CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63/416,765, filed October 17, 2022, the content of which is incorporated by reference in their entirety herein.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named 51771-0009W01_SL_ST26.xml. The XML file, created on October 13, 2023, is 39,746 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

FIELD

[0003] 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.

BACKGROUND OF THE INVENTION

[0004] Alpha 2 Macroglobulin (A2M) functions as a broad-spectrum protease-binding protein, which acted as a major component of the innate immune system, and has a dual role as a panprotease inhibitor and a scavenger protein. More recently, it has been reported that numerous growth factors, cytokines and hormones bind to A2M through diverse mechanisms. It is a highly abundant protein that can interact with a range of molecules, resulting in its ability to influence many biological processes such as cytokine clearance and the opsonization of bacteria. There is evidence to suggest that A2M may have an important role in the neuro- inflammatory response to Alzheimer’s disease (AD) pathogenesis (Varma et al., Mol Psychiatry. (2017) 22: 1; Wu et al., J Immunol. (1998) 161(8):4356-65). [0005] Klotho, also called Klotho-a, is the founding member of the Klotho 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). Klotho is expressed in areas concerned with calcium regulation, predominantly in the kidney distal convoluted tubules, but also in the brain choroid plexus (which produces cerebrospinal fluid) and the parathyroid (Nabeshima Y, Imura H. Sci. Aging Knowl. (2008) 28:455-464). The phenotype of Klotho-deficient mice resembles premature aging, including arteriosclerosis, osteoporosis, skin atrophy, infertility, emphysema and premature death (Kuro-o, M. et al., Nature. (1997) 390 (6655):45-51).

[0006] Klotho exists in two main forms: full-length transmembrane Klotho and soluble Klotho, the latter being formed by proteolytic cleavage of membrane Klotho. The role of soluble Klotho in disease and repair is not fully understood. Emerging evidence suggest that soluble Klotho exhibits humoral activity as Klotho deficient mice have functional deficits in organs which do not express Klotho. Other reports demonstrate that soluble Klotho may act as an on-demand coreceptor for fibroblast growth factor 23 (FGF-23) or it may act independently of FGF23 and interact with monosialogangliosides in lipid rafts of the plasma membrane, altering their lipid organization and modulating biological pathways that regulate oxidative stress, apoptosis, stem cell renewal, fibrogenesis and angiogenesis. Soluble Klotho reduces oxidative stress by promoting forkhead transcription factor activation and nuclear translocation, leading to upregulation of manganese superoxide dismutase (MnSoD). It also suppresses fibrosis by binding to type II transforming growth factor-beta 1 (TGF-P) receptor, preventing TGF-0 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).

SUMMARY OF THE INVENTION

[0007] This disclosure provides A2M proteins and sKL proteins and related methods of making same, polynucleotides (e.g., codon-optimized polynucleotides), vectors, and cells (e.g., CHO cells, CHOK1 cells, CHOK1 GenS cells). This disclosure also provides compositions (e.g., pharmaceutical compositions) comprising both an A2M protein and an sKL protein and a pharmaceutically acceptable carrier.

[0008] Provided herein is a polynucleotide (e.g., a codon-optimized polynucleotide) 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 instances, the polynucleotide is codon-optimized. In some instances, the A2M protein comprises the sequence set forth in any one of SEQ ID NOs:2, 4, and 6. In some instances, the promoter is a heterologous promoter. In some instances, the heterologous promoter is a cytomegalovirus (CMV) promoter.

[0009] Also provided herein is a vector comprising a polynucleotide (e.g., a codon-optimized polynucleotide) encoding an A2M protein described herein. In some instances, the vector comprises a glutamine synthetase gene.

[0010] Also provided herein is a cell (e.g., CHO cell, CHOK1 cell, CHOK1 GenS cell) comprising a polynucleotide (e.g., a codon- optimized polynucleotide) described herein encoding an A2M protein. In some instances, the cell is a mammalian cell. In some instances, the cell is a Chinese hamster ovary (CHO) cell. In some instances, the cell is a CHO KI cell. In some instances, the cell is a CHO KI GenS cell. In some instances, the cell stably expresses the A2M protein.

[0011] Also provided herein is a cell comprising a vector described herein comprising a polynucleotide (e.g., a codon-optimized polynucleotide) encoding an A2M protein. In some instances, the cell is a mammalian cell. In some instances, the cell is a CHO cell. In some instances, the cell is a CHO KI cell. In some instances, the cell is a CHO KI GenS cell. In some instances, the cell stably expresses the A2M.

[0012] Also provided herein is a method of producing an A2M protein, the method comprising (a) culturing a cell (e.g., CHO cell, CHOK1 cell, CHOK1 GenS cell) 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 instances, the method further comprises formulating the isolated A2M protein as a sterile pharmaceutical composition comprising the isolated A2M and a pharmaceutically acceptable carrier. In some instances, the isolated A2M is in dimeric form. In some instances, 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.

[0013] Also provided herein is a polynucleotide (e.g., a codon-optimized polynucleotide) comprising a nucleic acid sequence encoding a soluble klotho (sKL) protein, wherein the nucleic acid sequence encoding the sKL protein is operably linked to a promoter (e.g., a heterologous promoter). In some instances, the polynucleotide is codon- optimized. In some instances, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some instances, the promoter is a heterologous promoter. In some instances, the heterologous promoter is a CMV promoter. [0014] 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 instances, the vector comprises a glutamine synthetase gene. In some instances, the vector is a PiggyBac vector. In some instances, 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.

[0015] Also provided herein is a cell comprising a polynucleotide (e.g., a codon-optimized polynucleotide) described herein encoding an sKL protein. In some instances, the cell is a mammalian cell. In some instances, the cell is a CHO cell. In some instances, the cell is a CHO KI cell. In some instances, the cell is a CHO KI GenS cell. In some instances, the cell stably expresses the sKL protein.

[0016] Also provided herein is a cell comprising a vector (e.g., a PiggyBac vector) described herein comprising a polynucleotide encoding an sKL protein. In some instances, the cell is a mammalian cell. In some instances, the cell is a CHO cell. In some instances, the cell is a CHO KI cell. In some instances, the cell is a CHO KI GenS cell. In some instances, the cell stably expresses the sKL protein.

[0017] 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 instances, the method further comprises formulating the isolated sKL protein as a sterile pharmaceutical composition comprising the isolated sKL and a pharmaceutically acceptable carrier. In some instances, 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. [0018] Also provided herein is a method of 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 (e.g., a codon-optimized nucleic acid sequence) encoding an sKL protein described herein, and a 3 nucleic acid sequence PiggyBac transposon-specific ITR, and wherein the second vector comprises a nucleic acid sequence encoding a PiggyBac transposase.

BREIF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1A depicts an exemplary amino acid sequence of human A2M (GenBank Accession No. NP_000005.2) (SEQ ID NO: 1). Lowercase, bold residues are the signal peptide.

[0020] FIG. IB depicts an exemplary amino acid sequence of mature human A2M (amino acids 24-1,474 of SEQ ID NO: 1) (SEQ ID NO:2).

[0021] FIG. 1C depicts an exemplary amino acid sequence of human A2M (GenBank Accession No. NP_000005.3) (SEQ ID NO:3). Lowercase residues are the signal peptide.

[0022] FIG. ID depicts an exemplary amino acid sequence of mature human A2M (amino acids 24-1,474 of SEQ ID NO:3) (SEQ ID NO:4).

[0023] FIG. IE depicts an exemplary amino acid sequence of human A2M (GenBank Accession No. AAA51551.1) (SEQ ID NO:5). Lowercase, bold residues are the signal peptide.

[0024] FIG. IF depicts an exemplary amino acid sequence of mature human A2M (amino acids 24-1,474 of SEQ ID NO: 5) (SEQ ID NO:6).

[0025] FIG. 2A-FIG. 2B depict an exemplary mRNA sequence encoding human A2M (GenBank Accession No. NM_000014.6) (SEQ ID NO:7).

[0026] FIG. 3A-FIG. 3G depict an alignment of exemplary mouse (SEQ ID NO: 8), rat (SEQ ID NO:9), cow (SEQ ID NOTO), macaque (SEQ ID NO: 11), human (SEQ ID NO: 1), and orangutan (SEQ ID NO: 12) A2M proteins.

[0027] FIG. 3H depicts the percent identity matrix for the alignment of FIG. 3A-FIG. 3G.

[0028] FIG. 4A depicts an exemplary amino acid sequence of human klotho (GenBank Accession No. NP_004786.2) (SEQ ID NO: 13). Lowercase, bold residues are the signal peptide; lowercase italicized residues are the transmembrane and cytoplasmic domains.

[0029] FIG. 4B depicts an exemplary amino acid sequence of mature human Klotho (amino acids 34-1,012 of SEQ ID NO: 13) (SEQ ID NO: 14). [0030] FIG. 4C depicts an exemplary amino acid sequence of human soluble Klotho (amino acids 34-981 of SEQ ID NO: 13) (SEQ ID NO: 15).

[0031] FIG. 5A-FIG. 5B depict an exemplary mRNA sequence encoding human Klotho (GenBank Accession No. NM_004795.4) (SEQ ID NO: 16).

[0032] FIG. 6A-FIG. 6E depict an alignment of exemplary mouse (SEQ ID NO: 17), rat (SEQ ID NO: 18), cow (SEQ ID NO: 19), human (SEQ ID NO: 14), and macaque (SEQ ID NO:20) Klotho proteins.

[0033] FIG. 6F depicts the percent identity matrix for the alignment of FIG. 6A-FIG. 6E.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure relates generally to polynucleotides (e.g., codon- optimized polynucleotides), vectors, and cells (e.g., host cells, e.g., CHO cells, CHOK1 cells, CHOK1 GenS cells) for producing an alpha 2 macroglobulin (A2M) protein, methods of making an A2M protien, and compositions (e.g., pharmaceutical compositions) comprising an A2M protein. The present disclosure also relates generally to polynucleotides (e.g., codon-optimized polynucleotides), vectors (e.g., PiggyBac vectors), and cells (e.g., host cells, e.g., CHO cells, CHOK1 cells, CHOK1 GenS cells)for producing a soluble klotho (sKL) protein, methods of making an sKL protein, and compositions (e.g., pharmaceutical compositions) comprising an sKL protein. The present disclosure also relates to methods of making compositions (e.g., pharmaceutical compositions) comprising an A2M protein and an sKL protein and compositions comprising an A2M protein and an sKL protein, and reagents for said methods of making.

A2M

[0035] The present disclosure provides an A2M protein, methods of making the A2M protein, including polynucleotides (e.g., codon-optimized polynucleotides), vectors, and cells (e.g., CHO cells, CHO KI cells, CHO KI GenS cells), and compositions (e.g., pharmaceutical compositions comprising the A2M protein. In some instances, the compositions comprising an A2M protein further comprise an sKL protein. In some instances, the A2M protein (or compositions comprising the A2M protein) are for use in combination with an sKL protein (or a composition comprising an sKL protein). Also provided herein are methods of making an A2M protein, including related polynucleotides, vectors, and cells.

[0036] A2M (also known in the art as C3 and PZP-like alpha-2-macroglobulin domaincontaining protein 5 (CPAMD5)) is a 1,474-amino acid (including the 23-amino acid signal peptide) protein that functions as a broad-spectrum protease-binding protein. Human A2M is a 720 kDa homotetramer comprised of four 180 kDa subunits. The subunits are paired by disulfide bonds to form covalently-linked dimers, which non-covalently associate to form the cage-like quaternary structure of A2M.

[0037] Exemplary amino acid sequences of full-length human A2M are provided in SEQ ID NOs:l, 3, and 5 (FIG. 1A, FIG. 1C, FIG. IE). Exemplary amino acid sequences of full-length mature human A2M are provided in SEQ ID NOs:2, 4, and 6 (FIG. IB, FIG. ID, FIG. IF). An exemplary nucleic acid sequence of an mRNA encoding human A2M is provided in SEQ ID NO:7 (FIG. 2A-FIG. 2B)

[0038] A2M proteins among different species have high homology. See FIG. 3A-FIG. 3G for an alignment of exemplary amino acid sequences for A2M proteins from human, mouse, rat, macaque, orangutan, and bovine. In some instances, the A2M protein is a human A2M protein (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6). The skilled artisan will understand that region(s) or amino acid(s) amenable to variation (e.g., substitution, insertion, or deletion) may be identified by comparing a reference A2M sequence to a homolog or known variant and determining region(s) or amino acid(s) that are not conserved (see, e.g., FIG. 3A-FIG. 3G or compare SEQ ID NOs:2, 4, and 6).

[0039] In certain instances, the A2M protein comprises a modified amino acid sequence of a wild type A2M protein (e.g., a modified amino acid sequence of wild type human A2M protein, e.g., SEQ ID NO:2). In certain instances, the A2M protein described herein binds trypsin.

[0040] In certain instances, 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), e.g., 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 certain instances, these substitution(s) are of amino acids that directly interact with an A2M protein binding partner known in the art or described herein (e.g., FGF1, FGF2, FGF-4, FGF-6, IFN-gamma, IL- 1 beta, IL2, IL-4, IL-6, IL-8, IL- 18, NGF-beta, PDGF, TGF- betal, TGF-beta2, TNF-alpha, VEGF). In certain instances, these substitution(s) are of amino acids that do not directly interact with an A2M protein binding partner known in the art or described herein (e.g., FGF1, FGF2, FGF-4, FGF-6, IFN-gamma, IL-lbeta, IL2, IL-4, IL-6, IL-8, IL-18, NGF-beta, PDGF, TGF-betal, TGF-beta2, TNF-alpha, VEGF). In certain instances, these substitutions are of both 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 instances, the A2M protein comprises an aspartic acid (D) at the amino acid corresponding to position 616 of SEQ ID NO:2. In some instances, 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 instances, the A2M protein comprises an aspartic acid (D) at the amino acid corresponding to position 616 of SEQ ID NO:2 and a valine (V) at the amino acid corresponding to position 977 of the amino acid sequence set forth in SEQ ID NO:2. In certain instances, the A2M protein described herein binds trypsin.

[0041] In some instances, the substitutions are conservative amino acid substitutions. In some instances, the substitutions are non-conservative amino acid substitutions. In some instances, the substitutions are with conservative amino acid substitutions and non-conservative amino acid substitutions. A “conservative amino acid substitution” means that the substitution replaces one amino acid with another amino acid residue having a similar side chain. Families of amino acid residues having 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and acidic side chains and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). [0042] In certain instances, the A2M protein described herein has 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 certain instances, the A2M protein described herein binds trypsin. The variability in the amino acid sequence may be in region(s) that directly interact with an A2M protein binding partner known in the art or described herein and/or in region(s) that do not directly interact with an A2M protein binding partner known in the art or described herein. Methods for determining percent identity between sequences are known in the art. For example, the sequences are aligned for optimal comparison purposes (e.g., 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). In a preferred instance, the length of a reference sequence aligned for comparison purposes is at least 30%, 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% 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. The determination of percent identity between two amino acid sequences is accomplished using the BLAST 2.0 program. Sequence comparison is performed using an ungapped alignment and using the default parameters (Blossom 62 matrix, gap existence cost of 11, per residue gapped cost of 1, and a lambda ratio of 0.85). The mathematical algorithm used in BLAST programs is described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).

[0043] In certain instances, 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- and/or C-terminus of the A2M protein. In certain instances, 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 ammo acids in length. In certain instances, the A2M protein is 1,451 amino acids in length. In certain instances, the A2M protein described herein binds trypsin.

[0044] In certain instances, the A2M protein described herein comprises the amino acid sequence of SEQ ID NO:2. In certain instances, the A2M protein described herein consists of the amino acid sequence of SEQ ID NO: 2. In certain instances, the A2M protein is part of a composition (e.g., a pharmaceutical composition). In certain instances, the A2M protein is part of a composition (e.g., a pharmaceutical composition) further comprising an sKL protein (e.g., an sKL protein described herein). In certain instances, the A2M protein (or a composition comprising the A2M protein) is for use in combination with an sKL protein (or a composition, e.g., a pharmaceutical composition, comprising an sKL protein, e.g., an sKL protein described herein). In some instances, the sKL protein comprises the amino acid sequence of SEQ ID NO: 15. In some instances, the sKL protein consists of the amino acid sequence of SEQ ID NO: 15.

[0045] In certain instances, the A2M protein described herein comprises the amino acid sequence of SEQ ID NO:4. In certain instances, the A2M protein described herein consists of the amino acid sequence of SEQ ID NO: 4. In certain instances, the A2M protein is part of a composition (e.g., a pharmaceutical composition). In certain instances, the A2M protein is part of a composition (e.g., a pharmaceutical composition) further comprising an sKL protein (e.g., an sKL protein described herein). In certain instances, the A2M protein (or a composition comprising the A2M protein) is for use in combination with an sKL protein (or a composition, e.g., a pharmaceutical composition, comprising an sKL protein, e.g., an sKL protein described herein). In some instances, the sKL protein comprises the amino acid sequence of SEQ ID NO: 15. In some instances, the sKL protein consists of the amino acid sequence of SEQ ID NO: 15.

[0046] In certain instances, the A2M protein described herein comprises the amino acid sequence of SEQ ID NO: 6. In certain instances, the A2M protein described herein consists of the amino acid sequence of SEQ ID NO: 6. In certain instances, the A2M protein is part of a composition (e.g., a pharmaceutical composition). In certain instances, the A2M protein is part of a composition (e.g., a pharmaceutical composition) further comprising an sKL protein (e.g., an sKL protein described herein). In certain instances, the A2M protein (or a composition comprising the A2M protein) is for use in combination with an sKL protein (or a composition, e.g., a pharmaceutical composition, comprising an sKL protein, e.g., an sKL protein described herein). In some instances, the sKL protein comprises the amino acid sequence of SEQ ID NO: 15. In some instances, the sKL protein consists of the amino acid sequence of SEQ ID NO: 15.

[0047] In some instances, the A2M protein described herein is in monomeric form, dimeric form, or tetrameric form. In some instances, the A2M protein described herein is in tetrameric form. In some instances, the A2M protein described herein is in dimeric form. Methods of determining A2M protein is in monomeric, dimeric, or tetrameric form are known in the art, such as, e.g., polyacrylamide gel electrophoresis. Methods of obtaining A2M protein in monomeric, dimeric, or tetrameric form are known in the art, such as, e.g., exposure of tetrameric A2M to an oxidant (e.g., hypochlorite) to produce dimeric A2M.

[0048] 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 lease 90%, or 100% compared to cathepsin L activity in the absence of the A2M protein). Methods of determining whether A2M protein inhibits cathepsin L activity are known in the art (see, e.g., the Examples described herein).

[0049] In certain instances, the A2M protein inhibits binding between ACE2 and SARS-CoV-2 S protein (e.g., reduces binding between ACE2 and SARS-CoV-2 S protein 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 lease 90%, or 100% compared to cathepsin L activity in the absence of the A2M protein).

Methods of determining whether A2M protein inhibits binding between ACE2 and SARS-CoV-2 S protein (see, e.g., the Examples described herein).

[0050] In certain instances, the A2M protein described herein binds trypsin. Methods of determining whether an A2M protein binds trypsin are known in the art, e.g., co-localization assays, co-immunoprecipitation assays, ELISA, Biacore, SPR, the trypsin binding assay described in Wyatt et al., 2015, PLOS ONE, D01:10.1371/journal.pone.0130036, which is incorporated by reference herein in its entirety, and Trypsin Activity Colorimetric Assay Kit (AssayGenie, Catalog #BN00984).

[0051] In certain instances, the A2M protein described herein binds FGF23. Methods of determining whether an A2M protein binds FGF23 are known in the art, e.g., co-localization assays, co-immunoprecipitation assays, ELISA, Biacore, and SPR.

[0052] In certain instances, the A2M protein described herein binds FGF2. Methods of determining whether an A2M protein binds FGF2 are known in the art, e.g., co-localization assays, co-immunoprecipitation assays, ELISA, Biacore, and SPR. sKL

[0053] The present disclosure provides an sKL protein, methods of making the sKL protein, including polynucleotides (e.g., codon-optimized polynucleotides), vectors (e.g., PiggyBac vectors), and cells (e.g., CHO cells, CHO KI cells, CHO KI GenS cells), and compositions (e.g., pharmaceutical compositions) comprising the sKL protein. In some instances, the compositions comprising an sKL protein further comprise an A2M protein (e.g., as described herein). In some instances, the sKL protein (or compositions comprising the sKL protein) are for use in combination with an A2M protein (or a composition comprising an A2M protein). Also provided herein are methods of making an sKL protein, including related polynucleotides, vectors, and cells.

[0054] KL is a 1,012-amino acid protein (including the 33 amino acid signal peptide) that is a member of the glycosidase- 1 superfamily and hydrolyzes steroid P-glucuronides. KL may act as a co-receptor for FGF23. KL exists in two main forms: full-length, membrane-bound KL (e.g., SEQ ID NO: 14) and sKL (e.g., SEQ ID NO: 15). sKL is formed from proteolytic cleavage of membrane-bound KL. sKL does not include the cytoplasmic domain (e.g., amino acids 1,003- 1,012 of SEQ ID NO: 13) nor the transmembrane domain (e.g., amino acids 982-1,002 of SEQ ID NO: 13).

[0055] 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 of an mRNA encoding human KL is provided in SEQ ID NO: 16 (FIG. 5A-FIG. 5B).

[0056] KL proteins among different species have high homology. See FIG. 6A-FIG. 6F for an alignment of exemplary amino acid sequences for KL proteins from mouse, rat, cow, human, and macaque. In some instances, the sKL protein is a human sKL protein (e.g., SEQ ID NO: 15). The skilled artisan will understand that region(s) or amino acid(s) amenable to variation (e.g., substitution, insertion, or deletion) may be identified by comparing a reference KL sequence to a homolog and determining region(s) or amino acid(s) that are not conserved (see, e.g., FIG. 6A- FIG. 6F)

[0057] In certain instances, the sKL protein comprises a modified amino acid sequence of a wild type sKL protein (e.g., a modified amino acid sequence of wild type human sKL protein, e.g., the amino acid sequence set forth in SEQ ID NO: 15).

[0058] In certain instances, the sKL protein comprises a valine (V) at the amino acid corresponding to position 12 of SEQ ID NO: 15. [0059] In certain instances, the sKL protein comprises a phenylalanine (F) at the amino acid corresponding to position 12 of SEQ ID NO: 15.

[0060] In some instances, the sKL protein comprises the amino acids corresponding to positions 34-503 of SEQ ID NO: 13. In some instances, the sKL protein comprises the amino acids corresponding to positions 515-956 of SEQ ID NO:13. In some instances, the sKL protein comprises the amino acids corresponding to positions 34-503 of SEQ ID NO: 13 and the amino acids corresponding to positions 515-956 of SEQ ID NO: 13. In some instances, the sKL protein comprises the amino acids corresponding to positions 504-515 of SEQ ID NO: 13. In some instances, the sKL protein comprises the amino acids corresponding to positions 967-981 of SEQ ID NO: 13. In some instances, the sKL protein comprises the amino acids corresponding to positions 957-981 of SEQ ID NO: 13. In some instances, the sKL protein does not comprise the amino acids corresponding to positions 967-981 of SEQ ID NO: 13. In some instances, the sKL comprises the amino acids corresponding to positions 34-981 of SEQ ID NO: 13. In some instances, the sKL comprises the amino acids corresponding to positions 34-966 of SEQ ID NO: 13. In some instances, the C-terminus of the sKL protein corresponds to position 966 of SEQ ID NO: 13. In some instances, the C-terminus of the sKL protein corresponds to position 981 of SEQ ID NO: 13.

[0061] In certain instances, the sKL 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 a wild type sKL protein sequence (e.g., SEQ ID NO: 15)), e.g., 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 certain instances, these substitution(s) are of amino acids that directly interact with an sKL protein binding partner known in the art or described herein (e.g., FGF23). In certain instances, these substitution(s) are of amino acids that do not directly interact with an sKL protein binding partner known in the art or described herein (e.g., FGF23). In certain instances, these substitutions are of both amino acids that directly interact with an sKL protein binding partner known in the art or described herein and amino acids that do not directly interact with an sKL protein binding partner known in the art or described herein.

[0062] In some instances, the substitutions are conservative amino acid substitutions. In some instances, the substitutions are non-conservative amino acid substitutions. In some instances, the substitutions are with conservative amino acid substitutions and non-conservative amino acid substitutions.

[0063] In certain instances, the sKL protein described herein has 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 may be in region(s) that directly interact with an sKL protein binding partner known in the art or described herein and/or in region(s) that do not directly interact with an sKL protein binding partner known in the art or described herein.

[0064] In certain instances, the sKL 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- and/or C- terminus of the sKL protein. In certain instances, the sKL protein is 750-1200, 750-1100, 750- 1000, 850-1200, 840-1100, 850-1000, or 900-1000 amino acids in length. In certain instances, the sKL protein is 948 amino acids in length. In some instances, the sKL is 948 amino acids in length, 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.

[0065] In certain instances, the sKL protein described herein comprises the amino acid sequence of SEQ ID NO: 15. In certain instances, the sKL protein described herein consists of the amino acid sequence of SEQ ID NO: 15. In certain instances, the sKL protein is part of a composition (e.g., a pharmaceutical composition). In certain instances, the sKL protein is part of a composition (e.g., a pharmaceutical composition) further comprising an A2M protein (e.g., an A2M protein described herein). In certain instances, the sKL protein (or a composition comprising the sKL protein) is for use in combination with an A2M protein (or a composition, e.g., a pharmaceutical composition, comprising an A2M protein, e.g., an A2M protein described herein). In some instances, the A2M protein comprises the amino acid sequence of SEQ ID NO:2. In some instances, the A2M protein comprises the amino acid sequence of SEQ ID NO:4. In some instances, the A2M protein comprises the amino acid sequence of SEQ ID NO: 6. In some instances, the A2M protein consists of the amino acid sequence of SEQ ID NO:2. In some instances, the A2M protein consists of the amino acid sequence of SEQ ID NO:4. In some instances, the A2M protein consists of the amino acid sequence of SEQ ID NO: 6. [0066] In certain instances, the sKL protein described herein binds FGF23. Methods of determining whether an sKL protein binds FGF23 are known in the art, e.g., co-localization assays, co-immunoprecipitation assays, ELISA, Biacore, and SPR.

[0067] In certain instances, the sKL protein described herein induces ERK1/2 phosphorylation (e.g., in a cell assay). Methods of determining whether an sKL protein induces ERK1/2 phosphorylation (e.g., in a cell assay) are known in the art, e.g., western blot, ELISA, immunofluorescence. See, e.g., Zhong et al., 2020, JBC, 295(10:3115-3133, which is incorporated by reference herein in its entirety, for a description of an exemplary ERK1/2 phosphorylation assay.

[0068] In certain instances, the sKL protein described herein induces ERK1/2 phosphorylation (e.g., in a cell assay). Methods of determining whether an sKL protein induces ERK1/2 phosphorylation (e.g., in a cell assay) are known in the art, e.g., western blot, ELISA, immunofluorescence. See, e.g., Zhong et al., 2020, JBC, 295(10:3115-3133, which is incorporated by reference herein in its entirety, for a description of an exemplary ERK1/2 phosphorylation assay. See, also, the Phospho(Thr202/Tyr204; Thrl84/Tyrl87)/Total ERK1/2 Assay (Mesoscale, Catalog No. K15107D-1).

POLYNUCLEOTIDES, VECTORS, AND CELLS

Polynucleotides

[0069] Provided herein are polynucleotides (e.g., codon-optimized polynucleotides) encoding A2M and polynucleotides encoding sKL. In some instances, the polynucleotide (e.g., codon- optimized polynucleotide) encodes an A2M protein described herein. In some instances, the polynucleotide (e.g., codon- optimized polynucleotide) encodes an sKL protein described herein. In some instances, the polynucleotide (e.g., codon-optimized polynucleotide) encodes an A2M protein and an sKL protein described herein.

[0070] In some instances, the polynucleotides are codon-optimized. Methods for producing codon-optimized polynucleotides are known in the art. Codon optimization may be used to improve expression of an A2M protein or an sKL protein described herein and to increase the translational efficiency of a polynucleotide encoding an A2M protein or an sKL protein, e.g., as described herein, by accommodating codon bias of the host organism or of a desired cell type (e.g., a CHO cell). 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 instances, the polynucleotide is codon-optimized for expression and translation in a CHO cell (e.g., a CHO KI cell, a CHO KI GenS cell).

[0071] In some instances, the polynucleotide is a codon-optimized polynucleotide comprising a nucleic acid sequence encoding an A2M protein. In some instances, the A2M protein comprises the sequence set forth in any one of SEQ ID NOs:2, 4, and 6. In some instances, the nucleic acid sequence encoding the A2M protein is operably linked to a heterologous promoter. A heterologous promoter relative to a nucleic acid sequence encoding an A2M protein comprises a promoter not found associated with a nucleic acid sequence encoding A2M in nature. In some instances, the heterologous promoter is a CMV promoter. In some instances, the sequence encoding the A2M protein comprises an SV40 poly(A) signal.

[0072] In some instances, the polynucleotide is a codon-optimized polynucleotide comprising a nucleic acid sequence encoding an sKL protein. In some instances, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some instances, the nucleic acid sequence encoding the sKL protein is operably linked to a heterologous promoter. A heterologous promoter relative to a nucleic acid sequence encoding an sKL protein comprises a promoter not found associated with a nucleic acid sequence encoding sKL in nature. In some instances, the heterologous promoter is a CMV promoter. In some instances, the sequence encoding the sKL protein comprises an SV40 poly(A) signal.

[0073] Promoters useful in the polynucleotides described herein are known in the art. The choice of a promoter generally depends upon the choice of host. Useful promoters for eukaryotic hosts include, for example, promoters from CMV, SV40, bovine papilloma virus, adenovirus. In some instances, the promoter is a CMV promoter.

Vectors

[0074] Also provided herein are vectors (e.g., expression vectors, PiggyBac vectors) comprising a polynucleotide (e.g., codon-optimized polynucleotide) encoding an A2M protein described herein and/or a polynucleotide (e.g., codon-optimized polynucleotide) encoding an sKL protein described herein. In some instances, the vector comprises a polynucleotide encoding an A2M protein described herein. In some instances, the vector comprises a polynucleotide encoding an sKL protein described herein. In some instances, 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, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes. In some instances, the vector is a PiggyBac vector.

[0075] A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences (e.g., promoter(s)) from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as Ml 3 and other filamentous single-stranded DNA phages. [0076] In some instances, the vector is a vector described in US Patent No. 10,889,822, which is incorporated by reference herein in its entirety.

[0077] In some instances, the vector is a PiggyBac vector. The PiggyBac transposon is a mobile genetic element that efficiently transposes between vectors and host cell chromosomes via a “cut and paste” transposition mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both the 5' and 3' ends of the transposon- based vector and moves the vector contents between the 5' and 3' ITRs from the original vector sites and integrates them into TTAA chromosomal sites. 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 by reference herein in its entirety. In some instances, the vector comprises, in 5' to 3' order, a 5' PiggyBac transposonspecific ITR sequence, a nucleic acid sequence (e.g., a nucleic acid sequence) encoding a protein described herein (e.g., an sKL protein), and a 3' PiggyBac transposon-specific ITR.

Cells

[0078] Also provided herein are cells expressing an A2M protein and/or an sKL protein described herein. [0079] In some instances, the cell expresses an A2M protein described herein. In some instances, the cell comprises a polynucleotide comprising a sequence encoding an A2M protein operably linked to a promoter. In some instances, the cell comprises a vector comprising a sequence encoding an A2M protein operably linked to a promoter. In some instances, the cell stably integrates a polynucleotide comprising a sequence encoding an A2M protein operably linked to a promoter. In some instances, the A2M protein comprises the sequence set forth in any one of SEQ ID NOs:2, 4, and 6. In some instances, the cell is a CHO cell. In some instances, the cell is a CHOKl-GenS cell.

[0080] In some instances, the cell expresses an sKL protein described herein. In some instances, the cell comprises a polynucleotide comprising a sequence encoding an sKL protein operably linked to a promoter. In some instances, the cell comprises a vector comprising a sequence encoding an sKL protein operably linked to a promoter. In some instances, the cell stably integrates a polynucleotide comprising a sequence encoding an sKL protein operably linked to a promoter. In some instances, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some instances, the cell is a CHO cell. In some instances, the cell is a CHOKl-GenS cell.

[0081] In some instances, the cell expresses an A2M protein and an sKL protein described herein. In some instances, the cell comprises 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 instances, the cell comprises a first vector comprising a sequence encoding an A2M protein operably linked to a promoter and a second vector comprising a sequence encoding an sKL protein operably linked to a promoter. In some instances, the cell comprises a vector comprising a first sequence encoding an A2M protein operably linked to a promoter and a second sequence encoding an sKL protein operably linked to a promoter. In some instances, 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 instances, the A2M protein comprises the sequence set forth in any one of SEQ ID NOs:2, 4, and 6. In some instances, the sKL protein comprises the sequence set forth in SEQ ID NO: 15. In some instances, the cell is a CHO cell. In some instances, the cell is a CHOKl-GenS cell. [0082] Various cells for expressing or encoding an A2M protein and/or an sKL protein are known in the art. For example, the cell may be an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that do not otherwise produce human A2M (e.g., an A2M described herein) or human sKL (e.g., an sKL described herein).

[0083] Various mammalian culture systems may be used to express an A2M protein and/or an sKL protein described herein. Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly 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 mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary- derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. In some isntances, the cell is a CHO cell (e.g., a CHOK1 cell or a CHOK1 GenS cell). Mammalian expression vectors can 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 nontranscribed sequences, and 5' or 3' non- translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

METHODS OF MAKING A2M AND SKL

[0084] Also provided herein are methods of making an A2M protein described herein. [0085] Also provided herein are methods of making an sKL protein described herein. [0086] Polynucleotides (e.g., codon-optimized), vectors (e.g., expression vectors, PiggyBac vectors), and cells (e.g., CHO cells, CHO KI cells, CHO KI GenS cells) useful in the methods of making an A2M protein and/or an sKL protein described herein are also provided herein. Thus, provided herein are polynucleotides encoding an A2M protein and/or an sKL protein described herein, vectors comprising said polynucleotides, and cells comprising said polynucleotides or vectors. An exemplary nucleic acid encoding wild type human A2M protein is provided in SEQ ID NO:7 (GenBank Accession No. NM_000014.6; FIG. 2A-FIG. 2B). An exemplary nucleic acid encoding wild type human KL protein is provided in SEQ ID NO: 16 (GenBank Accession No. NM_004795.4; FIG. 5A-FIG. 5B). In some instances, the polynucleotide is codon-optimized for use in mammalian (e.g., human, Chinese hamster ovary) cells. Methods of generating polynucleotides and codon-optimization are well-known in the art. [0087] Suitable host cells for expression of an A2M protein or an sKL protein described herein are known in the art and include, e.g., prokaryotes (e.g., bacteria, e.g., E. coli yeast cells, insect cells, and higher eukaryotic (e.g., mammalian) cells. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production are well-known in the art. In some instances, the cells are CHO cells. In some instances, the cells are CHO KI cells. In some instances, the cells are CHO KI cells.

[0088] In some instances, a method of making an A2M protein described herein comprises: (a) culturing a cell comprising a polynucleotide (e.g., codon-optimized) or vector encoding the A2M protein (e.g., under conditions that permit expression of the A2M protein), and (b) isolating the A2M protein. In some instances, the method further comprises formulating the A2M protein as a pharmaceutical composition described herein (e.g., a sterile pharmaceutical composition) comprising the A2M protein and a pharmaceutically acceptable carrier. In some instances, the concentration of 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 instances, the method further comprises formulating the A2M protein as a pharmaceutical composition described herein further comprising an sKL protein.

[0089] In some instances, a method of making an sKL protein described herein comprises: (a) culturing a cell comprising a polynucleotide (e.g., codon-optimized) or vector encoding the sKL protein (e.g., under conditions that permit expression of the sKL protein), and (b) isolating the sKL protein. In some instances, the method further comprises formulating the sKL protein as a pharmaceutical composition described herein (e.g., a sterile pharmaceutical composition) comprising the sKL protein and a pharmaceutically acceptable carrier. In some instances, 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 instances, the method further comprises formulating the sKL protein as a pharmaceutical composition described herein further comprising an A2M protein.

[0090] In some instances, a PiggyBac transposon-based vector system is used to make an 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 by reference herein in its entirety.

[0091] In some instances, the method of making an sKL protein comprises: (a) transfecting a cell (e.g., a CHO cell, e.g., a CHO KI or CHO KI GenS cell) 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 transposase, (b) culturing the cell, and (c) isolating the sKL protein, wherein the first vector comprises, in 5' to 3' orientation, a 5' ITR, the nucleic acid sequence encoding an sKL protein, and a 3' ITR. In some instances, the method further comprises formulating the sKL protein as a pharmaceutical composition described herein (e.g., a sterile pharmaceutical composition) comprising the sKL protein and a pharmaceutically acceptable carrier. In some instances, 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 instances, the method further comprises formulating the sKL protein as a pharmaceutical composition described herein further comprising an A2M protein.

EXAMPLES

Example 1: A2M Codon Optimization

[0092] This example summarizes the development of CHOKl-GenS cell line expressing A2M. [0093] The A2M open reading frame (ORF) was codon optimized for expression in CHO cells. [0094] The codon optimized A2M genes were synthesized and cloned into the mammalian expression vector pGenHTl 0-DGV

[0095] The pGenHTl .0-DGV contains a MCS for the protein and the promoters of MCS is Cytomegalovirus (CMV). The vector contains a 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 pGenHTl.0-DGV vector (restriction enzyme site: Ascl-Fsel). Then, this plasmid was amplified and linearized by Pvul restriction enzyme for transfection with a concentration of Ipg/pl.

[0096] Plasmids were transfected into CHOKl-GenS cells. CHOKl-GenS cells were cultured in growth medium (CD CHO + 6mM L-Glutamine + Anti-clumping agent (400*)) at 37.0°C, 5.0% CO2 and 120 rpm shaking speed. [0097] The plasmids were prepared for transfection and transfected into CHOKl-GenS cells followed by cell pools screening under the MSX pressure. Based on the performance, top 2 cell pools of each transfection were mixed into a new cell pool, and the mix cell pools were selected for single clone screening in 96-well plates using the limiting dilution method. Monoclonality was confirmed based on images captured by Cell MetricTM CLD (data not shown). The single cell clones with good confluency and higher titer were selected for fed-batch culture evaluation.

Example 2: Generation of A2M Vector

[0098] Codon optimized A2M genes were synthesized and cloned into the mammalian expression vector pGenHTl.O-DGV. The pGenHTl.O-DGV vector contains a multi-cloning site (MCS) for the protein. The promoter of MCS is Cytomegalovirus (CMV). Meanwhile, the vector contains a 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 plasmid was amplified for transfection with a concentration of Ipg/pl.

Example 3: Cells Expressing A2M

[0099] To engineer cells stably expressing A2M, CHOKl-GenS cells (European Collection of Authenticated Cell Cultures) were thawed from liquid nitrogen into growth medium (CD CHO medium (Gibco; Cat No. 10743-029), 6mM L-Glutamine + Anti-clumping agent (400*) (Gibco; Cat. No. 01-0057DG)), then the cells were maintained in a 37.0°C incubator with 5.0% CO2. The cells were resuspended at the density of 0.50 10 ⁶ cells/ml into 18 ml growth medium in a 125 ml shaker flask, then the cells were maintained in the incubator with 37.0°C, 5.0% CO2 and 120 rpm shaking speed. CHOKl-GenS cells were cultivated every 24-72 hours.

[00100] Vectors encoding sKL (see Example 2: Generation of A2M Vector) were transfected into CHOKl-GenS. The transfected cells were seeded into 24- well plates with the selection medium (CD CHO medium + 25 pM L-Methionine Sulfoximine&lG (MSX; Sigma, Cat. No. M5379-1G) + Anti-clumping agent (400 )) for cell pool screening. Cells were subcultured with selection medium every 4-6 days. After screening 26 days, the conditioned media from a 6-day culture of pools were collected for dot blot analysis (data not shown). Cell 1 pools were compared and the top 2 cell pools in titer were mixed into new cell pools, which were selected for fed-batch culture evaluation. Culture supernatants were analyzed by ELISA and SDS PAGE. Based on the performance of cell pool culture fed-batch, the top pools were selected for single clone screening.

[00101] Finally, based on the productivity and growth performance, top 6 clones were selected for primary cell bank (PCB) construction. The monoclonality and titer (mg/L) of the top 6 clones is provided in Table 1.

[00102] Table 1 CHOKl-GenS A2M clones. Data in the monoclonality row represent the number of cells observed through cell-imaging system. Image were captured 2 times on day

1. The data demonstrate single cell seeding on day 0 and cellular division status on day 1 and day

2.

[00103] Clones 1-6 were thawed and tested for viability confirmation (Table 2) and mycoplasma test. Each of the tested clones was mycoplasma- free.

[00104] Table 2. Recovery Data

Example 4: sKL Codon Optimization

[00105] Codon optimized polynucleotides encoding sKL (SEQ ID NO: 15) were prepared for expression in CHO cells. Example 5: Generation of sKL Vector

[00106] Codon optimized sKL genes were synthesized and cloned into the mammalian expression vector pGenHTl.O-DGV. The pGenHTl.O-DGV vector contains a multi-cloning site (MCS) for the protein. The promoter of MCS is Cytomegalovirus (CMV). Meanwhile, the vector contains a 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 plasmid was amplified for transfection with a concentration of Ipg/pl.

Example 6: Cells expressing sKL

[00107] To engineer cells stably expressing sKL, CHOKl-GenS cells (European Collection of Authenticated Cell Cultures) were thawed from liquid nitrogen into growth medium (CD CHO medium (Gibco; Cat No. 10743-029), 6mM L-Glutamine + Anti-clumping agent (400 ) (Gibco; Cat. No. 01-0057DG)), then the cells were maintained in a 37.0°C incubator with 5.0% CO2. The cells were resuspended at the density of 0.40 10 ⁶ cells/ml into 18.5 ml growth medium in a 125 ml shaker flask, then the cells were maintained in the incubator with 37.0°C, 5.0% CO2 and 120 rpm shaking speed. CHOKl-GenS cells were cultivated every 24-72 hours.

[00108] Vectors encoding sKL (see Example 5: Generation of sKL Vector) were transfected into CHOKl-GenS cells and the supernatants at 48 hours post-transfection were harvested for Western blot test. No significant signal were detected in the samples of 48 hours post-transfection by western blot test (data not shown).

[00109] The transfected cells were seeded into 24-well plates with the selection medium (CD CHO medium + 25 pM L-Methionine Sulfoximine&lG (MSX; Sigma, Cat. No. M5379- 1G) + Anti-clumping agent (400 )) for cell pool screening. Cells were subcultured with selection medium every 4-6 days. After screening 20 days, 12 cell pools were selected for fed- batch culture evaluation according to the recovery state of the cell pool. Based on the performance of cell pool culture fed-batch, the top 3 cell pools were selected for single clone screening in 96-well plates using the limiting dilution method. Cell MetricTM CLD Imager was used to record and confirm the monoclonality of single cell clones (data not shown). 36 single clones with higher titer and good confluency were selected for fed-batch culture evaluation.

Finally, based on the productivity and growth performance, top 6 clones were selected for primary cell bank (PCB) construction. The monoclonality and titer (mg/L) of the top 6 clones is provided in Table 3.

[00110] Table 3 CHOKl-GenS sKL clones. Data in the monoclonality row represent the number of cells observed through cell-imaging system. Image were captured 2 times on day 1. The data demonstrate single cell seeding on day 0 and cellular division status on day 1 and day 2.

[00111] Clones 1-6 were thawed and tested for viability confirmation (Table 4) and mycoplasma test. Each of the tested clones was mycoplasma- free.

[00112] Table 4. Recovery Data

Example 7: PiggyBac Vector Encoding sKL

[00113] To engineer cells stably expressing an sKL protein, CHOKl-GenS cells (European Collection of Authenticated Cell Cultures) were transfected with a PiggyBac vector encoding an sKL protein. An exemplary resultant clone yielded a titer of 1228.18 mg/L of sKL protein.

OTHER INSTANCES [00114] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.