, the composition comprising A2M protein further comprises sKL protein. In some cases, A2M protein (or a composition comprising A2M protein) is used in combination with sKL protein (or a composition comprising sKL protein). Also provided herein are methods for preparing A2M protein, including related polynucleotides, vectors and cells.

A2M (also referred to in the art as C3 and PZP-like alpha-2-macroglobulin domain-containing protein 5 (CPAMD5)) is a 1,474 amino acid (including a 23 amino acid signal peptide) protein that functions as a broad spectrum protease binding protein. Human A2M is a 720 kDa homotetramer that contains four 180 kDa subunits. The subunits pair by disulfide bonds to form covalently linked dimers that non-covalently associate to form the cage-like quaternary structure of A2M.

Exemplary amino acid sequences of full-length human A2M are provided in SEQ ID NOs: 1, 3, and 5 ( FIG. 1A , FIG. 1C , FIG. 1E ). Exemplary amino acid sequences of full-length mature human A2M are provided in SEQ ID NOs: 2, 4, and 6 ( FIG. 1B , FIG. 1D , FIG. 1F ). Exemplary nucleic acid sequences encoding mRNA of human A2M are provided in SEQ ID NO: 7 ( FIG. 2A to FIG. 2B ).

A2M proteins in different species have high homology. See Figures 3A to 3G , which show an alignment of exemplary amino acid sequences of A2M proteins in humans, mice, rats, macaques, orangutans, and bovine animals. In some cases, the A2M protein is a human A2M protein (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6). Those skilled in the art will appreciate that regions or amino acids that have undergone changes (e.g., substitutions, insertions, or deletions) can be identified by comparing a reference A2M sequence with homologs or known variants and determining regions or amino acids that are not retained (see, e.g., Figures 3A to 3G or comparing SEQ ID NOs: 2, 4, and 6).

In some cases, the A2M protein comprises a modified amino acid sequence of a wild-type A2M protein (eg, a modified amino acid sequence of a wild-type human A2M protein, such as SEQ ID NO: 2). In some cases, the A2M proteins described herein bind to trypsin.

In some cases, the A2M protein described herein may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acid substitutions (relative to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6), such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) conservative and/or non-conservative amino acid substitutions. In some cases, the substitutions are substitutions of amino acids that interact directly with A2M protein binding partners known in the art or described herein (e.g., FGF1, FGF2, FGF-4, FGF-6, IFN-γ, IL-1β, IL2, IL-4, IL-6, IL-8, IL-18, NGF-β, PDGF, TGF-β1, TGF-β2, TNF-α, VEGF). In some cases, the substitutions are substitutions of amino acids that do not interact directly with A2M protein binding partners known in the art or described herein (e.g., FGF1, FGF2, FGF-4, FGF-6, IFN-γ, IL-1β, IL2, IL-4, IL-6, IL-8, IL-18, NGF-β, PDGF, TGF-β1, TGF-β2, TNF-α, VEGF). In some cases, the substitutions are both substitutions of amino acids that directly interact with an A2M protein binding partner known in the art or described herein and amino acids that do not directly interact with an A2M protein binding partner known in the art or described herein. In some cases, the A2M protein comprises an aspartic acid (D) at the amino acid corresponding to position 616 of SEQ ID NO: 2. In some cases, the A2M protein comprises a valine (V) at the amino acid corresponding to position 977 of the amino acid sequence set forth in SEQ ID NO: 2. In some cases, the A2M protein comprises aspartic acid (D) at the amino acid corresponding to position 616 of SEQ ID NO: 2, and valine (V) at the amino acid corresponding to position 977 of the amino acid sequence set forth in SEQ ID NO: 2. In certain cases, the A2M proteins described herein bind trypsin.

In some cases, the substitution is a conservative amino acid substitution. In some cases, the substitution is a non-conservative amino acid substitution. In some cases, the substitution is made with a conservative amino acid substitution and with a non-conservative amino acid substitution. "Conservative amino acid substitution" means that the substitution replaces one amino acid with another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine), and amino acids with , isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) and acidic side chains and their amides (e.g., aspartic acid, glutamine, asparagine, glutamine).

In some cases, the A2M proteins described herein have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity to an A2M protein sequence known in the art or described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6). In some cases, the A2M proteins described herein bind to trypsin. The variability in the amino acid sequence can be in regions that directly interact with A2M protein binding partners known in the art or described herein and/or in regions that do not directly interact with A2M protein binding partners known in the art or described herein. Methods for determining percent identity between sequences are known in the art. For example, for the purpose of optimal comparison, the sequences are aligned (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal comparison, and non-homologous sequences can be ignored for comparison purposes). In a preferred case, the length of the reference sequence aligned for comparison purposes is at least 30% of the length of the reference sequence, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90% or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are consistent at that position. The determination of the percent identity between the two amino acid sequences is completed using the BLAST 2.0 program. Sequence comparisons were performed using ungapped alignments with default parameters (Blossom 62 matrix, gap penalty of 11, gap penalty per residue of 1, and lambda ratio of 0.85). The mathematical algorithm used in the BLAST program is described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).

In some cases, the A2M protein described herein may also contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) deletions from the N-terminus and/or C-terminus of the A2M protein. In some cases, the A2M protein is 1,000-1,474, 1,000-1,400, 1,000-1,300, 1,000-1,200, 1,200-1,500, 1,300-1,500, or 1,400-1,500 amino acids in length. In some cases, the A2M protein is 1,451 amino acids in length. In certain instances, the A2M proteins described herein bind trypsin.

In some cases, the A2M protein described herein comprises the amino acid sequence of SEQ ID NO: 2. In some cases, the A2M protein described herein consists of the amino acid sequence of SEQ ID NO: 2. In some cases, the A2M protein is part of a composition (e.g., a pharmaceutical composition). In some cases, the A2M protein is part of a composition (e.g., a pharmaceutical composition) that further comprises an sKL protein (e.g., an sKL protein described herein). In some cases, the A2M protein (or a composition comprising the A2M protein) is used in combination with an sKL protein (or a composition comprising an sKL protein, e.g., an sKL protein described herein, e.g., a pharmaceutical composition). In some cases, the sKL protein comprises the amino acid sequence of SEQ ID NO: 15. In some cases, the sKL protein consists of the amino acid sequence of SEQ ID NO: 15.

In some cases, the A2M protein described herein comprises the amino acid sequence of SEQ ID NO: 4. In some cases, the A2M protein described herein consists of the amino acid sequence of SEQ ID NO: 4. In some cases, the A2M protein is part of a composition (e.g., a pharmaceutical composition). In some cases, the A2M protein is part of a composition (e.g., a pharmaceutical composition) that further comprises an sKL protein (e.g., an sKL protein described herein). In some cases, the A2M protein (or a composition comprising the A2M protein) is used in combination with an sKL protein (or a composition comprising an sKL protein, e.g., an sKL protein described herein, e.g., a pharmaceutical composition). In some cases, the sKL protein comprises the amino acid sequence of SEQ ID NO: 15. In some cases, the sKL protein consists of the amino acid sequence of SEQ ID NO: 15.

In some cases, the A2M protein described herein comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the A2M protein described herein consists of the amino acid sequence of SEQ ID NO: 6. In some cases, the A2M protein is part of a composition (e.g., a pharmaceutical composition). In some cases, the A2M protein is part of a composition (e.g., a pharmaceutical composition) that further comprises an sKL protein (e.g., an sKL protein described herein). In some cases, the A2M protein (or a composition comprising the A2M protein) is used in combination with an sKL protein (or a composition comprising an sKL protein, e.g., an sKL protein described herein, e.g., a pharmaceutical composition). In some cases, the sKL protein comprises the amino acid sequence of SEQ ID NO: 15. In some cases, the sKL protein consists of the amino acid sequence of SEQ ID NO: 15.

In some cases, the A2M protein described herein is in monomeric form, dimeric form, or tetrameric form. In some cases, the A2M protein described herein is in tetrameric form. In some cases, the A2M protein described herein is in dimeric form. Methods for determining whether an A2M protein is in monomeric, dimeric, or tetrameric form are known in the art, such as polyacrylamide gel electrophoresis. Methods for obtaining an A2M protein in monomeric, dimeric, or tetrameric form are known in the art, such as exposing a tetrameric A2M to an oxidizing agent (e.g., hypochlorite) to produce a dimeric A2M.

In certain instances, the A2M protein inhibits cathepsin L activity (e.g., reduces cathepsin L activity by 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 100% compared to cathepsin L activity in the absence of the A2M protein). Methods for determining whether an A2M protein inhibits cathepsin L activity are known in the art (see, e.g., the examples described herein).

In certain instances, the A2M protein inhibits the binding between ACE2 and the SARS-CoV-2 S protein (e.g., compared to cathepsin L activity in the absence of the A2M protein, the binding between ACE2 and the SARS-CoV-2 S protein is reduced by 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 100%). Methods for determining whether an A2M protein inhibits the binding between ACE2 and the SARS-CoV-2 S protein (see, e.g., the examples described herein).

In certain instances, the A2M proteins described herein bind to trypsin. Methods for determining whether an A2M protein binds to trypsin are known in the art, such as colocalization assays, coimmunoprecipitation assays, ELISA, Biacore, SPR, trypsin binding assays, and a trypsin activity colorimetric assay kit (AssayGenie, catalog # BN00984) described in Wyatt et al., PLOS ONE, DOI: 10.1371/journal.pone.0130036, 2015, which is incorporated herein by reference in its entirety.

In certain instances, the A2M proteins described herein bind to FGF23. Methods for determining whether an A2M protein binds to FGF23 are known in the art, such as co-localization assays, co-immunoprecipitation assays, ELISA, Biacore, and SPR.

In certain instances, the A2M proteins described herein bind to FGF2. Methods for determining whether an A2M protein binds to FGF2 are known in the art, such as co-localization assays, co-immunoprecipitation assays, ELISA, Biacore, and SPR. sKL

The present invention provides sKL proteins, methods of preparing sKL proteins, including polynucleotides (e.g., codon-optimized polynucleotides), vectors (e.g., PiggyBac vectors), and cells (e.g., CHO cells, CHO K1 cells, CHO K1 GenS cells), and compositions (e.g., pharmaceutical compositions) comprising sKL proteins. In some cases, the composition comprising the sKL protein further comprises an A2M protein (e.g., as described herein). In some cases, the sKL protein (or a composition comprising the sKL protein) is used in combination with the A2M protein (or a composition comprising the A2M protein). Also provided herein are methods of preparing sKL proteins, including related polynucleotides, vectors, and cells.

KL is a 1,012 amino acid protein (including a 33 amino acid signal peptide) that is a member of the glycosidase-1 superfamily and hydrolyzes steroid β-glucuronides. KL can act as a co-receptor for FGF23. KL exists primarily in two forms: full-length, membrane-bound KL (e.g., SEQ ID NO: 14) and sKL (e.g., SEQ ID NO: 15). sKL is formed by proteolytic cleavage of membrane-bound KL. sKL includes neither a cytoplasmic domain (e.g., amino acids 1,003-1,012 of SEQ ID NO: 13) nor a transmembrane domain (e.g., amino acids 982-1,002 of SEQ ID NO: 13).

An exemplary amino acid sequence of full-length human KL is provided in SEQ ID NO: 13 ( FIG. 4A ). An exemplary amino acid sequence of full-length mature human KL is provided in SEQ ID NO: 14 ( FIG. 4B ). An exemplary amino acid sequence of full-length mature human sKL is provided in SEQ ID NO: 15 ( FIG. 4C ). An exemplary nucleic acid sequence encoding mRNA of human KL is provided in SEQ ID NO: 16 ( FIG. 5A to FIG. 5B ).

KL proteins in different species have high homology. See Figures 6A to 6F , which show an alignment of exemplary amino acid sequences of KL proteins in mice, rats, cattle, humans, and macaques. In some cases, the sKL protein is a human sKL protein (e.g., SEQ ID NO: 15). Those skilled in the art will appreciate that regions or amino acids that have undergone changes (e.g., substitutions, insertions, or deletions) can be identified by comparing a reference KL sequence with a homolog and determining regions or amino acids that are not retained (see, e.g., Figures 6A to 6F ).

In certain instances, the sKL protein comprises a modified amino acid sequence of a wild-type sKL protein (eg, a modified amino acid sequence of a wild-type human sKL protein, such as the amino acid sequence set forth in SEQ ID NO: 15).

In certain instances, the sKL protein comprises a valine (V) at the amino acid corresponding to position 12 of SEQ ID NO: 15.

In certain instances, the sKL protein comprises a phenylalanine (F) at the amino acid corresponding to position 12 of SEQ ID NO: 15.

In some cases, the sKL protein comprises amino acids corresponding to positions 34 to 503 of SEQ ID NO: 13. In some cases, the sKL protein comprises amino acids corresponding to positions 515 to 956 of SEQ ID NO: 13. In some cases, the sKL protein comprises amino acids corresponding to positions 34 to 503 of SEQ ID NO: 13 and amino acids corresponding to positions 515 to 956 of SEQ ID NO: 13. In some cases, the sKL protein comprises amino acids corresponding to positions 504 to 515 of SEQ ID NO: 13. In some cases, the sKL protein comprises amino acids corresponding to positions 967 to 981 of SEQ ID NO: 13. In some cases, the sKL protein comprises amino acids corresponding to positions 957 to 981 of SEQ ID NO: 13. In some cases, the sKL protein does not comprise amino acids corresponding to positions 967 to 981 of SEQ ID NO: 13. In some cases, sKL comprises amino acids corresponding to positions 34 to 981 of SEQ ID NO: 13. In some cases, sKL comprises amino acids corresponding to positions 34 to 966 of SEQ ID NO: 13. In some cases, the C-terminus of the sKL protein corresponds to position 966 of SEQ ID NO: 13. In some cases, the C-terminus of the sKL protein corresponds to position 981 of SEQ ID NO: 13.

In some cases, the sKL proteins described herein may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acid substitutions (relative to the wild-type sKL protein sequence (e.g., SEQ ID NO: 15)), such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) conservative and/or non-conservative amino acid substitutions. In some cases, these substitutions are substitutions of amino acids that directly interact with sKL protein binding partners (e.g., FGF23) known in the art or described herein. In some cases, the substitutions are substitutions of amino acids that do not directly interact with sKL protein binding partners known in the art or described herein (e.g., FGF23). In some cases, the substitutions are substitutions of both amino acids that directly interact with sKL protein binding partners known in the art or described herein and amino acids that do not directly interact with sKL protein binding partners known in the art or described herein.

In some cases, the substitution is a conservative amino acid substitution. In some cases, the substitution is a non-conservative amino acid substitution. In some cases, the substitution is performed with a conservative amino acid substitution and with a non-conservative amino acid substitution.

In certain instances, the sKL proteins described herein have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity to an sKL protein sequence known in the art or described herein (e.g., SEQ ID NO: 15). The variability in the amino acid sequence can be in regions that directly interact with sKL protein binding partners known in the art or described herein and/or in regions that do not directly interact with sKL protein binding partners known in the art or described herein.

In some cases, the sKL proteins described herein may also contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) deletions from the N-terminus and/or C-terminus of the sKL protein. In some cases, the sKL protein is 750-1200, 750-1100, 750-1000, 850-1200, 840-1100, 850-1000, or 900-1000 amino acids in length. In some cases, the sKL protein is 948 amino acids in length. In some cases, the length of sKL is 948 amino acids, the N-terminus of the sKL protein corresponds to position 34 of SEQ ID NO: 13, and the C-terminus of the sKL protein corresponds to position 981 of SEQ ID NO: 13.

In some cases, the sKL protein described herein comprises the amino acid sequence of SEQ ID NO: 15. In some cases, the sKL protein described herein consists of the amino acid sequence of SEQ ID NO: 15. In some cases, the sKL protein is part of a composition (e.g., a pharmaceutical composition). In some cases, the sKL protein is part of a composition (e.g., a pharmaceutical composition) that further comprises an A2M protein (e.g., an A2M protein described herein). In some cases, the sKL protein (or a composition comprising the sKL protein) is used in combination with an A2M protein (or a composition comprising an A2M protein, e.g., an A2M protein described herein, e.g., a pharmaceutical composition). In some cases, the A2M protein comprises the amino acid sequence of SEQ ID NO: 2. In some cases, the A2M protein comprises the amino acid sequence of SEQ ID NO: 4. In some cases, the A2M protein comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the A2M protein consists of the amino acid sequence of SEQ ID NO: 2. In some cases, the A2M protein consists of the amino acid sequence of SEQ ID NO: 4. In some cases, the A2M protein consists of the amino acid sequence of SEQ ID NO: 6.

In certain instances, the sKL proteins described herein bind to FGF23. Methods for determining whether an sKL protein binds to FGF23 are known in the art, such as co-localization assays, co-immunoprecipitation assays, ELISA, Biacore, and SPR.

In certain cases, the sKL protein described herein induces ERK1/2 phosphorylation (e.g., in a cell assay). Methods for determining whether an sKL protein induces ERK1/2 phosphorylation (e.g., in a cell assay) are known in the art, such as Western blot, ELISA, immunofluorescence. For a description of an exemplary ERK1/2 phosphorylation assay, see, e.g., Zhong et al., JBC, 295 (10: 3115-3133), 2020, which is incorporated herein by reference in its entirety.

In certain cases, the sKL protein described herein induces ERK1/2 phosphorylation (e.g., in a cell assay). Methods for determining whether the sKL protein induces ERK1/2 phosphorylation (e.g., in a cell assay) are known in the art, such as Western blot, ELISA, and immunofluorescence. For a description of an exemplary ERK1/2 phosphorylation assay, see, for example, Zhong et al., JBC, 295 (10:3115-3133), 2020, which is incorporated herein by reference in its entirety. See also phospho (Thr202/Tyr204; Thr184/Tyr187)/total ERK1/2 assay (Mesoscale, Catalog No. K15107D-1). Polynucleotides, Vectors, and Cell Polynucleotides

Provided herein are polynucleotides encoding A2M (e.g., codon-optimized polynucleotides) and polynucleotides encoding sKL. In some cases, the polynucleotides (e.g., codon-optimized polynucleotides) encode an A2M protein described herein. In some cases, the polynucleotides (e.g., codon-optimized polynucleotides) encode an sKL protein described herein. In some cases, the polynucleotides (e.g., codon-optimized polynucleotides) encode an A2M protein described herein and an sKL protein.

In some cases, the polynucleotide is codon optimized. Methods for generating codon optimized polynucleotides are known in the art. Codon optimization can be used to improve the expression of the A2M protein or sKL protein described herein and to increase the translation efficiency of the polynucleotide encoding the A2M protein or sKL protein by adapting the codon bias of the host organism or the desired cell type (e.g., CHO cells). Codon optimization tools, algorithms, and services are known in the art. Non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park CA). In some cases, the polynucleotide is codon optimized for expression and translation in CHO cells (e.g., CHO K1 cells, CHO K1 GenS cells).

In some cases, the polynucleotide is a codon-optimized polynucleotide comprising a nucleic acid sequence encoding an A2M protein. In some cases, the A2M protein comprises a sequence as set forth in any one of SEQ ID NOs: 2, 4, and 6. In some cases, the nucleic acid sequence encoding the A2M protein is operably linked to a heterologous promoter. A heterologous promoter relative to the nucleic acid sequence encoding the A2M protein comprises a promoter not found in nature to be related to the nucleic acid sequence encoding the A2M. In some cases, the heterologous promoter is a CMV promoter. In some cases, the sequence encoding the A2M protein comprises an SV40 poly(adenylic acid) signal.

In some cases, the polynucleotide is a codon-optimized polynucleotide comprising a nucleic acid sequence encoding an sKL protein. In some cases, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some cases, the nucleic acid sequence encoding the sKL protein is operably linked to a heterologous promoter. A heterologous promoter relative to the nucleic acid sequence encoding the sKL protein comprises a promoter not found in nature related to the nucleic acid sequence encoding sKL. In some cases, the heterologous promoter is a CMV promoter. In some cases, the sequence encoding the sKL protein comprises an SV40 poly(adenylic acid) signal.

Promoters suitable for use with the polynucleotides described herein are known in the art. The choice of promoter generally depends on the choice of the host. Promoters suitable for use with eukaryotic hosts include, for example, promoters from CMV, SV40, bovine papilloma virus, adenovirus. In some cases, the promoter is a CMV promoter. Vectors

Also provided herein are vectors (e.g., expression vectors, piggyBac vectors) comprising a polynucleotide encoding an A2M protein described herein (e.g., a codon-optimized polynucleotide) and/or a polynucleotide encoding an sKL protein described herein (e.g., a codon-optimized polynucleotide). In some cases, the vector comprises a polynucleotide encoding an A2M protein described herein. In some cases, the vector comprises a polynucleotide encoding an sKL protein described herein. In some cases, the vector comprises a first polynucleotide encoding an A2M protein described herein and a second polynucleotide encoding an sKL protein described herein. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with a cation condenser, and DNA or RNA expression vectors encapsulated in liposomes. In some cases, the vector is a PiggyBac vector.

A wide variety of expression host/vector combinations can be employed. Expression vectors suitable for eukaryotic hosts include, for example, vectors comprising expression control sequences (e.g., promoters) from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors suitable for bacterial hosts include known bacterial plasmids, such as those from Escherichia coli, including pCR1, pBR322, pMB9, and derivatives thereof; and broader host range plasmids, such as M13 and other filamentous single-stranded DNA bacteriophages.

In some cases, the carrier is a carrier described in U.S. Patent No. 10,889,822, which is incorporated herein by reference in its entirety.

In some cases, the vector is a PiggyBac vector. The PiggyBac transposon is a mobile genetic element that is efficiently translocated between the vector and the host cell chromosome via a "cut and paste" transposition mechanism. During transposition, the PB transposase recognizes the transposon-specific inverted terminal repeat sequences (ITRs) located on the 5' and 3' ends of the transposon-based vector and moves the vector content from the original vector site to between the 5' ITR and 3' ITR and integrates it into the TTAA chromosomal site. PiggyBac vectors and methods of using PiggyBac vectors are known in the art, see, e.g., U.S. Patent Nos. 6,962,810, 7,105,343, 7,129,083, 8,592,211, 9,670,503, 9,840,718, 10,087,463, 11,098,310, 11,186,847, 11,261,462, and 11,434,503, each of which is incorporated herein by reference in its entirety. In some cases, the vector comprises, in 5' to 3' order, a 5' PiggyBac transposon-specific ITR sequence, a nucleic acid sequence (eg, a nucleic acid sequence) encoding a protein described herein (eg, an sKL protein), and a 3' PiggyBac transposon-specific ITR.

Also provided herein are cells expressing the A2M protein and/or sKL protein described herein.

In some cases, the cell expresses an A2M protein described herein. In some cases, the cell comprises a polynucleotide comprising a sequence encoding an A2M protein operably linked to a promoter. In some cases, the cell comprises a vector comprising a sequence encoding an A2M protein operably linked to a promoter. In some cases, the cell stably integrates a polynucleotide comprising a sequence encoding an A2M protein operably linked to a promoter. In some cases, the A2M protein comprises a sequence as set forth in any one of SEQ ID NOs: 2, 4, and 6. In some cases, the cell is a CHO cell. In some cases, the cell is a CHOK1-GenS cell.

In some cases, the cell expresses an sKL protein described herein. In some cases, the cell comprises a polynucleotide comprising a sequence encoding an sKL protein operably linked to a promoter. In some cases, the cell comprises a vector comprising a sequence encoding an sKL protein operably linked to a promoter. In some cases, the cell stably integrates a polynucleotide comprising a sequence encoding an sKL protein operably linked to a promoter. In some cases, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some cases, the cell is a CHO cell. In some cases, the cell is a CHOK1-GenS cell.

In some cases, the cell expresses the A2M protein and the sKL protein described herein. In some cases, the cell comprises a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a sequence encoding the A2M protein operably linked to a promoter, the second polynucleotide comprising a sequence encoding the sKL protein operably linked to a promoter. In some cases, the cell comprises a first vector and a second vector, the first vector comprising a sequence encoding the A2M protein operably linked to a promoter, the second vector comprising a sequence encoding the sKL protein operably linked to a promoter. In some cases, the cell comprises a vector comprising a first sequence encoding the A2M protein operably linked to a promoter and a second sequence encoding the sKL protein operably linked to a promoter. In some cases, the cell stably integrates a first polynucleotide comprising a sequence encoding an A2M protein operably linked to a promoter and a second polynucleotide comprising a sequence encoding an sKL protein operably linked to a promoter. In some cases, the A2M protein comprises the sequence set forth in any one of SEQ ID NOs: 2, 4, and 6. In some cases, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some cases, the cell is a CHO cell. In some cases, the cell is a CHOK1-GenS cell.

Various cells for expressing or encoding A2M protein and/or sKL protein are known in the art. For example, the cell can be an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce human A2M (e.g., A2M described herein) or human sKL (e.g., sKL described herein).

A variety of mammalian culture systems can be used to express the A2M proteins and/or sKL proteins described herein. Expression of recombinant proteins in mammalian cells can be desirable because such proteins are generally accurately folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney derived), L-929 (murine fibroblast derived), C127 (murine breast tumor derived), 3T3 (murine fibroblast derived), CHO (Chinese hamster ovary derived), HeLa (human cervical carcinoma derived), BHK (hamster kidney fibroblast derived), HEK-293 (human embryonic kidney derived) cell lines and variants thereof. In some cases, the cell is a CHO cell (e.g., a CHOK1 cell or a CHOK1 GenS cell). The mammalian expression vector may comprise non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-transcribed sequences, such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites and transcription termination sequences. Methods for preparing A2M and SKL

Also provided herein are methods of making the A2M proteins described herein.

Also provided herein are methods of making the sKL proteins described herein.

Also provided herein are polynucleotides (e.g., codon optimization), vectors (e.g., expression vectors, piggyBac vectors), and cells (e.g., CHO cells, CHO K1 cells, CHO K1 GenS cells) suitable for use in the methods of making the A2M proteins and/or sKL proteins described herein. Thus, provided herein are polynucleotides encoding the A2M proteins and/or sKL proteins described herein, vectors comprising the polynucleotides, and cells comprising the polynucleotides or vectors. An exemplary nucleic acid encoding a wild-type human A2M protein is provided in SEQ ID NO: 7 (GenBank Accession No. NM_000014.6; FIG. 2A to FIG. 2B ). An exemplary nucleic acid encoding a wild-type human KL protein is provided in SEQ ID NO: 16 (GenBank Accession No. NM_004795.4; FIG. 5A to FIG. 5B ). In some cases, the polynucleotide is codon optimized for use in mammalian (e.g., human, Chinese hamster ovary) cells. Methods for generating polynucleotides and codon optimization are well known in the art.

Host cells suitable for expressing the A2M protein or sKL protein described herein are known in the art and include, for example, prokaryotes (e.g., bacteria, such as E. coli), yeast cells, insect cells, and higher eukaryotic (e.g., mammalian) cells. Cell-free translation systems may also be employed. Appropriate selection of expression vectors and methods for protein production for bacterial, fungal, yeast, and mammalian cell hosts are well known in the art. In some cases, the cell is a CHO cell. In some cases, the cell is a CHO K1 cell. In some cases, the cell is a CHO K1 cell.

In some cases, the method of preparing an A2M protein described herein comprises: (a) culturing a cell comprising a polynucleotide (e.g., codon optimized) or a vector encoding an A2M protein (e.g., under conditions that allow expression of the A2M protein), and (b) isolating the A2M protein. In some cases, the method further comprises formulating the A2M protein into a pharmaceutical composition described herein (e.g., a sterile pharmaceutical composition) comprising the A2M protein and a pharmaceutically acceptable carrier. In some cases, the concentration of the isolated A2M is at least 5,000 mg/L, at least 6,000 mg/L, at least 7,000 mg/L, at least 8,000 mg/L, or at least 9,000 mg/L. In some cases, the method further comprises formulating the A2M protein into a pharmaceutical composition described herein, which further comprises an sKL protein.

In some cases, methods of preparing an sKL protein described herein comprise: (a) culturing cells comprising a polynucleotide (e.g., codon optimized) or a vector encoding an sKL protein (e.g., under conditions that allow expression of the sKL protein), and (b) isolating the sKL protein. In some cases, the method further comprises formulating the sKL protein into a pharmaceutical composition described herein (e.g., a sterile pharmaceutical composition) comprising the sKL protein and a pharmaceutically acceptable carrier. In some cases, the concentration of the isolated sKL protein is at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, or at least 1,1000 mg/L. In some cases, the method further comprises formulating the sKL protein into a pharmaceutical composition described herein, which further comprises an A2M protein.

In some cases, a PiggyBac transposon-based vector system is used to prepare sKL protein. PiggyBac vectors and methods of using PiggyBac vectors are known in the art, see, e.g., U.S. Patent Nos. 6,962,810, 7,105,343, 7,129,083, 8,592,211, 9,670,503, 9,840,718, 10,087,463, 11,098,310, 11,186,847, 11,261,462, and 11,434,503, each of which is incorporated herein by reference in its entirety.

In some cases, the method of preparing sKL protein comprises: (a) transfecting cells (e.g., CHO cells, e.g., CHO K1 or CHO K1 GenS cells) with a first vector comprising a nucleic acid sequence encoding an sKL protein described herein and a second vector comprising a nucleic acid sequence encoding a piggyBac translocase, (b) culturing the cells, and (c) isolating the sKL protein, wherein the first vector comprises a 5' ITR, a nucleic acid sequence encoding the sKL protein, and a 3' ITR in a 5' to 3' orientation. In some cases, the method further comprises formulating the sKL protein into a pharmaceutical composition described herein (e.g., a sterile pharmaceutical composition) comprising the sKL protein and a pharmaceutically acceptable carrier. In some cases, the concentration of the isolated sKL protein is at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, or at least 1,1000 mg/L. In some cases, the method further comprises formulating the sKL protein into a pharmaceutical composition described herein, which further comprises an A2M protein. Examples Example 1 : A2M codon optimization

This example outlines the development of a CHOK1-GenS cell line expressing A2M.

The A2M open reading frame (ORF) was codon-optimized for expression in CHO cells.

The codon-optimized A2M gene was synthesized and cloned into the mammalian expression vector pGenHT1.0-DGV.

pGenHT1.0-DGV contains the MCS for proteins, and the promoter of the MCS is cellular giant virus (CMV). The vector contains the glutamine synthetase (GS) gene driven by the simian virus 40 (SV40) early promoter. The codon-optimized A2M gene was inserted into the upstream MCS region of the pGenHT1.0-DGV vector (restriction enzyme sites: AscI-FseI). Subsequently, the plasmid was amplified and linearized by the PvuI restriction enzyme and transfected at a concentration of 1 μg/μl.

The plasmid was transfected into CHOK1-GenS cells. CHOK1-GenS cells were cultured in growth medium (CD CHO + 6 mM L-glutamine + anti-agglutinating agent (400×)) at 37.0°C, 5.0% CO ₂ and 120 rpm shaking speed.

Plasmids were prepared for transfection and transfected into CHOK1-GenS cells, followed by cell pool screening under MSX pressure. Based on potency, two cell pools were combined into new cell pools before each transfection, and the combined cell pool was selected for single cell line screening using limiting dilution in 96-well plates. Single cell lines were confirmed based on images captured by Cell MetricTM CLD (data not shown). Single cell lines with good confluence and relatively high titers were selected for batch feed culture evaluation. Example 2 : Generation of A2M vectors

The codon-optimized A2M gene was synthesized and cloned into the mammalian expression vector pGenHT1.0-DGV. The pGenHT1.0-DGV vector contains a multiple cloning site (MCS) for the protein. The promoter of the MCS is the cytomegalovirus (CMV). At the same time, the vector contains the glutamine synthetase (GS) gene driven by the simian virus 40 (SV40) early promoter. After codon optimization, the target gene was synthesized and inserted into the upstream MCS region of the vector. Next, the plasmids were expanded and transfected at a concentration of 1 μg/μl. Example 3 : Cells expressing A2M

To engineer cells that stably express A2M, CHOK1-GenS cells (European Collection of Authenticated Cell Culture) were thawed from liquid nitrogen into growth medium (CD CHO medium (Gibco; catalog number 10743-029), 6 mM L-glutamine + antiagglutinant (400×) (Gibco; catalog number 01-0057DG)), and then the cells were maintained in a 37.0°C incubator with 5.0% _CO2 . The cells were resuspended in 18 ml of growth medium in a 125 ml shake flask at a density of 0.50 × 10 ⁶ cells/ml and then maintained in an incubator at 37.0°C, 5.0% CO ₂ and 120 rpm shaking. CHOK1-GenS cells were cultured every 24 to 72 hours.

The vector encoding sKL (see Example 2 : Generation of A2M vector) was transfected into CHOK1-GenS. The transfected cells were inoculated into a 24-well plate with selection medium (CD CHO medium + 25 μM L-methionine-sulfenimide & 1G (MSX; Sigma, catalog number M5379-1G) + anti-agglutinant (400×)) for cell pool screening. The cells were subcultured with selection medium every 4 to 6 days. After 26 days of screening, the modified medium was collected from the 6-day culture of the pool for dot blot analysis (data not shown). Compare cell pools and combine the top 2 titer pools into new pools that are selected for fed-batch culture evaluation. Analyze culture supernatants by ELISA and SDS PAGE. Based on the performance of cell pool cultures in fed-batch, select the top pools for simplex screening.

Finally, based on productivity and growth efficiency, the top 6 clones were selected for primary cell bank (PCB) construction. The individuality and titer (mg/L) of the top 6 clones are provided in Table 1.

Table 1. CHOK1-GenS A2M clones. Data in the single cell column represent the number of cells observed by the cell imaging system. Images were captured twice on day 1. Data show single cell inoculation on day 0 and cell division status on days 1 and 2. Pure line ID Pure line 1 Pure 2 Pure 3 Pure 4 Pure 5 Pure 6 Individual plant (D0-D1-D1-D2) 1-2-2-7 1-1-2-4 1-2-2-8 1-2-2-4 1-1-1-2 1-2-2-4 Potency (mg/L) 9185.06 9104.32 8904.04 8757.9 7807.13 3510.7 Peak VCD (×10 ⁶ cells / mL ) 21.7 16.6 21.7 21.0 9/16 18.3

Clones 1 to 6 were thawed and subjected to viability confirmation ( Table 2 ) and mycoplasma testing. Each of the tested clones was free of mycoplasma.

Table 2. Recovered data Pure line ID Viable cell density (×10 ⁶ cells / mL ) Survival rate (%) Pure 1 1.04 91.87 Pure 2 0.84 92/64 Pure 3 1.19 95.94 Pure 4 0.96 94.43 Pure 5 1.26 97.7 Pure 6 1.39 93.96 Example 4 : sKL Codon Optimization

A codon-optimized polynucleotide encoding sKL (SEQ ID NO: 15) was prepared for expression in CHO cells. Example 5 : Generation of sKL vector

The codon-optimized sKL gene was synthesized and cloned into the mammalian expression vector pGenHT1.0-DGV. The pGenHT1.0-DGV vector contains a multiple cloning site (MCS) for the protein. The promoter of the MCS is the cytomegalovirus (CMV). At the same time, the vector contains the glutamine synthetase (GS) gene driven by the simian virus 40 (SV40) early promoter. After codon optimization, the target gene was synthesized and inserted into the upstream MCS region of the vector. Next, the plasmids were expanded and transfected at a concentration of 1 μg/μl. Example 6 : Cells expressing sKL

To engineer cells that stably express sKL, CHOK1-GenS cells (European Collection of Authenticated Cell Culture) were thawed from liquid nitrogen into growth medium (CD CHO medium (Gibco; catalog number 10743-029), 6 mM L-glutamine + antiagglutinant (400×) (Gibco; catalog number 01-0057DG)), and then the cells were maintained in a 37.0°C incubator with 5.0% _CO2 . The cells were resuspended in 18.5 ml of growth medium in a 125 ml shake flask at a density of 0.40 × 10 ⁶ cells/ml and then maintained in an incubator at 37.0°C, 5.0% CO ₂ and 120 rpm shaking. CHOK1-GenS cells were cultured every 24 to 72 hours.

The vector encoding sKL (see Example 5 : Generation of sKL vector) was transfected into CHOK1-GenS cells and the supernatant was collected 48 hours after transfection for Western blot analysis. No significant signal was detected in the sample 48 hours after transfection by Western blot analysis (data not shown).

Transfected cells were seeded into 24-well plates with selection medium (CD CHO medium + 25 μM L-methionine-sulfenimide & 1G (MSX; Sigma, catalog number M5379-1G) + anti-agglutinant (400×)) for cell pool screening. Cells were subcultured with selection medium every 4 to 6 days. After 20 days of screening, 12 cell pools were selected for batch feed culture evaluation based on the recovery status of the cell pools. Based on the efficiency of the batch feed of the cell pool culture, the top 3 cell pools were selected for single line screening using limiting dilution method in 96-well plates. The Cell MetricTM CLD imager was used to record and confirm the individuality of single cell lines (data not shown). 36 single lines with high titers and good confluence were selected for batch feed culture evaluation. Finally, based on productivity and growth efficiency, the top 6 lines were selected for primary cell bank (PCB) construction. The individuality and titer (mg/L) of the top 6 lines are provided in Table 3.

Table 3. CHOK1-GenS sKL clones. Data in the single cell column represent the number of cells observed by the cell imaging system. Images were captured twice on day 1. Data show single cell inoculation on day 0 and cell division status on day 1 and day 2. Pure line ID Pure 1 Pure 2 Pure 3 Pure 4 Pure 5 Pure 6 Individual plant (D0-D1-D1-D2) 1-1-2-4 1-2-2-8 1-2-2-4 1-2-4-8 1-1-2-4 1-2-2-8 Potency (mg/L) 1125.75 1228.18 1118.11 684.54 1091.67 838.65

Clones 1 to 6 were thawed and subjected to viability confirmation ( Table 4 ) and mycoplasma testing. Each of the tested clones was free of mycoplasma.

Table 4. Recovered data Pure line ID Viable cell density (×10 ⁶ cells / mL ) Survival rate (%) Pure line 1 1.19 98.43 Pure 2 1.09 97.89 Pure 3 1.06 98.38 Pure 4 1.35 95.93 Pure 5 1.18 99.02 Pure 6 1.52 98.57 Example 7 : Encoding sKL in a PiggyBac vector

To engineer cells that stably express sKL protein, CHOK1-GenS cells (European Collection of Authenticated Cell Culture) were transfected with a piggyBac vector encoding sKL protein. The resulting clones produced a titer of 1228.18 mg/L of sKL protein.

Although the invention has been described in conjunction with its embodiments, the foregoing description is intended to illustrate and not limit the scope of the invention as defined by the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Figure 1A depicts an exemplary amino acid sequence (SEQ ID NO: 1) of human A2M (GenBank Accession No. NP_000005.2). The residues in lowercase bold are signal peptides.

FIG. 1B depicts an exemplary amino acid sequence (SEQ ID NO: 2) of mature human A2M (amino acids 24-1,474 of SEQ ID NO: 1).

Figure 1C depicts an exemplary amino acid sequence (SEQ ID NO: 3) of human A2M (GenBank Accession No. NP_000005.3). The residues in lowercase are signal peptides.

Figure 1D depicts an exemplary amino acid sequence (SEQ ID NO: 4) of mature human A2M (amino acids 24-1,474 of SEQ ID NO: 3).

Figure 1E depicts an exemplary amino acid sequence (SEQ ID NO: 5) of human A2M (GenBank Accession No. AAA51551.1). The residues in lowercase bold are signal peptides.

FIG. 1F depicts an exemplary amino acid sequence (SEQ ID NO: 6) of mature human A2M (amino acids 24-1,474 of SEQ ID NO: 5).

Figures 2A - 2B depict an exemplary mRNA sequence (SEQ ID NO: 7) encoding human A2M (GenBank Accession No. NM_000014.6).

Figures 3A - 3G depict alignments of exemplary mouse (SEQ ID NO: 8), rat (SEQ ID NO: 9), bovine (SEQ ID NO: 10), macaque (SEQ ID NO: 11), human (SEQ ID NO: 1), and orangutan (SEQ ID NO: 12) A2M proteins.

Figure 3H depicts the percent identity matrix for the alignment of Figures 3A to 3G .

Figure 4A depicts an exemplary amino acid sequence (SEQ ID NO: 13) of human Klossol (GenBank Accession No. NP_004786.2). Residues in lowercase bold are signal peptides; residues in lowercase italics are transmembrane domains and cytoplasmic domains.

FIG. 4B depicts an exemplary amino acid sequence (SEQ ID NO: 14) of mature human Klossol (amino acids 34-1,012 of SEQ ID NO: 13).

FIG. 4C depicts an exemplary amino acid sequence (SEQ ID NO: 15) of human soluble Crosby (amino acids 34-981 of SEQ ID NO: 13).

5A - 5B depict an exemplary mRNA sequence encoding human Crosby (GenBank Accession No. NM_004795.4) (SEQ ID NO: 16).

Figures 6A - 6E depict an alignment of exemplary mouse (SEQ ID NO: 17), rat (SEQ ID NO: 18), bovine (SEQ ID NO: 19), human (SEQ ID NO: 14), and macaque (SEQ ID NO: 20) Crosbylin proteins.

FIG6F depicts the percent identity matrix for the alignment of FIG6A - 6E .

TW202426480A_112139684_SEQL.xml

Claims (31)

A codon-optimized polynucleotide comprising a nucleic acid sequence encoding an alpha 2 macroglobulin (A2M) protein, wherein the A2M protein comprises the sequence set forth in any one of SEQ ID NOs: 2, 4, and 6, and wherein the nucleic acid sequence encoding the A2M protein is operably linked to a heterologous promoter. The codon-optimized polynucleotide of claim 1, wherein the heterologous promoter is a cytomegalovirus (CMV) promoter. The codon-optimized polynucleotide of claim 1 or 2, wherein the nucleic acid sequence encoding the A2M protein comprises a simian virus 40 (SV40) poly(adenylic acid) (poly(A)) signal. A vector comprising the codon-optimized polynucleotide of any one of claims 1 to 3. The vector of claim 4, comprising a glutamine synthetase (GS) gene. A cell comprising the codon-optimized polynucleotide of any one of claims 1 to 3. A cell comprising the vector of claim 4 or 5. The cell of claim 6 or 7, which is a mammalian cell. The cell of claim 6 or 7, which is a Chinese hamster ovary (CHO) cell. The cell of claim 6 or 7, which is a CHO K1 cell or a CHO K1 GenS cell. A cell according to any one of claims 6 to 10, which stably expresses the A2M. A method for producing an A2M protein, the method comprising (a) culturing the cell of any one of claims 6 to 11, and (b) isolating the A2M protein. The method of claim 12, further comprising formulating the isolated A2M into a sterile pharmaceutical composition comprising the isolated A2M and a pharmaceutically acceptable carrier. The method of claim 12 or 13, wherein the isolated A2M is in a dimeric form. The method of any one of claims 12 to 14, wherein the concentration of the isolated A2M protein is at least 5,000 mg/L, at least 6,000 mg/L, at least 7,000 mg/L, at least 8,000 mg/L, or at least 9,000 mg/L. A codon-optimized polynucleotide comprising a nucleic acid sequence encoding a soluble klotho (sKL) protein, wherein the sKL protein comprises the sequence set forth in SEQ ID NO: 15, and wherein the nucleic acid sequence encoding the sKL protein is operably linked to a heterologous promoter. The codon-optimized polynucleotide of claim 16, wherein the heterologous promoter is a CMV promoter. The codon-optimized polynucleotide of claim 16 or 17, wherein the nucleic acid sequence encoding the sKL protein comprises a simian virus 40 (SV40) poly(A) signal. A vector comprising the codon-optimized polynucleotide of any one of claims 16 to 18. The vector of claim 19, comprising a glutamine synthetase (GS) gene. A vector comprising, in 5' to 3' order, a 5' piggyBac transposon-specific inverted terminal repeat (ITR) sequence, a codon-optimized polynucleotide according to any one of claims 16 to 18, and a 3' piggyBac transposon-specific ITR. A cell comprising the codon-optimized polynucleotide of any one of claims 16 to 18. A cell comprising the vector of any one of claims 19 to 21. The cell of claim 22 or 23, which is a mammalian cell. The cell of claim 22 or 23, which is a CHO cell. The cell of claim 22 or 23, which is a CHO K1 cell or a CHO K1 GenS cell. The cell of any one of claims 22 to 26, which stably expresses the sKL protein. A method for producing sKL protein, the method comprising (a) culturing the cell of any one of claims 22 to 27, and (b) isolating the sKL protein. A method for producing sKL protein, the method comprising: (a) transfecting cells with the vector of claim 21 and a second vector comprising a nucleic acid sequence encoding a piggyBac translocase, (b) culturing the cells, and (c) isolating the sKL protein. The method of claim 28 or 29, further comprising formulating the isolated sKL into a sterile pharmaceutical composition comprising the isolated sKL and a pharmaceutically acceptable carrier. The method of any one of claims 28 to 30, wherein the concentration of the isolated sKL is at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, or at least 1,1000 mg/L.