Parathyroid hormone variants

Abstract

The present invention relates to parathyroid hormone (PTH) variants and pharmaceutical compositions comprising the same. The present invention further relates to PTH compositions with improved serum half-lives and peak / trough ratios, and methods for controlling serum calcium levels using the PTH variants and compositions of the present invention. The present invention further relates to methods for treating hypoparathyroidism and / or hypocalcemia resulting from hypoparathyroidism using the PTH variants and compositions of the present invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 798,113, filed January 29, 2019, U.S. Provisional Application No. 62 / 800,744, filed February 4, 2019, and U.S. Provisional Application No. 62 / 884,730, filed August 9, 2019, the disclosures of which are incorporated herein in their entireties. [Technical field]

[0002] The present invention relates to parathyroid hormone variants and compositions comprising the same. The present invention further relates to parathyroid hormone variants and compositions with improved serum half-lives, and methods of controlling serum calcium levels using the parathyroid hormone compositions of the present invention. [Background technology]

[0003] Parathyroid hormone (PTH) is an 84-amino acid product secreted by the mammalian parathyroid gland that controls serum calcium levels through its actions on various tissues, including bone. Human studies with certain forms of PTH have demonstrated anabolic effects on bone, generating great interest in its use for the treatment of osteoporosis and related bone disorders, as well as hypoparathyroidism.

[0004] The first 34 N-terminal amino acids are known to have the same activation as full-length PTH (1-84) at the only known receptor for PTH, "PTH1R." See, for example, Non-Patent Documents 1-3. Furthermore, it has been shown that C-terminal fragments such as 39-84 and 53-84 do not compete with the 1-34 fragment for receptor binding. Furthermore, these C-terminal fragments do not activate adenylate cyclase, all of which led to the conclusion that the C-terminal portion of the PTH peptide is irrelevant. See References 4-6.

[0005] However, even though the C-terminus of PTH is thought not to be related to biological activity, it has been found that the C-terminal portion of the peptide is necessary for normal transport and processing. See, for example, Non-Patent Documents 7-12. In particular, there is evidence that full-length PTH is cleaved into C-terminal fragments in the parathyroid gland, and that these fragments are secreted in response to an increase in calcium ion concentration in the blood. There is no evidence to date that N-terminal fragments are stored in or secreted from the gland, except in the form of intact PTH. See, Non-Patent Documents 13-33.

[0006] Furthermore, it is now apparent that the C-terminal region of PTH has a novel receptor specific for this region of the hormone. See, e.g., J. Neurosci. 1999, 143: 1311-1321 (2002). Thus, full-length PTH has biological properties that differ from N-terminal PTH analogs.

[0007] However, the importance and effects of full-length parathyroid hormone on bone growth, calcium physiology, and replenishment are perhaps not yet readily understood, as are the effects on calcium, for example. The normal daily rise and fall of PTH levels in the blood has a profound effect on bone, and injections of PTH can stimulate new bone growth in cases where bone has been lost to osteoporosis.

[0008] Hypoparathyroidism is a lifelong disease characterized by insufficient production of parathyroid hormone (PTH) by the parathyroid glands. Because PTH is important in regulating calcium and phosphate levels, loss of PTH leads to low calcium levels and high phosphate levels in the blood and bones (hypocalcemia and hyperphosphatemia). Hypocalcemia leads to symptoms such as paresthesia, muscle spasms, laryngospasm (which can result in the inability to speak and alert health care providers to an underlying medical condition, leading to delayed or inaccurate treatment), and neuromuscular irritability, potentially including tetany and seizures.

[0009] Unlike other successfully formulated proteins, PTH is particularly susceptible to various forms of degradation. For example, oxidation can occur at the methionine residues at positions 8 and 18, giving rise to the oxidized PTH species ox-M(8)-PTH and ox-M(18)-PTH, while deamidation can occur at the asparagine at position 16, giving rise to d16-PTH. The polypeptide chain is shortened by peptide bond cleavage at both the N- and C-termini. In addition, PTH can also adsorb to surfaces, form nonspecific aggregates, and / or precipitate, thus reducing the available concentration of the drug. All these degradation reactions, and their combinations, lead to partial or complete loss of the biological activity of PTH. Thus, formulations of PTH must prevent these degradation reactions.

[0010] Teriparatide (PTH(1-34) sold by Eli Lilly and Co under the brand name Forteo®) has been identified as an effective alternative to calcitriol therapy for hypoparathyroidism, allowing the maintenance of normal serum calcium levels without hypercalciuria.

[0011] Full-length PTH(1-84) has recently been approved as a safe and effective treatment for hypoparathyroidism (sold under the brand name NATPARA® by Shire-NPS Pharmaceuticals). It is the first specific hormone replacement for hypoparathyroidism and is a once-daily subcutaneous injectable taken as a supplement to calcium and vitamin D. However, due to its short half-life in vivo, the pharmacokinetic profile of NATPARA in humans shows fast clearance and a high peak-to-trough ratio, which leads to large diurnal variations in serum calcium and poor control of calciuria, a condition associated with elevated urinary calcium levels.

[0012] Thus, there is a need for improved PTH receptor agonists, particularly those that have long acting activity at the PTH receptor and a more steady-state physiological profile. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Potts et al., Amer. Journ. of Med., 50: 639-649 (1971) [Non-Patent Document 2] Potts et al., Proceed. of the 3rd Intern. Symp. of Endocrin., London, pp 333-349 (1971) [Non-Patent Document 3] Tregear et al., Endocrin., 93: 1349-1353 (1973) [Non-Patent Document 4] Segre et al., Journ. of Bio. Chem., 254: 6980-6986 (1979) [Non-Patent Document 5] Nissenson et al., Journ. of Bio. Chem., 254: 1469-1475 (1979) [Non-Patent Document 6] Potts et al., Adv. in Prot. Chem., 35: 323-396 (1982) [Non-Patent Document 7] Kemper et al., Proceed. of the Nat. Acad. of Sciences, 71: 3731-3735 (1974) [Non-Patent Document 8] Freeman et al., Molec. Endocrin., 1: 628-638 (1987) [Non-Patent Document 9] Wiren et al., Journ. of Bio. Chem., 263: 19771-19777 (1988)

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[0014] Various non-limiting aspects and embodiments of the present invention are described below.

[0015] In one aspect, a parathyroid hormone (PTH) variant is provided. In some embodiments, the PTH variant according to the present invention may be a PTH-Fc fusion protein comprising PTH or a variant thereof chemically bound to the Fc region of a human IgG antibody or a derivative thereof. In one embodiment, the PTH or variant thereof bound to the Fc may be full-length PTH (i.e., PTH(1-84)), or a truncated PTH, e.g., C-terminal truncated PTH, or a variant thereof. In one embodiment, the PTH bound to the Fc may be PTH(1-74), or PTH(1-64), or PTH(1-54), or PTH(1-44). In one embodiment, the PTH bound to the Fc is not PTH(1-34). In one embodiment, the PTH bound to the Fc may be full-length PTH(1-84), e.g., mutated PTH(1-84).

[0016] In one embodiment, the PTH bound to Fc can be any length from PTH(1-35) to PTH(1-83). In one embodiment, the PTH bound to Fc can be PTH(1-33).

[0017] In one embodiment, the PTH linked to Fc is PTH(1-84), or PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH(1-73). ), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH(1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1-61), or PTH(1-60), or PTH(1-59), or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH(1-48), or PTH(1-47), or can be PTH(1-46), or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH(1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1-36), or PTH(1-35), or PTH(1-33).

[0018] In one embodiment, the amino acid sequence of PTH bound to Fc comprises further modification selected from amino acid substitution, addition, or deletion. In one embodiment, PTH bound to Fc comprises F34A substitution, F34D substitution, V35S substitution, or V35T substitution, or a combination thereof. In one embodiment, PTH bound to Fc may be further modified, for example, pegylated (PEGylated), glycosylated, etc. In one embodiment, PTH bound to Fc is PEGylated. In one embodiment, PTH bound to Fc is glycosylated. In one embodiment, Fc is a native Fc form. In another embodiment, Fc comprises further modification selected from amino acid substitution, addition, or deletion. In one embodiment, Fc is hFcLALA, comprising L234A and L235A substitutions. In other embodiments, the Fc may be modified to increase half-life extension, for example as described in Yang et al, mAbs, 2017, 9, 1105, or by other methods commonly known in the art.

[0019] In one embodiment, there is provided an Fc-binding PTH variant having the amino acid sequence of SEQ ID NO:8-11.

[0020] In one embodiment, the Fc-binding PTH variant according to the invention is provided in a bivalent structure, ie with two copies of PTH and one copy of Fc.

[0021] In another embodiment, the Fc-linked PTH variant according to the invention is provided as a monovalent structure, i.e., with one copy of PTH and one copy of Fc. In one embodiment, the monovalent PTH-Fc variant can be prepared using heavy chain mutations known in the art (e.g., knobs and holes, etc.). One method known in the art is described, for example, in U.S. Pat. No. 8,679,785. In another embodiment, the monovalent PTH-Fc variant can be prepared using a monomeric Fc, and the two Fc moieties can be connected via a linker (e.g., an amino acid linker).

[0022] In one embodiment, the serum half-life of the PTH-Fc fusion protein of the invention is longer than the serum half-life of PTH(1-84). In one embodiment, the serum half-life of the PTH-Fc fusion protein of the invention is 2 times longer, or 3 times longer, or 5 times longer, or 10 times longer, or 20 times longer, or 30 times longer, or 40 times longer than the serum half-life of PTH(1-84). In one embodiment, the PTH-Fc fusion protein of the invention has a serum half-life 40 times longer than the serum half-life of PTH(1-84).

[0023] In another embodiment, the peak / trough ratio of the PTH-Fc fusion protein is lower than that of exogenous PTH(1-84). In one embodiment, the PTH-Fc fusion protein of the invention has a peak / trough ratio of 1 / 2 or less than that of PTH(1-84). In one embodiment, the PTH-Fc fusion protein of the invention has a peak / trough ratio of 1 / 10 or less than that of PTH(1-84).

[0024] In some embodiments, the PTH variant is processed from a PTH variant precursor polypeptide that includes a signal peptide directly linked to the PTH variant. The signal peptide on the polypeptide can facilitate secretion of the PTH variant from the mammalian host cell used to generate the PTH variant, and the signal peptide is cleaved from the PTH variant after secretion. Any number of signal peptides can be used. In one embodiment, the signal peptide can have the following sequence (IgK signal peptide): METPAQLLFLLLLWLPDTTG (SEQ ID NO: 3). In another embodiment, the signal peptide can have the following sequence (IgG heavy chain signal peptide): MEFGLSWLFLVAILKGVQC (SEQ ID NO: 4). In yet another embodiment, the signal peptide can have the following sequence (CD33 signal peptide): MPLLLLLPLLWAGALA (SEQ ID NO: 5). In one particular embodiment, the CD33 signal peptide with the sequence MPLLLLLPLLWAGALA (SEQ ID NO: 5) can provide optimal signal peptide cleavage, leaving position 1 of the PTH variant intact.

[0025] In another aspect, there is provided a pharmaceutical composition comprising at least one PTH-Fc fusion protein of the invention and a pharma- ceutically acceptable carrier.

[0026] In another aspect, there is provided a pharmaceutical dosage form comprising at least one PTH-Fc fusion protein of the invention or a pharmaceutical composition of the invention. In one embodiment, the pharmaceutical formulation of the invention is a liquid dosage form. In one embodiment, the pharmaceutical formulation is suitable for administration by injection or infusion.

[0027] In another aspect, there is provided a nucleic acid encoding a PTH variant according to the invention.In one embodiment, there is provided a nucleic acid encoding a PTH-Fc fusion variant protein.

[0028] In another aspect, there is provided an expression vector comprising a nucleic acid encoding a PTH variant according to the invention.In one embodiment, there is provided an expression vector comprising a nucleic acid encoding a PTH-Fc fusion variant protein.

[0029] In another aspect, a host cell is provided that comprises an expression vector or a nucleic acid encoding a PTH variant according to the present invention. In one embodiment, a host cell is provided that comprises an expression vector or a nucleic acid encoding a PTH-Fc fusion variant protein. In one embodiment, the host cell is a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell. In one embodiment, the host cell is a CHO cell.

[0030] In another aspect, there is provided a method of controlling serum calcium and phosphate levels in a subject in need thereof, the method comprising administering to the subject a PTH variant of the invention, or a pharmaceutical composition comprising a PTH variant of the invention, or a pharmaceutical formulation comprising a PTH variant of the invention.

[0031] In another aspect, there is provided a method of controlling urinary calcium levels in a subject in need thereof, the method comprising administering to the subject a PTH variant of the invention, or a pharmaceutical composition comprising a PTH variant of the invention, or a pharmaceutical formulation comprising a PTH variant of the invention.

[0032] In another embodiment, a method of treating a subject having hypoparathyroidism is provided, the method comprising administering to the subject an effective amount of a PTH variant of the invention, or a pharmaceutical composition comprising a PTH variant of the invention, or a pharmaceutical formulation comprising a PTH variant of the invention.

[0033] In another embodiment, a method of treating a subject having hypoparathyroidism is provided, the method comprising administering to the subject an effective amount of a PTH variant of the invention, or a pharmaceutical composition comprising a PTH variant of the invention, or a pharmaceutical formulation comprising a PTH variant of the invention.

[0034] In some embodiments, the PTH variants of the invention are administered twice daily, or once daily, or every 2 days, or every 3 days, or every 5-8 days. In one embodiment, the PTH variants of the invention are administered once daily. In one embodiment, the PTH variants of the invention are administered once weekly. In one embodiment, the PTH variants of the invention are administered subcutaneously.

[0035] In some embodiments, the dose of the PTH variant of the invention may need to be titrated for the individual due to population variation. In one embodiment, the PTH variant of the invention is administered in an amount of about 1 μg / day to about 500 μg / day, or about 2 μg / day to about 250 μg / day, or about 5 μg / day to about 100 μg / day, or about 10 μg / day to about 80 μg / day, or about 20 μg / day to about 100 μg / day, or about 50 μg / day to about 100 μg / day, or about 50 μg / day, or about 60 μg / day, or about 70 μg / day, or about 80 μg / day, or about 90 μg / day, or about 100 μg / day.

[0036] These and other aspects of the present invention will become apparent to those of ordinary skill in the art after reading the following detailed description of the invention, including the appended claims.

[0037] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0038] [Figure 1] Comparison of serum concentrations as a function of time of various C-terminal truncated PTH-Fc fusion variants following a single subcutaneous administration to rats. [Figure 2A] FIG. 1 shows serum calcium concentrations as a function of time for PTH-66 (PTH(1-74)-Fc fusion) compared to vehicle (phosphate buffer) control following a single subcutaneous administration to mice. [Figure 2B]FIG. 1 shows urinary calcium concentrations as a function of time for PTH-66 (PTH(1-74)-Fc fusion) compared to vehicle (phosphate buffer) control following a single subcutaneous administration to mice. [Figure 3A] PK data of PTH-66 (PTH(1-74)-Fc) fusion at various concentrations in TxPTx rats after a single subcutaneous injection. [Figure 3B] FIG. 1 shows PD data of PTH-66 (PTH(1-74)-Fc) fusion at various concentrations on serum calcium in TxPTx rats after a single subcutaneous injection. [Figure 4A] Single dose PK study data in TPTx rats comparing PTH-66 and PTH(1-84) are shown. [Figure 4B] 1 shows single-dose PD study data in TPTx rats comparing PTH-66 with PTH(1-84). [Figure 5A] 1 shows multiple dose PK study data with daily subcutaneous administration of PTH-66 in TPTx rats. [Figure 5B] 1 shows multiple dose PD study data with daily subcutaneous administration of PTH-66 in TPTx rats. [Figure 5C] 1 shows multiple dose PD study data with daily subcutaneous administration of PTH-66 in TPTx rats. [Figure 5D] 1 shows multiple dose PD study data with daily subcutaneous administration of PTH-66 in TPTx rats. [Figure 6A] 1 shows single-dose PK study data in wild-type cynomolgus monkeys comparing PTH-66 with PTH(1-84). [Figure 6B] 1 shows data from a single-dose PD study in wild-type cynomolgus monkeys comparing PTH-66 with PTH(1-84). [Figure 7] PK and PD study data in wild-type cynomolgus monkeys comparing PTH-66 to vehicle after daily subcutaneous administration are shown. [Figure 8A]Shown is a trace of chromatographic purification of approximately 900 mL of CM expressing PTH-66 using 2x5 mL MabSelect Sure columns. [Figure 8B] 1 is a flow chart outlining the purification steps of PTH-66, according to an embodiment of the present invention. [Figure 9A] Mab-selected crude elution fractions of PTH-66 are shown. [Figure 9B] Coomassie blue staining of Mab-selected crude elution fractions of PTH-66 is shown. [Figure 10] SDS-PAGE Coomassie staining of the final PTH-66 product is shown. [Figure 11] 1 shows the downstream process flow of PTH-66. [Figure 12] 1 is a flow chart of the manufacturing process for PTH-66 that achieves a goal of less than 10% fragmentation. [Figure 13A] 1 is a flow chart of the development of the polishing (final purification) step of PTH-66 purification. [Figure 13B] 1 is a flowchart of a process flow. [Figure 14A] 13 is a plot of Capto MMC ImpRes high throughput screening. [Figure 14B] 13 is a plot of high throughput screening of Capto Adhere ImpRes to determine optimal binding conditions. [Figure 14C] The chemical structure of Capto MMC. [Figure 14D] The chemical structure of Capto Adhere. [Figure 15] The process flow of Capto MMC ImpRes is shown. [Figure 16] Shows the Capto Adhere ImpRes process flow. [Figure 17A] 1 is a plot of gradient elution of PTH-66 using Capto MMC ImpRes. [Figure 17B]FIG. 13 is a flow-through plot of PTH-66 using Capto Adhere ImpRes. [Figure 18A] 1 shows the design of a polishing step downstream of PTH-66. [Figure 18B] 1 is a graph showing the robustness of the process with stable pools based on impurity clearance and process yield. [Figure 19A] 1 shows the step yields of the pilot plant operation (PPO) for purification of PTH-66 compared to the process development (PD) yields for pilot scale production of PTH-66. [Figure 19B] 1 shows the step yields of the pilot plant operation (PPO) for purification of PTH-66 compared to the process development (PD) yields for pilot scale production of PTH-66. [Figure 19C] 1 shows the step yields of the pilot plant operation (PPO) for purification of PTH-66 compared to the process development (PD) yields for pilot scale production of PTH-66. [Figure 19D] 1 shows the step yields of the pilot plant operation (PPO) for purification of PTH-66 compared to the process development (PD) yields for pilot scale production of PTH-66. [Figure 19E] 1 shows the step yields of the pilot plant operation (PPO) for purification of PTH-66 compared to the process development (PD) yields for pilot scale production of PTH-66. [Figure 19F] 1 shows the step yields of the pilot plant operation (PPO) for purification of PTH-66 compared to the process development (PD) yields for pilot scale production of PTH-66. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the present invention, which may be embodied in various forms. Moreover, each example given in connection with various embodiments of the present invention is intended to be illustrative and not limiting. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a representative basis for teaching those skilled in the art how to use the present invention in various ways.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0042] The term "treat" or "treatment" of a condition, disorder or state includes: (1) preventing, delaying or reducing the likelihood of the onset and / or appearance of at least one clinical or asymptomatic symptom of the condition, disorder or state developing in a subject who may be afflicted with or predisposed to the condition, disorder or state, but who has not yet experienced or displayed a clinical or asymptomatic symptom of the condition, disorder or state; or (2) inhibiting the condition, disorder or state, i.e., arresting, reducing or delaying the progression of the disease or its recurrence, or at least one clinical or asymptomatic symptom thereof; or (3) palliating the disease, i.e., causing regression of the condition, disorder or state, or at least one clinical or asymptomatic symptom thereof. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0043] As used herein, "subject" or "patient" or "individual" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0044] As used herein, the term "effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce a desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would have been effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or method of administration used, etc.

[0045] The phrase "pharmacologically acceptable" as used in connection with the compositions of the present invention refers to molecular entities and other components of such compositions that are physiologically tolerable and do not normally produce adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmacologically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more specifically, humans.

[0046] As used herein, the terms "carrier" and "diluent" refer to a pharma- ceutically acceptable (e.g., safe and non-toxic for administration to humans) carrier or diluent substance useful in the preparation of a pharmaceutical formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0047] As used herein, the term "fusion protein" or "chimeric protein" refers to a protein made by the joining of two or more originally separate proteins or portions thereof. In some embodiments, a linker or spacer will be present between each protein.

[0048] As used herein, the term "half-life" is the time required for a quantity, such as a protein concentration or activity, to fall to half of the value measured at the beginning of the period.

[0049] As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. A "control subject" is a subject who is about the same age as the treated subject and who is suffering from the same form of the disease as the treated subject.

[0050] As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, and the like.

[0051] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0052] As used herein, the term "linker" refers to an amino acid sequence other than that which appears in a fusion protein at a specific position in its native protein or derivative, and is generally designed to be flexible or to place a structure such as an α-helix between two protein moieties. A linker is also called a spacer. A linker or spacer usually has no biological function by itself.

[0053] The term "polypeptide" as used herein refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but those skilled in the art will understand that the term is not limited to long chains and can also refer to a minimum chain that includes two amino acids linked together via peptide bonds. As known to those skilled in the art, polypeptides can be processed and / or modified. As used herein, the terms "polypeptide" and "peptide" are used interchangeably. The term "polypeptide" can also refer to a protein.

[0054] As used herein, the term "PTH variant" refers to any derivative of native PTH(1-84), including, but not limited to, PTH(1-84) derivatives containing amino acid additions, deletions, and / or substitutions, truncated (e.g., C-terminally truncated) PTH(1-84), PTH fused directly or via a linker or spacer to a peptide or protein or protein domain, and PTH post-translationally modified in any manner known in the art (e.g., glycosylation, PEGylation, etc.), or combinations thereof.

[0055] PTH Parathyroid hormone (PTH) is a polypeptide secreted by the parathyroid gland in mammals. Native PTH is 84 amino acids long and has the following sequence (SEQ ID NO:1): SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ, or TIFF0007601775000001.tif59134

[0056] As used herein, the term "human PTH" or "hPTH" is included within the term PTH or parathyroid hormone. Human PTH can be synthesized in vivo, recombinantly (intracellularly), or synthetically using standard techniques known in the art.

[0057] PTH-Fc fusion In some embodiments, the PTH variant of the present invention is a PTH-Fc fusion protein. As used herein, "Fc" refers to the Fc region of a human IgG antibody. Fc fusion proteins (also known as Fc chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins, and Fc-tag proteins) consist of the Fc domain of IgG chemically bound to a peptide or protein of interest (e.g., PTH).

[0058] Any IgG Fc domain can be used. As a non-limiting example, IgG1, or IgG2, or IgG3, or IgG4 Fc domains can be used, as well as various combinations of Fc domains from different IgGs, such as, for example, half IgG2 and half IgG4. In one non-limiting embodiment, the PTH-Fc fusion protein of the invention has the Fc domain of a human IgG1 antibody.

[0059] Silenced effector functions can be obtained by mutations in the Fc region of the antibody, and different IgGs can have different silencer regions known to those skilled in the art. For example, for IgG1, the following silencer regions are known and described in the literature: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69; Strohl, W., supra). An example of a silent Fc IgG1 antibody includes the so-called LALA mutant ("hFcLALA"), which contains L234A and L235A (EU numbering) mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody contains the D265A mutation. Another silent IgG1 antibody contains the N297A mutation, which results in an aglycosylated / non-glycosylated antibody.

[0060] As used herein, IgG1 hFcLALA has the following sequence, with the LALA mutation underlined (SEQ ID NO:2): DKTHTCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0061] The PTH sequence may be directly or indirectly linked to the Fc domain. In one embodiment, PTH is directly linked to the Fc domain. In another embodiment, PTH is linked to the Fc domain by an amino acid linker.

[0062] In some embodiments, the Fc region can be further modified to further extend the half-life of the fusion protein.In one non-limiting example, half-life extension technology, such as NHance technology by ArGenX, can be utilized in combination with the PTH-Fc variant of the present invention.NHance technology is described, for example, in U.S. Patent No. 8,163,881, the contents of which are incorporated herein by reference in their entirety.

[0063] In one embodiment, the Fc-binding PTH variant according to the invention is provided as a bivalent structure, ie having two copies of PTH and one copy of Fc.

[0064] In another embodiment, the Fc-linked PTH variant according to the invention is provided as a monovalent structure, i.e., with one copy of PTH and one copy of Fc. In one embodiment, the monovalent PTH-Fc variant can be prepared using heavy chain mutations known in the art (e.g., knobs and holes, etc.). One method known in the art is described, for example, in U.S. Pat. No. 8,679,785. In another embodiment, the monovalent PTH-Fc variant can be prepared using a monomeric Fc, and the two Fc moieties can be connected via a linker (e.g., an amino acid linker).

[0065] Signal peptides and protease tags In some embodiments, the PTH variants of the present invention are processed from a PTH variant precursor polypeptide that includes a signal peptide directly linked to the PTH variant. The signal peptide on the polypeptide can facilitate secretion of the PTH variant from the mammalian host cell used to produce the PTH variant, and the signal peptide is cleaved from the PTH variant after secretion. Any number of signal peptides can be used.

[0066] In some embodiments, an IgK leader sequence having the following sequence (SEQ ID NO:3) is present in a variant of the invention: METPAQLLFLLLLWLPDTTG

[0067] In some embodiments, an IgG heavy chain signal peptide having the following sequence (SEQ ID NO:4) is present in a variant of the invention: MEFGLSWLFLVAILKGVQC

[0068] In some embodiments, a CD33 signal peptide having the following sequence (SEQ ID NO:5) is present in a variant of the invention: MPLLLLLPLLWAGALA

[0069] In one particular embodiment, the CD33 signal peptide having the sequence MPLLLLLPLLWAGALA (SEQ ID NO:5) can provide optimal signal peptide cleavage, leaving position 1 of the PTH variant intact.

[0070] In some embodiments, a polyhistidine tag (HIS tag) is present in the variants of the invention to facilitate purification of the PTH protein using an affinity chromatography resin that recognizes and binds to the HIS tag. In one embodiment, a TEV-HIS tag is present at the C-terminus of the PTH variants of the invention. The TEV component is a protease recognition site that allows the HIS tag to be removed after protein purification. In one embodiment, the sequence of the TEV-HIS tag is ENLYFQSHHHHHH (SEQ ID NO: 6). In one embodiment, ENLYFQ (SEQ ID NO: 7), which is part of the TEV recognition sequence, remains at the end of the protein and does not affect the activity of the protein.

[0071] Protein Glycosylation Glycosylation, as used herein, is the reaction by which carbohydrates (glycans) are attached to peptides or proteins. Different classes of glycans are recognized, the two most important being N-linked glycans, which are attached to the nitrogen or arginine side chains, and O-linked glycans, which are attached to the hydroxyl oxygen of the side chains of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline.

[0072] In some embodiments, amino acid substitutions are introduced into the PTH variants of the present invention to insert glycosylation sites. In some embodiments, these amino acid substitutions can introduce serine moieties into the PTH variants. In some embodiments, the PTH variants of the present invention can include F34A, F34D, and / or V35S, or V35T mutations to insert glycan sites into the PTH variants.

[0073] In some embodiments, amino acid changes can be made at F34 (e.g., F34A or F34D) and / or V35 (e.g., V35S or V35T) to minimize protein degradation observed in this region during expression of the molecule in certain cell types, e.g., CHO cells. The F34A and F34D mutations reduce protein degradation. The combination of the F34A and V35S (or V35T) mutations further reduces protein degradation significantly. These mutations create a consensus N-linked glycosylation site that includes position N33. This allows glycosylation at position N33, greatly reducing protein degradation at this position.

[0074] In some embodiments, the insertion of a glycan site at N33 reduces protein cleavage during host cell expression. The glycan introduces additional size and bulk to the PTH variant. The glycan site may also improve the solubility of the PTH variant. The glycan site may also extend the half-life (T1 / 2) of the PTH variant. The insertion of a glycan at the protein mutation site may also reduce the potential immunogenicity of the mutated sequence. In some embodiments, the amino acid substitution to insert a glycosylation site may be combined with other mutations or sequence truncations or post-translational modifications of the PTH variant.

[0075] In some embodiments, an amino acid change can be made at L59 (e.g., L59S or L59T). This mutation creates a glycosylation site at N57 and can reduce cleavage of the protein during host cell expression.

[0076] Other variants: In some embodiments, amino acid substitutions are introduced into the PTH variant to improve developability properties. In some embodiments, these amino acid substitutions result in reduced cleavage of the PTH variant during expression. In some embodiments, these amino acid substitutions result in improved solubility of the PTH variant. In some embodiments, these amino acid substitutions result in glycan insertions. In some embodiments, these amino acid substitutions may introduce aspartic acid into the sequence.

[0077] In some embodiments, the PTH variants of the invention may contain the F34D mutation to reduce protein truncation during expression. In some embodiments, the PTH variants containing F34D may be combined with other mutations or sequence truncations or post-translational modifications of the PTH variant.

[0078] Typically, suitable PTH variants have an in vivo half-life of about 2 hours or more, 3 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, 22 hours or more, 24 hours or more, 26 hours or more, 28 hours or more, 30 hours or more, 32 hours or more, 34 hours or more, 36 hours or more, 38 hours or more, 40 hours or more, 42 hours or more, 44 hours or more, 46 hours or more, or 48 hours or more. In some embodiments, the PTH variant has an in vivo half-life of between 2-48 hours, between 2-44 hours, between 2-40 hours, between 3-36 hours, between 3-32 hours, between 3-28 hours, between 4-24 hours, 6-24 hours, or 6-18 hours.

[0079] List of selected parathyroid hormone variants The following abbreviations are used in the list below and throughout the specification: PTH(1-N) refers to parathyroid hormone peptide residues 1 through N, where N is 84 (for native-length PTH and variants) or less than 84 (for C-terminally truncated PTH variants). For example, PTH(1-84) refers to residues 1 through 84 (full-length sequence) of the parathyroid hormone peptide; PTH(1-74) refers to residues 1 through 74 of the parathyroid hormone peptide, i.e., the C-terminally truncated variant of PTH(1-84), in which the last 10 residues of PTH are removed, i.e., the first 74 residues; PTH(1-34) refers to residues 1 through 34 of the parathyroid hormone peptide, i.e., the first 34 residues, with the last 50 residues removed; and so forth.

[0080] Point mutations in the variant sequences are designated according to standard rules; for example, F34A means that the phenylalanine amino acid at position 34 is replaced by an alanine amino acid; F34D means that the phenylalanine amino acid at position 34 is replaced by an aspartic acid residue, and V35S means that the valine at position 35 is replaced by a serine.

[0081] Δ means [deleted sequence], for example, ΔLys means deletion of a lysine residue. In the constructs included herein, the Lys that is deleted is in the C-terminal region of the Fc molecule, which, among other things, improves homogeneity of the pharmaceutical product.

[0082] In one embodiment, the PTH linked to Fc can be full length PTH(1-84), e.g., mutant PTH(1-84).

[0083] In one embodiment, the PTH bound to Fc can be any truncated PTH from PTH(1-35) to PTH(1-83). In one embodiment, the PTH bound to Fc can be PTH(1-33).

[0084] In some embodiments of the invention, the PTH linked to Fc is PTH(1-84), or PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH(1-75). H(1-73), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH(1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1-61), or PTH(1- 60), or PTH(1-59), or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH(1-48), or PTH(1-47), or PTH(1-46), or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH(1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1-36), or PTH(1-35), or PTH(1-33).

[0085] In the following description of exemplary embodiments of the invention, the signal peptide is marked in italics, the PTH domain is in bold with any mutations therein underlined, and any fusion sequences are marked with a wavy underline.

[0086] Molecular designation "PTH-66" (SEQ ID NO: 8) Description: PTH(1-74)-[F34A, V35S]-hFcLALA-ΔLys Signal peptide, PTH1-74, Mutations to insert glycan sites , Fc TIFF0007601775000002.tif48161

[0087] Molecular designation "PTH-67" (SEQ ID NO: 9) Description: PTH(1-64)-[F34A, V35S]-hFcLALA-ΔLys Signal peptide, PTH1-64, Mutations to insert glycan sites , Fc TIFF0007601775000003.tif48162

[0088] Molecular designation "PTH-68" (SEQ ID NO: 10) Description: PTH(1-54)-[F34A, V35S]-hFcLALA-ΔLys Signal peptide, PTH1-54, Mutations to insert glycan sites , Fc TIFF0007601775000004.tif49162

[0089] Molecular designation "PTH-69" (SEQ ID NO: 11) Description: PTH(1-44)-[F34A, V35S]-hFcLALA-ΔLys Signal peptide, PTH1-44, Mutations to insert glycan sites , Fc TIFF0007601775000005.tif48161

[0090] Molecular designation "PTH-11" (SEQ ID NO: 12) Description:PTH(1-84)-hFcLALA TIFF0007601775000006.tif47162

[0091] Other PTH variants In one embodiment, the PTH variants according to the invention may be conjugated to albumin or a domain of albumin, either directly or via a linker. In one embodiment, the PTH-albumin fusions according to the invention may be prepared by the method described in US Pat. No. 7,592,010. The PTH-albumin variants may be monovalent, i.e., have one copy of PTH and one copy of albumin or a domain of albumin. In one embodiment, the serum half-life of the PTH-albumin fusion protein is longer than that of PTH(1-84). In one embodiment, the PTH-albumin fusion proteins of the invention have a serum half-life that is 2-fold longer, or 3-fold longer, or 5-fold longer, or 10-fold longer, or 20-fold longer, or 30-fold longer, or 40-fold longer than that of PTH(1-84). In one embodiment, the PTH-Fc fusion proteins of the invention have a serum half-life that is 40-fold longer than that of PTH(1-84).

[0092] In one embodiment, the PTH bound to albumin is a variant PTH(1-84), or PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH (1-73), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH(1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1-61), or PTH(1-6 0), or PTH(1-59), or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH(1-48), or PTH(1-47), or or PTH(1-46), or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH(1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1-36), or PTH(1-35), or PTH(1-33).

[0093] nucleic acid In another aspect, a nucleic acid is provided that comprises a sequence encoding a PTH variant as described herein. The sequence may have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of SEQ ID NOs: 8-12. In some embodiments, the nucleic acid may comprise additional non-coding sequences. The nucleic acid may further comprise a particular fragment, variant or consensus sequence thereof, or a deposited vector comprising at least one of these sequences. The nucleic acid molecule may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning, synthetically produced, via gene therapy (e.g., using AAV), or any combination thereof. The DNA may be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, also known as the anti-sense strand.

[0094] In some embodiments, the nucleic acid encoding the transgene may be modified to provide increased expression of the encoded PTH variant, also referred to as codon optimization. For example, the nucleic acid encoding the transgene may be modified by changing the open reading frame of the coding sequence. As used herein, the term "open reading frame" is synonymous with "ORF" and refers to any nucleotide sequence that can potentially code for a protein or a portion of a protein. An open reading frame usually begins with a start codon (represented in standard codes, e.g., AUG for RNA molecules and ATG for DNA molecules) and is read in codon triplets until the frame ends with a stop codon (represented in standard codes, e.g., UAA, UGA or UAG for RNA molecules and TAA, TGA or TAG for DNA molecules). As used herein, the term "codon" refers to a sequence of three nucleotides in a nucleic acid molecule that specifies a particular amino acid during protein synthesis; also referred to as a triplet or codon triplet. For example, of the 64 possible codons in the standard genetic code, two codons, GAA and GAG, code for the amino acid glutamine, and the codons, AAA and AAG, specify the amino acid lysine. In the standard genetic code, three codons are stop codons and do not specify an amino acid. As used herein, the term "synonymous codon" refers to all codons that code for a single amino acid. With the exception of methionine and tryptophan, amino acids are coded for by two to six synonymous codons. For example, in the standard genetic code, the four synonymous codons that code for the amino acid alanine are GCA, GCC, GCG, and GCU, the two synonymous codons that specify glutamine are GAA and GAG, and the two synonymous codons that code for lysine are AAA and AAG.

[0095] The nucleic acid encoding the open reading frame of PTH variant can be modified using standard codon optimization methods.Various commercially available algorithms for codon optimization are available and can be used to implement the present invention.Usually, codon optimization does not change the encoded amino acid sequence.

[0096] The nucleotide changes may alter synonymous codons in the open reading frame to match the endogenous codon usage found in the particular heterologous cell selected to express the PTH variant. Alternatively, or in addition, the nucleotide changes may change the G+C content in the open reading frame to better match the average G+C content of the open reading frame found in the endogenous nucleic acid present in the heterologous host cell. The nucleotide changes may also alter polymononucleotide regions or internal regulatory or structural sites found within the PTH variant sequence. Thus, a variety of modified or optimized nucleotide sequences are envisioned, including, but not limited to, nucleic acid sequences that provide increased expression of PTH variants in prokaryotic, yeast, insect, and mammalian cells.

[0097] As provided herein, the polynucleotide may further include additional sequences, such as coding sequences for at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, along with additional non-coding sequences, including, but not limited to, non-coding 5' and 3' sequences, such as transcribed, non-translated sequences that play a role in transcription and mRNA processing (e.g., ribosome binding and stability of mRNA), including splicing and polyadenylation signals; additional coding sequences that encode additional amino acids, such as amino acids that provide additional functionality; Thus, the sequence encoding the PTH variant or particular portion can be fused to a marker sequence, such as, for example, a sequence encoding a peptide that facilitates purification of the fused PTH variant or particular portion (including a fragment or portion of the PTH variant).

[0098] The nucleic acid may further comprise a sequence in addition to the polynucleotide of the present invention. For example, a multi-cloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, a translatable sequence may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexahistidine marker sequence provides a convenient means for purifying the protein of the present invention. The nucleic acid of the present invention (excluding the coding sequence) is optionally a vector, adapter, or linker for cloning and / or expressing the polynucleotide of the present invention.

[0099] The coding region of the transgene may contain one or more silent mutations to optimize codon usage for a particular cell type. For example, the codons of the PTH variant may be optimized for expression in vertebrate cells. In some embodiments, the codons of the PTH variant may be optimized for expression in mammalian cells. In some embodiments, the codons of the PTH variant may be optimized for expression in human cells. In some embodiments, the codons of the PTH variant may be optimized for expression in CHO cells.

[0100] The nucleic acid sequence encoding the PTH variant described in this application may be molecularly cloned (inserted) into a vector suitable for propagation or expression in a host cell, or in some embodiments, the entire plasmid may be synthesized as well. For example, a PTH variant sequence including a signal peptide effective to secrete the PTH variant from a host cell, such as a sequence selected from SEQ ID NOs: 8-12, is inserted into a suitable vector. A wide variety of expression vectors may be used to practice the invention, including, but not limited to, prokaryotic expression vectors, yeast expression vectors, insect expression vectors, and mammalian expression vectors. Exemplary vectors suitable for the invention include, but are not limited to, viral-based vectors (e.g., AAV-based vectors, retroviral-based vectors, plasmid-based vectors). In some embodiments, the nucleic acid sequence encoding the PTH variant may be inserted into a suitable vector. Typically, the nucleic acid encoding the PTH variant is operably linked to various regulatory sequences or elements.

[0101] A variety of regulatory sequences or elements can be incorporated into expression vectors suitable for the present invention. Exemplary regulatory sequences or elements include, but are not limited to, promoters, enhancers, repressors or suppressors, 5' non-translated (or non-coding) sequences, introns, 3' non-translated (or non-coding) sequences.

[0102] As used herein, a "promoter" or "promoter sequence" is a DNA regulatory region that can bind (e.g., directly or through other promoter-binding proteins or substances) RNA polymerase in a cell and initiate transcription of a coding sequence. A promoter sequence generally binds at its 3' end to a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. A promoter can be operably associated or operably linked to an expression control sequence, including enhancer and repressor sequences, or to the nucleic acid to be expressed. In some embodiments, a promoter can be inducible. In some embodiments, an inducible promoter can be unidirectional or bidirectional. In some embodiments, a promoter can be a constitutive promoter. In some embodiments, a promoter can be a hybrid promoter, in which the sequence containing the transcriptional regulatory region is obtained from one source and the sequence containing the transcriptional initiation region is obtained from a second source. Systems for linking control elements to coding sequences in transgenes are well known in the art (general molecular biology and recombinant DNA techniques are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, which is incorporated herein by reference). Commercially available vectors suitable for inserting transgenes for expression in a variety of host cells under a variety of growth and induction conditions are also well known in the art.

[0103] In some embodiments, the SRa promoter (Takebe et al., Molec. and Cell. Bio. 8:466-472 (1988)), the human cytomegalovirus (CMV) immediate early promoter (Boshart et al., Cell 41:521-530 (1985); Foecking et al., Gene 45:101-105 (1986)), human CMV promoter, human CMV5 promoter, mouse CMV immediate early promoter, EF1-α-promoter, hybrid CMV promoters for liver-specific expression (e.g., created by combining the CMV immediate early promoter with transcription promoter elements of either the human α-1-antitrypsin (HAT) or albumin (HAL) promoters), or promoters for hepatocellular carcinoma-specific expression (e.g., the transcription promoter elements of either human albumin (HAL; approx. 1000 bp) or human α-1-antitrypsin (HAT; approx. 2000 bp) combined with the 145-long enhancer elements of the human α-1-microglobulin and bikunin precursor genes (AMBP); HAL-AMBP and HAT-AMBP); SV40 early promoter region (Benoist at al., Nature 290:304-310 (1981)), Orgyia pseudotsugata immediate early promoter, herpes thymidine kinase promoter (Wagner at al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981); or the regulatory sequences of metallothionein genes (Brinster et al., Nature 296:39-42 (1982)). Specific promoters can be used to control the expression of transgenes in mammalian host cells. In some embodiments, the mammalian promoter is a constitutive promoter, such as, but not limited to, the hypoxanthine phosphoribosyltransferase (HPTR) promoter, the adenosine deaminase promoter, the pyruvate kinase promoter, the beta-actin promoter, as well as other constitutive promoters known to those skilled in the art.

[0104] In some embodiments, the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)), the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)), T7 promoter, T3 promoter, M13 promoter, or M16 promoter, can be used to control the expression of a transgene in a prokaryotic host cell; and specific promoters such as, but not limited to, the GAL1, GAL4, or GAL10 promoter, the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, the glyceraldehyde-3-phosphate dehydrogenase III (TDH3) promoter, the glyceraldehyde-3-phosphate dehydrogenase II (TDH2) promoter, the glyceraldehyde-3-phosphate dehydrogenase I (TDH1) promoter, pyruvate kinase (PYK), enolase (ENO), or triosephosphate isomerase (TPI) can be used to control the expression of a transgene in a yeast host cell.

[0105] In some embodiments, the promoter may be a viral promoter, many of which are capable of regulating the expression of transgenes in several host cell types, including mammalian cells. Viral promoters that have been shown to drive constitutive expression of coding sequences in eukaryotic cells include, for example, simian virus promoters, herpes simplex virus promoters, papilloma virus promoters, adenovirus promoters, human immunodeficiency virus (HIV) promoters, Rous sarcoma virus promoters, cytomegalovirus (CMV) promoters, Moloney murine leukemia virus and other retrovirus long terminal repeats (LTRs), herpes simplex virus thymidine kinase promoters, and other viral promoters known to those skilled in the art.

[0106] In some embodiments, the genetic control elements of the expression vector may also include 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, such as TATA box, capping sequence, CAAT sequence, Kozak sequence, etc. Optionally, enhancer elements may be used to increase the expression level of the expressed polypeptide or protein. Examples of enhancer elements that have been shown to function in mammalian cells include the SV40 early gene enhancer described in Dijkema et al., EMBO J. (1985) 4: 761, and the enhancer / promoter derived from the Rous sarcoma virus (RSV) described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and the long terminal repeat (LTR) of human cytomegalovirus described in Boshart et al., Cell (1985) 41:521. The genetic control elements of the expression vector may also include 3' non-transcribed and 3' non-translated sequences involved in the termination of transcription and translation. Each of these signals is a polyadenylation (poly A) signal for stabilizing and processing the 3' end of the mRNA transcribed from the promoter. Exemplary poly A signals include, for example, rabbit beta globin poly A signal, bovine growth hormone poly A signal, chicken beta globin terminator / poly A signal, and SV40 late poly A region.

[0107] The expression vector preferably, but optionally, includes at least one selection marker. In some embodiments, the selection marker is a nucleic acid sequence encoding a resistance gene operably linked to one or more genetic regulatory elements to confer to the host cell the ability to maintain viability when grown in the presence of cytotoxic chemicals and / or drugs. In some embodiments, a selectable agent may be used to maintain the retention of the expression vector in the host cell. In some embodiments, a selectable agent may be used to prevent modification (i.e., methylation) and / or silencing of the transgene sequence in the expression vector. In some embodiments, a selectable agent is used to maintain episomal expression of the vector in the host cell. In some embodiments, a selectable agent is used to promote stable integration of the transgene sequence into the host cell genome. In some embodiments, the drug and / or resistance genes include methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos. 4,399,216, 4,634,665, 4,656,134, 4,956,288, 5,149,636, 5,179,017), ampicillin, neomycin (G418), zeomycin, mycophenolic acid, or glycerol for eukaryotic host cells. These may include, but are not limited to, glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464, 5,770,359, 5,827,739); for prokaryotic host cells, tetracycline, ampicillin, kanamycin or chlorampenichol; and for yeast host cells, URA3, LEU2, HIS3, LYS2, HIS4, ADE8, CUP1 or TRP1.

[0108] An expression vector can be transfected, transformed, or transduced into a host cell. As used herein, the terms "transfection", "transformation" and "transduction" all refer to the introduction of an exogenous nucleic acid sequence into a host cell. In some embodiments, an expression vector comprising a nucleic acid sequence encoding a PTH Fc fusion is transfected, transformed, or transduced into a host cell simultaneously. In some embodiments, an expression vector comprising a nucleic acid sequence encoding a PTH variant is transfected, transformed, or transduced into a host cell sequentially.

[0109] Examples of transformation, transfection and transduction methods well known in the art include liposome delivery, i.e., the Lipofectamine™ (Gibco BRL) method of Hawley-Nelson, Focus 15:73 (1193), electroporation, the CaPO4 delivery method of Graham and van der Erb, Virology, 52:456-457 (1978), DEAE-dextran mediated delivery, microinjection, biolistic particle delivery, polybrene mediated delivery, cation mediated lipid delivery, transduction, and viral infection, e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus and baculovirus (insect cells).

[0110] When introduced into a cell, the expression vector can be stably integrated into the genome or exist as an extrachromosomal construct.The vector can also be amplified, and exist in multiple copies or integrated into the genome.In some embodiments, the cell of the present invention can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more copies of the nucleic acid encoding the PTH variant.

[0111] host cell In another aspect, a host cell is provided that includes a polynucleotide described herein (eg, a polynucleotide that enables expression of a PTH variant in a host cell). The host cell may be a mammalian cell, non-limiting examples of which include BALB / c mouse myeloma cell line (NSO / 1, ECACC number: 85110503); human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (HEK293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); CHO EBNA (Daramola O. et al., Biotechnol. Prog., 2014, 30(1):132-41) and CHO GS (Fan L. et al., Biotechnol. Bioeng. 2012, 109(4):1007-15; Chinese hamster ovary cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatocellular carcinoma line (Hep G2).In one embodiment, the host cell may be a Chinese hamster ovary cell.

[0112] The polynucleotide may be in an expression plasmid. The expression plasmid may have any number of origins of replication known to those skilled in the art. The polynucleotide or expression plasmid may be introduced into the host cell by any number of methods known to those skilled in the art. For example, the polynucleotide or expression plasmid may be introduced into the host cell using a flow electroporation system such as the MaxCyte GT®, MaxCyte VLX®, or MaxCyte STX® transfection system.

[0113] In various embodiments, the host cells express the nucleic acid. The host cells may express the PTH variant at levels sufficient for fed-batch cell culture scale or other large scale. Alternative methods for large-scale production of recombinant PTH variants include roller bottle culture, bioreactor batch culture, and perfusion. In some embodiments, the recombinant PTH variant protein is produced by cells in suspension culture. In some embodiments, the recombinant PTH variant protein is produced by adherent cells.

[0114] manufacturing Recombinant PTH variants can be produced by any available means. For example, recombinant PTH variants can be produced recombinantly by utilizing a host cell line engineered to express nucleic acid encoding recombinant PTH variants. Alternatively, or in addition, recombinant PTH variants can be produced by activating endogenous genes. Alternatively, or in addition, recombinant PTH variants can be partially or completely prepared by chemical synthesis. Alternatively, recombinant PTH variants can be produced in vivo by mRNA therapy or AAV / lentivirus gene therapy.

[0115] In some embodiments, the recombinant PTH variant is produced in mammalian cells. Non-limiting examples of mammalian cells that may be used according to the present invention include BALB / c mouse myeloma cell line (NSO / 1, ECACC number: 85110503); human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (HEK293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); CHO EBNA (Daramola O. et al., Biotechnol. Prog., 2014, 30(1):132-41) and CHO GS (Fan L. et al., Biotechnol. Bioeng. Chinese hamster ovary cells + / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980), including 2012, 109(4):1007-15; mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); mouse kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatocellular carcinoma line (Hep G2).

[0116] In some embodiments, the recombinant PTH variant is produced from human cells, hi some embodiments, the recombinant PTH variant is produced from CHO cells or HT1080 cells.

[0117] In certain embodiments, host cells are selected to generate cell lines based on certain preferred attributes or growth under the particular conditions selected for culturing the cells. It will be understood by those skilled in the art that such attributes can be ascertained based on known characteristics and / or properties of established lines (i.e., characterized commercially available cell lines) or through empirical evaluation. In some embodiments, cell lines can be selected for their ability to grow on a feeder cell layer. In some embodiments, cell lines can be selected for their ability to grow in suspension. In some embodiments, cell lines can be selected for their ability to grow as an adherent monolayer of cells. In some embodiments, cell lines can be selected for preferential post-translational modifications (e.g., glycosylation). In some embodiments, such cells can be used in any tissue culture vessel or any vessel treated with an appropriate adhesive substrate. In some embodiments, the suitable adhesive substrate is selected from the group consisting of collagen (e.g., collagen I, II, II, or IV), gelatin, fibronectin, laminin, vitronectin, fibrinogen, BD Matrigel™, basement membrane matrix, dermatan sulfate proteoglycan, poly-D-lysine, and / or combinations thereof. In some embodiments, the adherent host cells can be selected and modified under specific growth conditions to grow in suspension. Such methods of modifying adherent cells to grow in suspension are known in the art. For example, cells can be conditioned to grow in suspension culture (or floating culture) by gradually removing animal serum from the growth medium over time.

[0118] Typically, the cell engineered to express recombinant PTH variants may contain a transgene encoding the recombinant PTH variants described herein.It should be understood that the nucleic acid encoding the recombinant PTH variants may contain regulatory sequences, gene control sequences, promoters, non-coding sequences, and / or other suitable sequences for expressing recombinant PTH variants.Typically, the coding region is operably linked to one or more of these nucleic acid components.

[0119] In some embodiments, the PTH variants are expressed using a batch culture method. In some embodiments, the batch culture period can be 7-14 days. In some embodiments, the batch culture can be 14-21 days. In some embodiments, the PTH variants are expressed using a perfusion culture method (daily collection of culture medium over time). In some embodiments, the PTH variants are expressed using a pseudoperfusion culture method (daily collection of culture medium at a single time point with replacement with fresh medium). In some embodiments, a specific feeding regimen / media can be used to promote optimal PTH variant production (improved glycans, reduced clipping). In some embodiments, the cell density can be controlled / maintained to promote optimal PTH variant production (improved glycans / reduced clipping).

[0120] In some embodiments, recombinant PTH variants are produced in vivo by mRNA therapy. An mRNA encoding the PTH variant is prepared and administered to a patient in need of the PTH variant. The mRNA can include sequences corresponding to the DNA sequences of SEQ ID NOs: 8-12. A variety of routes of administration can be used, including injection, pulmonary aerosolization, and subcutaneous electroporation. The mRNA can be encapsulated within a viral or non-viral vector. Exemplary non-viral vectors include liposomes, cationic polymers, and cubosomes.

[0121] Recovery and purification Various means can be used to purify PTH variants from cells. Various methods can be used to purify or isolate PTH variants produced according to the various methods described herein. In some embodiments, the expressed protein is secreted into the medium, and thus cells and other solids can be removed, for example, by centrifugation or filtration, as an initial step in the purification process. Alternatively, or in addition, the expressed protein is bound to the surface of the host cell. In this embodiment, the host cell expressing the polypeptide or protein is lysed for purification. Lysis of mammalian host cells can be achieved by any number of means well known to those skilled in the art, including physical disruption with glass beads, the use of detergents, and exposure to high pH conditions. In some embodiments, the PTH variant can be expressed in an insoluble fraction (inclusion bodies). In such embodiments, the cells are collected, for example, by centrifugation, and lysed using denaturants commonly known in the art.

[0122] PTH variants can be isolated and purified by standard methods, including but not limited to chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation, or other techniques available for protein purification.See, for example, Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997, all of which are incorporated herein by reference. In particular, in the case of immunoaffinity chromatography, a protein may be isolated by binding antibodies obtained against the protein to an affinity column immobilized on a solid support. Alternatively, affinity tags such as influenza coat sequence, polyhistidine, or glutathione-S-transferase may be attached to the protein by standard recombinant techniques to allow for easy purification by passage through an appropriate affinity column. To reduce or eliminate degradation of the polypeptide or protein during the purification process, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, or aprotinin may be added at any or all stages.Protease inhibitors are particularly desirable when cells must be lysed to isolate and purify the expressed polypeptide or protein.

[0123] PTH variants or specific portions can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, mixed-mode chromatography (e.g., MEP Hypercel™), hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2003).

[0124] Neutralization after Protein A purification must be performed carefully to avoid aggregation / precipitation of the PTH-Fc fusion protein.

[0125] The PTH variants or specific portions of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Depending on the host and the particular PTH variant used in the recombinant production procedure, the PTH variants or specific portions of the present invention may be glycosylated or non-glycosylated.

[0126] formulation In some embodiments, the pharmaceutical composition described herein further comprises a carrier. Suitable acceptable carriers include, but are not limited to, water, salt solution (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin; carbohydrates such as lactose, amylose or starch; sugars such as mannitol, sucrose or others; dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oils, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc., and combinations thereof. Pharmaceutical preparations can be mixed with auxiliary agents (e.g., diluents, buffers, lipophilic solvents, preservatives, adjuvants, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances and / or aromatic substances, etc.) that do not adversely react with the active compounds and do not interfere with the activity of the active compounds, as required. In some embodiments, a water-soluble carrier suitable for intravenous administration is used.

[0127] Pharmaceutically acceptable excipients are preferred. Non-limiting examples of such sterile solutions and methods for preparing them can be found in Gennaro, Ed., Remington's Pharmaceutical Sciences, 18 thEdition, Mack Publishing Co. (Easton, Pa.) 1990, and the like. Pharmaceutically acceptable carriers suitable for the method of administration, solubility and / or stability of the PTH variant composition can be routinely selected, as known in the art or as described herein. For example, sterile saline and phosphate buffered saline at slightly acidic or physiological pH can be used. The pH buffer can be phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, the preferred buffers being histidine, arginine, lysine, or acetate, or mixtures thereof. The preferred buffer range is pH 4-8, pH 6.5-8, and more preferably pH 7-7.5. Preservatives such as para-, meta-, and ortho-cresol, methyl and propyl paraben, phenol, benzyl alcohol, sodium benzoate, benzoic acid, benzyl benzoate, sorbic acid, propanoic acid, esters of p-hydroxybenzoic acid, etc. Stabilizers to prevent oxidation, deamidation, isomerization, racemization, cyclization, peptide hydrolysis, etc., such as ascorbic acid, methionine, tryptophan, EDTA, asparagine, lysine, arginine, glutamine, and glycine, etc. Stabilizers to prevent aggregation, fibrillation, and precipitation, such as sodium dodecyl sulfate, polyethylene glycol, carboxymethylcellulose, cyclodextrin, etc. Organic modifiers for solubilization or to prevent aggregation, such as ethanol, acetic acid or acetates and their salts, may be provided in the pharmaceutical composition. Isotonicity makers, such as salts (eg, sodium chloride), or carbohydrates (eg, dextrose, mannitol, lactose, trehalose, sucrose, or mixtures thereof), may be provided in the pharmaceutical composition.

[0128] Pharmaceutical excipients and additives useful in the present compositions include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., saccharides including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides; derivatized saccharides such as alditols, aldonic acids, esterified sugars; and polysaccharides or sugar polymers), which may be present alone or in combination and may comprise 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / PTH variant components that can also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0129] Carbohydrate excipients can be used, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; as well as alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc.

[0130] The PTH variant composition may also include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or an organic base. Exemplary buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.

[0131] Additionally, the PTH variant compositions of the invention may include polymeric excipients / additives such as polyvinylpyrrolidone, ficoll (a sugar polymer), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavorings, antimicrobials, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN® 20" and "TWEEN® 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0132] These and additional known pharmaceutical excipients and / or additives suitable for use in the PTH variant compositions according to the invention are described, for example, in "Remington: The Science & Practice of Pharmacy", vol. st ed., Williams & Williams, (2005), and “Physician's Desk Reference”, 71 st ed., Medical Economics, Montvale, NJ (2017), the disclosures of which are incorporated herein by reference in their entireties. Preferred carrier or excipient materials are salts (e.g., sodium chloride), carbohydrates (e.g., mannitol) and buffers (e.g., citrate).

[0133] The pharmaceutical composition may be formulated as a solution suitable for administration by intravenous or subcutaneous injection or infusion. The solution may contain one or more solvents. Exemplary solvents include, but are not limited to, water; alcohols such as ethanol and isopropyl alcohol; vegetable oils; polyethylene glycol; propylene glycol; and glycerin; or mixtures and combinations thereof. A water-soluble carrier suitable for intravenous administration may be used. For example, in some embodiments, a composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, the ingredients are mixed separately or together in unit dosage form and are supplied, for example, as a lyophilized powder or water-free concentrate in an airtight container such as an ampoule or sachet indicating the quantity of active agent. If the composition is administered by infusion, the composition can be dispensed in an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0134] As mentioned above, the formulation may preferably include at least one PTH variant in a pharma- ceutically acceptable formulation, a physiological saline or a suitable buffer containing a selected salt, and an optional preserved solution and formulation containing a preservative, and a versatile preserved formulation suitable for pharmaceutical or veterinary use.The preserved formulation may include at least one known preservative in an aqueous diluent, and optionally the preservative is selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl paraben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. As known in the art, any suitable concentration or mixture can be used, such as 0.001-5% or any range or value therein, for example, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1. Such as, but not limited to, 2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein.Non-limiting examples include no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0. 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0135] PTH variants can be formulated for parenteral administration, and can include, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hardened naphthalene, and the like. Aqueous or oily suspensions for injection can be prepared by using suitable emulsifiers or wetting agents and suspending agents according to known methods. Injectables can be non-toxic parenterally administrable diluents, such as aqueous solutions, or sterile injection solutions or suspensions in solvents. As usable vehicles or solvents, water, Ringer's solution, isotonic saline, and the like are acceptable; as ordinary solvents or suspension solvents, sterile non-volatile oils can be used. For these purposes, any kind of non-volatile oils and fatty acids can be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic monoglycerides or diglycerides or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, the gas-pressure needle-free injection device described in U.S. Pat. No. 5,851,198, and the laser perforation device described in U.S. Pat. No. 5,839,446.

[0136] The pharmaceutical composition may be an extended release formulation. The pharmaceutical composition may also be formulated for sustained, extended, delayed or sustained release of PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO: 8). Extended release, also known as controlled release and sustained release, may be provided in the injectable formulation. Microspheres, nanospheres, implants, depots, and polymers may be used in combination with any of the compounds, methods, and formulations described herein to provide extended release (or sustained release) profiles.

[0137] For example, the PTH variant of the present invention comprising the amino acid sequence of SEQ ID NO: 8 may be formulated at a concentration of about 0.001 to about 100 mg / mL, or about 0.005 to about 50 mg / mL, or about 0.007 to about 20 mg / mL, or about 0.01 to about 10 mg / mL, or about 0.05 to about 5.0 mg / mL, or about 0.07 to about 2.0 mg / mL, or about 0.1 to about 1.0 mg / mL. In one embodiment, the PTH variant may be formulated at a concentration of about 0.01 to about 10 mg / mL. In one embodiment, the PTH variant may be formulated at a concentration of about 0.1 to about 1.0 mg / mL.

[0138] Formulations and compositions comprising PTH variants may optionally further comprise an effective amount of at least one compound or protein selected from at least one of diabetes or insulin metabolism related drugs, anti-infective drugs, cardiovascular (CV) system drugs, central nervous system (CNS) drugs, autonomic nervous system (ANS) drugs, respiratory drugs, gastrointestinal (GI) tract drugs, hormonal drugs, drugs for fluid or electrolyte balance, hematological drugs, anti-tumor drugs, immunomodulatory drugs, ophthalmic drugs, otologic drugs, topical drugs, nutritional drugs, etc. Such drugs are well known in the art, including the respective formulations, indications, dosing and administration presented herein (see, e.g., Nursing 2001 Handbook of Drugs, 21st Edition, each of which is incorporated herein by reference in its entirety). stedition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmacotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT).

[0139] PTH variants can also be formulated as sustained release implantation devices for long-term or sustained administration of PTH variants. Such sustained release formulations can be in the form of patches placed on the outside of the body. Examples of sustained release formulations include composites of biocompatible polymers such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, sialic acid, silicates, collagen, liposomes, etc. Sustained release formulations can be particularly interesting when it is desirable to provide high local concentrations of PTH variants.

[0140] PTH variant compositions and formulations can be provided to patients as clear solutions or as dual vials containing a vial of at least one lyophilized PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO:8) or specific portion or variant to be reconstituted and a second vial containing an aqueous diluent. The single solution vial or the dual vial requiring reconstitution can be reused multiple times and is sufficient for single or multiple cycle treatment of the patient, thus providing a more convenient treatment regimen than currently available.

[0141] Compositions and formulations of PTH variants can be provided indirectly to patients by providing a pharmacy, clinic, or other such facility and equipment with a clear solution or a dual vial containing a vial of at least one lyophilized PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO: 8) or specific portion or variant to be reconstituted and a second vial containing an aqueous diluent. In this case, the clear solution may be up to a liter or more in size, providing a large reservoir from which smaller portions of the solution of PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO: 8) or specific portion or variant can be withdrawn one or more times and transferred to smaller vials for provision to customers and / or patients from the pharmacy or clinic. Such products can include packaging materials. The packaging materials can provide the conditions under which the product can be used, in addition to information required by regulatory authorities. For a two-vial wet / dry product, the packaging material can provide instructions to the patient to reconstitute the PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO:8) or specified portion or variant with an aqueous diluent to form a solution and use the solution for 2-24 hours or more.

[0142] treatment In another embodiment, a method is provided for treating a subject with hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism, comprising treating the subject with a PTH variant of the present invention using a dosing regimen effective to control the subject's serum and urinary calcium levels. The PTH variants may be particularly effective in treating hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism because they have a longer half-life than native PTH(1-84). The longer half-life allows for daily administration and maintenance of the PTH variant at more physiologically relevant levels, allowing for more sustained control over both serum and urinary calcium.

[0143] In some embodiments, the method is effective in controlling the serum calcium level of a subject. In one embodiment, the method is effective in increasing the serum calcium level of a subject with hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism, compared to the serum calcium level of said subject in the absence of administration of a PTH variant of the invention. In one embodiment, the method is effective in maintaining a lower peak-to-trough ratio of serum calcium in a subject, compared to the serum calcium level of a subject receiving exogenous native PTH(1-84). In one embodiment, the method is effective in reducing the amount of calcium supplements in a subject with hypoparathyroidism.

[0144] In some embodiments, the method is effective in controlling urinary calcium secretion in a subject, hi one embodiment, the method is effective in reducing urinary calcium excretion to the level of healthy volunteers (reducing potential renal complications).

[0145] For example, the PTH variant comprising the amino acid sequence of SEQ ID NO:8 may be administered subcutaneously or intravenously. In various embodiments, multiple administrations are administered according to a dosing regimen. As used herein, the term "QD" or "qd" refers to administration once a day, "Q2D" refers to administration every 2 days, etc. "QW" refers to weekly (once a week) administration. "BID" refers to administration twice a day. Dosing may be, for example, BID, once a day (QD), Q2D, Q3D, Q4D, Q5D, Q6D, QW, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 2 weeks, once every 15 days, once every 16 days, or once every 17 days, once every 3 weeks, or once a month.

[0146] A PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO: 8) may be administered subcutaneously at between 0.001 μg and 1,000 μg twice daily, or once daily, or every other day, or every 5-8 days, or weekly (QW) according to a dosing regimen. Alternatively, the PTH variant may be administered every 3 weeks or once a month, such as for maintenance purposes.

[0147] PTH variants (e.g., comprising the amino acid sequence of SEQ ID NO: 8) may be administered subcutaneously according to a dosing regimen of about 1 μg / day to about 500 μg / day, or about 2 μg / day to about 250 μg / day, or about 5 μg / day to about 100 μg / day, or about 10 μg / day to about 80 μg / day, or about 20 μg / day to about 100 μg / day, or about 50 μg, or about 60 μg, or about 70 μg, or about 80 μg, or about 90 μg, or about 100 μg once daily (QD). PTH variants (e.g., comprising the amino acid sequence of SEQ ID NO: 8) may be administered at a concentration of 0.001 to 1,000 μg / mL.

[0148] The above dosing regimens may be administered for 1 month, or 2 months, or 6 months, or 1 year, or 2 years, or 5 years, or longer than 5 years to treat hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism. The PTH variant may be administered for the life of the subject for maintenance.

[0149] As used herein, the term "subcutaneous tissue" is defined as a layer of loose irregular connective tissue just below the skin. For example, subcutaneous administration may be performed by injecting the composition into areas including, but not limited to, the thigh, abdomen, buttocks, or shoulder blade. For such purposes, the formulation may be injected using a syringe. However, other devices for administration of the formulation are also available, such as injection devices (e.g., Inject-ease™ device and Genject™ device), injection pens (such as GenPen™), needleless devices (e.g., MediJector™ and BioJector™), and subcutaneous patch delivery systems. In some embodiments, the PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO: 8), or a pharmaceutical composition comprising the same, is administered intravenously.

[0150] In various embodiments, the above methods of treating hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism are used in combination with other methods of treating hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism. EXAMPLES

[0151] The following examples illustrate certain aspects of the present disclosure. The examples are merely intended to provide a more specific understanding and practice of the embodiments and their various aspects, and should not be construed as limiting.

[0152] Example 1: Production and purification of Fc-fused PTH variants (exemplified by PTH-66) PTH-66 was transiently expressed in CHO cells and the conditioned media was stored at -20°C until further use. The conditioned media was thawed in a 25°C water bath and filtered through a 0.2 μm bottle-top filter unit. PTH-66 protein was purified with Protein A derived resin MabSelect SuRe according to the manufacturer's protocol (Figure 8). The MabSelect SuRe column was pre-equilibrated with PBS. The thawed conditioned media was loaded onto the column and then washed extensively with PBS. PTH-66 protein was eluted by stepwise elution with 100 mM sodium citrate buffer pH 3.2. The eluted fractions were analyzed by SDS-PAGE (Figure 9). The pH of the pooled eluted fractions was brought to approximately pH 6 with 1 M Tris pH 10. The neutralized pooled elution fractions were dialyzed into PTH-66 storage buffer (20 mM sodium phosphate buffer, 50 mM sodium chloride, 26 mg / ml mannitol, pH 6.0) and stored at -80°C.

[0153] Figure 8A shows a chromatographic purification trace of approximately 900 mL of CM expressing PTH-66 using 2x5 mL MabSelect Sure columns, and Figure 8B is a flow chart outlining the purification steps of PTH-66 according to an embodiment of the invention.

[0154] Figure 9A shows Mab-selected crude elution fractions of PTH-66, and Figure 9B shows Coomassie blue staining of Mab-selected crude elution fractions of PTH-66.

[0155] Figure 10 shows the SDS-PAGE Coomassie stain of the final PTH-66 product. The final yield was approximately 75 mg / 900 mL cell culture medium (CM). The protein is at 3 mg / mL in PTH storage buffer. The protein was aliquoted and stored at -80°C.

[0156] Example 2: Effect of C-terminal truncation of PTH-Fc fusions PTH-Fc fusion variants containing various lengths of truncated PTH were prepared according to the methodology outlined in Example 1. The following PTH-Fc fusion variants were generated: PTH-66 (PTH(1-74)-Fc), PTH-67 (PTH(1-64)-Fc), PTH-68 (PTH(1-54)-Fc), PTH-69 (PTH(1-44)-Fc), and PTH(1-34)-Fc.

[0157] Serum concentrations of PTH after a single subcutaneous dose of 0.25 mg / kg with truncated variants and full-length PTH(1-84)-Fc in Spraque-Dawley rats were assessed using the Immunotopics bioactivity ELISA kit (60-3000) and the results are shown in Figure 1.

[0158] As shown in FIG. 1, C-terminal truncation of PTH surprisingly and unexpectedly improves the exposure and pharmacokinetics of PTH-Fc fusions.

[0159] As the figure shows, the truncated PTH-Fc fusion variants of the invention are superior to both native-length PTH(1-84)-Fc fusions and teriparatide-Fc (PTH(1-34)-Fc fusions). Even more surprisingly, PTH(1-74) has a particularly long half-life in vivo compared to native-length PTH(1-84)-Fc and other analogs.

[0160] Example 3: PTH-66 in normal mice PTH-66 was administered to normal mice at a dose of 50 nmol / kg. Samples were taken at 2, 8, 24, 48, and 72 hours after administration and calcium concentrations were plotted and compared to phosphate buffer (vehicle) controls.

[0161] Figure 2 shows serum calcium levels (Figure 2A) and urinary calcium levels (Figure 2B) as a function of time after administration of PTH-66 (50 nmol / kg dose) in mice. As shown in Figure 2A, PTH-66 induces a prolonged increase in serum calcium compared to PTH(1-84) and vehicle controls. As shown in Figure 2B, PTH-66 shows longer maintenance of urinary calcium levels than PTH(1-84). PTH(1-84) treatment results in a spike in urinary calcium within the first few hours, consistent with elevated serum calcium and transient exposure of the molecule. In contrast, despite elevated serum calcium levels, PTH-66 shows more long-term control of urinary calcium levels, maintaining levels close to vehicle for 12-24 hours. In summary, treatment with PTH-66 produces a sustained effect on calcium levels.

[0162] Example 4: PTH-66 in TxPTx rats Normal (intact) and thyroparathyroidectomized (TxPTx) rats were administered PTH-66 at various doses of 5, 20, and 60 nmol / kg. Serial samples were taken at 1, 6, 12, 18, 24, 36, 48, and 72 hours post-dose and compared with vehicle controls.

[0163] As shown in Figures 3A and 3B, TxPTx rats treated with PTH-66 exhibit a dose-linear PK / PD response with dose-dependent serum Ca++ levels that are elevated and maintained up to 72 hours post-dose. Figure 3B shows that TxPTx rats administered 5 nmol / kg PTH-66 have normalized serum calcium levels that return to those of intact rats, whereas treatment with 20 and 60 nmol / kg PTH-66 exhibits higher serum calcium levels than intact rats that received placebo vehicle at 12-48 hours post-dose.

[0164] Figure 4 (AB) shows single dose PK (Figure 4A) and PD (Figure 4B) study data from TPTx rats comparing PTH-66 with PTH(1-84). The TPTx rat data show clear PK prolongation over PTH(1-84) and demonstrate that 5 nmol / kg PTH-66 is able to return serum calcium to normal levels, in contrast to PTH(1-84), which shows only a very transient increase in serum calcium.

[0165] In another multiple-dose study, normal (intact) and thyroparathyroidectomized (TxPTx) rats were given PTH-66 subcutaneously at doses of 2 and 4 nmol / kg daily for 10 days. Samples were taken once daily before dosing and compared with vehicle controls.

[0166] Figure 5 (A-D) shows multiple dose PK (Figure 5A) and PD (Figure 5B-D) study data from daily subcutaneous administration of PTH-66 to TPTx rats. The PK data in Figure 5A shows a dose-linear PK response where near steady state levels of PTH-66 were achieved. As shown in Figure 5B, 2 and 4 nmol / kg PTH-66 return and maintain serum calcium in the normal range (intact) levels. Figure 5C shows that daily administration of PTH-66 results in normalization of serum phosphate levels. Figure 5D shows that while PTH-66 maintains normal serum calcium levels, the molecule also maintains normal urinary calcium levels, as shown in Figure 5A. These data show that daily subcutaneous administration of PTH-66 results in complete normalization of serum calcium and phosphate, and importantly, urinary calcium.

[0167] Example 5: PTH-66 in primates Wild-type cynomolgus monkeys were given a single dose of PTH-66 at a dosage of 2 mmol / kg, while other monkeys were given a single dose of Natpara (PTH(1-84)) at 5 mmol / kg. Serial samples were taken at 0, 1, 3, 6, 9, 12, 18, 24, 36, 48, and 72 hours post-dose and compared with Natpara and vehicle controls.

[0168] FIG. 6(AB) shows single-dose PK (FIG. 6A) and PD (FIG. 6B) study data in wild-type cynomolgus monkeys comparing PTH-66 with PTH(1-84).

[0169] The data in Figure 6 show a clear PK prolongation of PTH-66 over Natpara, indicating that 2 nmol / kg PTH-66 can stimulate and maintain serum calcium levels above those achieved with PTH(1-84). Compared to PTH-66, Natpara has a short-lived and more transient effect on serum calcium.

[0170] In another multiple-dose study, PTH-66 was administered subcutaneously to wild-type cynomolgus monkeys at a dose of 1 nmol / kg daily for 9 days. Samples were taken daily before dosing and 24, 48, 72, and 120 hours after the last dose and compared with vehicle controls.

[0171] Figure 7 shows multiple dose PK and PD study data for daily subcutaneous administration of 1 nmol / kg PTH-66 in wild type cynomolgus monkeys. The PK data show that a relatively flat PK profile of PTH-66 was achieved, resulting in increased serum calcium and decreased serum phosphate over the study period compared to vehicle controls.

[0172] Example 6: PTH cAMP Potency Assay Table 1 shows the cAMP potency (EC 50 ) assay results are summarized. Table 2 summarizes the PK data for the PTH variants.

[0173] [Table 1]

[0174] [Table 2]

[0175] As shown in Tables 1 and 2, all PTH-Fc samples had subnanomolar EC 50S It has activity at 100 mg / kg / day and has significantly improved PK properties over PTH(1-84).

[0176] Example 6: Multimodal chromatography in the purification of PTH-66 Protein A chromatography as the first capture step was performed according to the downstream platform shown in Figure 11. The eluate was kept at a low pH of 3.5 for 1 h for virus inactivation, then neutralized to pH 6 and frozen at -80 °C. Most of the impurities were sufficiently removed except for clipping species and host cell proteins. The polishing step described below was developed to remove residual host cell and process impurities.

[0177] The application of multimodal (also known as mixed-mode) chromatography is growing rapidly in antibody purification where media allow separation of compounds with high efficiency through various interactions. In downstream polishing development, two multimodal column steps were considered: cationic Capto™ MMC ImpRes and anionic Capto™ Adhere ImpRes. However, given the complex media-specific interactions, the purification process is determined by the buffer components, ionic strength, pH, and salt type. As optimal binding and elution conditions are difficult to estimate, the separation conditions for these two resins were determined through the steps described below to achieve optimal process performance.

[0178] PTH-66 (PTH(1-74)-Fc fusion) undergoes moderate fragmentation from the bioreactor, which may adversely affect its potency despite its increased circulating half-life. Addition of protease inhibitors to harvested cell cultures prior to downstream processing has been shown to prevent future product degradation. Fragmentation of recombinant proteins in cell culture may adversely affect drug safety and efficacy. In downstream process development, hydrophobic or charge-altered fragment isoforms resulting from the upstream process were successfully separated from the intact Fc fusion protein using multimodal chromatography. A flowchart of the manufacturing process achieving a fragmentation goal of less than 10% is shown in Figure 12.

[0179] Extensive high-throughput screening (HTS) of initial operating conditions for Capto MMC and Adhere ImpRes was performed using 6 μL PreDictor 96-well plates to evaluate a wide range of parameters, including salt concentration and pH (see Figures 13A and 13B).

[0180] Separation conditions for cation exchange (cation) Capto MMC ImpRes and anion exchange (anion) Capto Adhere ImpRes determined using the high-throughput process are shown in Figures 14A and 14B. Screened experimental conditions for Capto MMC ImpRes include acetate, MES, and phosphate buffer systems at pH 5.0-7.0 and salt (NaCl) concentrations of 0-750 mM, with a 50 g / L resin load and 1 hour incubation time. Screened experimental conditions for Capto Adhere ImpRes include acetate, MES, phosphate, and Tris buffer systems at pH 5.0-8.0 and salt (NaCl) concentrations of 0-1500 mM, with a 50 g / L resin load and 1 hour incubation time.

[0181] The optimal binding conditions for Capto MMC ImpRes, a cationic resin with the structure shown in Figure 14C, were determined to include 50 mM MES buffer at pH 6.0 without salt. The optimal flow-through conditions for Capto Adhere ImpRes, an anionic resin with the structure shown in Figure 14D, were determined to include 50 mM acetate buffer at pH 5.0 without salt.

[0182] The second step is to optimize the process at bench scale accelerated by the HTS static binding capacity results. Capto MMC ImpRes was run in bind and elute mode to maximize impurity clearance capacity. Wash steps were considered to reduce excess process- and product-related impurities without unnecessary product loss (Figure 15). The subsequent Capto Adhere ImpRes step was developed as a fast flow-through (FT) step that allows the process to meet product quality criteria (Figure 16). Bench-top confirmation experiments of MMC and Adhere were performed to verify the robustness of the process in terms of impurity clearance and step yields. Quantification of PTH-Fc relies on UV280 to track MMC and Adhere yields.

[0183] Conditions from the high-throughput studies were successfully transferred to bench scale, as shown in Figures 17-19. The Capto MMC ImpRes gradient elution trace is shown in Figure 14A. Better resolution was achieved with 50 mM MES, pH 6.0, 0-1 M NaCl gradient elution on a 0.66x10 cm (3.4 mL) column. MMC effectively results in removal of HCPs, DNA, HMW, and clipping species.

[0184] [Table 3]

[0185] An MMC wash step was considered to reduce other process-related and product-related impurities. The optimization of the MMC wash is shown in Table 4. The optimal wash conditions were determined to be 6 CV of 200 mM NaCl and 3 CV of 250 mM NaCl.

[0186] [Table 4]

[0187] The Capto Adhere ImpRes flow-through shown in Figure 17B and shown below in Table 5, demonstrates that it provides additional HCP and HMW removal capacity. Better resolution was achieved with the flow-through using 50 mM sodium acetate buffer at pH 5.0 on a 0.66x10 cm (3.4 mL) column.

[0188] [Table 5]

[0189] The design of the downstream polishing step is shown in Figure 18A. The robustness of the process with stable pools based on impurity clearance and process yield is shown in Figure 15B and Table 6 below.

[0190] [Table 6]

[0191] The product quality characteristics at pilot scale are shown in Figures 19A-19F. As shown in these figures, the step yields from the large-scale purification, pilot plant operation (PPO), are comparable to the yields from the small-scale lab-scale, process development (PD). By comparing these two yields, it can be concluded that the purification process was successfully scaled up using the above parameters.

[0192] The stability of in-process samples was determined by holding the MMC eluate and Adhere FT at ambient temperature and 4-8°C for 5 and 4 days, respectively, followed by analysis by SEC-HPLC and intact mass analysis.

[0193] Therefore, the issue of fragmentation of Fc fusion proteins was successfully addressed using multimodal chromatography, where a step elution from a linear gradient was established that achieves relatively high recovery without compromising robust impurity clearance.

[0194] Since various changes may be made to the above subject matter without departing from the scope and spirit of the invention, it is intended that all subject matter contained in the above description or defined in the appended claims be construed as illustrative and exemplary of the invention. Numerous modifications and variations of the invention are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variations that are within the scope of the appended claims.

[0195] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein. Finally, preferred embodiments of the present invention are described in sections. [Embodiment 1] A parathyroid hormone (PTH)-Fc fusion protein comprising a C-terminal mutated and / or truncated PTH or its variant chemically linked to the Fc region of a human IgG antibody or a derivative thereof, wherein the C-terminal mutated and / or truncated PTH or its variant is not PTH(1-34). [Embodiment 2] 2. The PTH-Fc fusion protein of embodiment 1, wherein the C-terminal mutated and / or truncated PTH or variant thereof is selected from mutant PTH(1-84), PTH(1-74), PTH(1-64), PTH(1-54), and PTH(1-44). [Embodiment 3] The C-terminal mutated and / or truncated PTH or variant thereof is mutated PTH(1-84), PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH( 1-73), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH(1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1-61), or PTH(1-60), or PTH (1-59), or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH(1-48), or PTH(1-47), or PTH(1-46), or PT 2. The PTH-Fc fusion protein of embodiment 1, selected from PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH(1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1-36), or PTH(1-35), or PTH(1-33). [Embodiment 4] 4. The PTH-Fc fusion protein according to any one of embodiments 1 to 3, wherein the C-terminal mutated and / or truncated PTH or variant thereof is PTH(1-74). [Embodiment 5] 5. The PTH-Fc fusion protein according to any one of embodiments 1 to 4, wherein the Fc region or derivative thereof is hFcLALA comprising the L234A and L235A substitutions. [Embodiment 6] 6. The PTH-Fc fusion protein of any one of embodiments 1 to 5, wherein the PTH or variant thereof comprises an F34A substitution, an F34D substitution, a V35S substitution, or a V35T substitution, or a combination thereof. [Embodiment 7] 7. The PTH-Fc fusion protein according to any one of embodiments 1 to 6, wherein the PTH or variant thereof is glycosylated. [Embodiment 8] 8. The PTH-Fc fusion protein according to any one of embodiments 1 to 7, which is PTH-66 having the amino acid sequence of SEQ ID NO:8. [Embodiment 9] 8. The PTH-Fc fusion protein according to any one of embodiments 1 to 7, which is PTH-67 having the amino acid sequence of SEQ ID NO:9. [Embodiment 10] 8. The PTH-Fc fusion protein according to any one of embodiments 1 to 7, which is PTH-68 having the amino acid sequence of SEQ ID NO:10. [Embodiment 11] 8. The PTH-Fc fusion protein according to any one of embodiments 1 to 7, which is PTH-69 having the amino acid sequence of SEQ ID NO:11. [Embodiment 12] A nucleic acid encoding the PTH-Fc fusion protein according to any one of embodiments 1 to 11. [Embodiment 13] An expression vector comprising the nucleic acid of embodiment 12. [Embodiment 14] A host cell comprising a nucleic acid according to embodiment 12 or an expression vector according to embodiment 13. [Embodiment 15] 15. The host cell of embodiment 14, wherein the host cell is a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell. [Embodiment 16] 16. The host cell of embodiment 14 or 15, wherein the host cell is a CHO cell. [Embodiment 17] 12. A PTH-Fc fusion protein according to any one of embodiments 1 to 11, which has a serum half-life longer than the serum half-life of PTH(1-84). [Embodiment 18] 12. A PTH-Fc fusion protein according to any of embodiments 1 to 11, which has a serum half-life at least two-fold longer than the serum half-life of PTH(1-84). [Embodiment 19] 12. A PTH-Fc fusion protein according to any of embodiments 1 to 11, which has a serum half-life at least 10 times longer than the serum half-life of PTH(1-84). [Embodiment 20] 12. A PTH-Fc fusion protein according to any of embodiments 1 to 11, which has a serum half-life at least 20 times longer than the serum half-life of PTH(1-84). [Embodiment 21] A pharmaceutical composition comprising the PTH-Fc fusion protein according to any one of embodiments 1 to 11 or 17 to 20 and a pharma- ceutically acceptable carrier. [Embodiment 22] A pharmaceutical formulation comprising a PTH-Fc fusion protein according to any one of embodiments 1 to 11 or 17 to 20, or a pharmaceutical composition according to embodiment 21. [Embodiment 23] 23. The pharmaceutical formulation of embodiment 22, which is a liquid dosage form suitable for administration by injection or infusion. [Embodiment 24] A method for controlling serum calcium levels in a subject in need thereof, comprising administering to said subject an effective amount of a PTH-Fc fusion protein of embodiments 1 to 11 or 17 to 20, a pharmaceutical composition of embodiment 21, or a pharmaceutical formulation of embodiment 22 or 23. [Embodiment 25] 25. The method of embodiment 24, wherein the subject has hypoparathyroidism. [Embodiment 26] 26. The method of embodiment 24 or 25, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is administered subcutaneously. [Embodiment 27] 27. The method according to any one of embodiments 24 to 26, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is administered once a day. [Embodiment 28] 28. The method according to any one of embodiments 24 to 27, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is administered once a week. [Embodiment 29] The method according to any one of embodiments 24 to 28, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is provided to the subject in an amount of about 20 μg / day to about 100 μg / day of PTH-Fc fusion protein. [Embodiment 30] A method for treating a subject having hypoparathyroidism, comprising administering to the subject an effective amount of a PTH-Fc fusion protein of embodiments 1 to 11 or 17 to 20, a pharmaceutical composition of embodiment 21, or a pharmaceutical formulation of embodiment 22 or 23. [Embodiment 31] The method of embodiment 30, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is administered subcutaneously. [Embodiment 32] 32. The method of embodiment 30 or 31, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is administered once a day. [Embodiment 33] The method according to any one of embodiments 30 to 32, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is provided to the subject in an amount of about 20 μg / day to about 100 μg / day of PTH-Fc fusion protein. [Embodiment 34] A method for treating a subject having hypocalcemia due to hypoparathyroidism, comprising administering to said subject an effective amount of a PTH-Fc fusion protein of embodiments 1 to 11 or 17 to 20, a pharmaceutical composition of embodiment 21, or a pharmaceutical formulation of embodiment 22 or 23. [Embodiment 35] The method of embodiment 34, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is administered subcutaneously. [Embodiment 36] The method of embodiment 34 or 35, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is administered once a day. [Embodiment 37] The method according to any one of embodiments 34 to 36, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical formulation is provided to the subject in an amount of about 20 μg / day to about 100 μg / day of PTH-Fc fusion protein. [Embodiment 38] A PTH-Fc fusion protein according to any of embodiments 1 to 11 or 17 to 20, a pharmaceutical composition according to embodiment 21, or a pharmaceutical formulation according to embodiment 22 or 23, wherein the PTH-Fc fusion protein comprises one copy of PTH and one copy of Fc. [Embodiment 39] A PTH-Fc fusion protein according to any of embodiments 1 to 11 or 17 to 20, a pharmaceutical composition according to embodiment 21, or a pharmaceutical formulation according to embodiment 22 or 23, wherein the PTH-Fc fusion protein comprises two copies of PTH and one copy of Fc. [Embodiment 40] 1. A method for preparing a C-terminally truncated parathyroid hormone (PTH)-Fc fusion protein, or a variant thereof, including PTH(1-74), comprising purifying said (PTH)-Fc fusion protein using a multimodal chromatography resin. [Embodiment 41] 41. The method of embodiment 40, wherein the multimodal chromatography resin comprises a cation exchange resin and an anion exchange resin. [Embodiment 42] 42. The method of embodiment 41, wherein the cation exchange resin is Capto MMC ImpRes. [Embodiment 43] The method of embodiment 42, wherein the (PTH)-Fc fusion protein is bound in 50 mM MES buffer at pH 6.0. [Embodiment 44] The method of embodiment 41, wherein the anion exchange resin is Capto Adhere ImpRes. [Embodiment 45] The method of embodiment 44, wherein the (PTH)-Fc fusion protein is eluted in 50 mM acetate buffer at pH 5.0. [Embodiment 46] 46. ​​The method of any one of embodiments 40 to 45, further comprising gradually eluting the (PTH)-Fc fusion protein from the multimodal chromatography resin. [Embodiment 47] 47. The method according to any one of embodiments 40 to 46, wherein the purified (PTH)-Fc fusion protein contains less than 10% total fragmentation impurities. 。

Claims

1. A parathyroid hormone (PTH)-Fc fusion protein comprising PTH chemically linked to the Fc region of a human IgG antibody or a derivative thereof comprising L234A and L235A substitutions, said PTH being a mutant or mutated and C-terminally truncated PTH comprising amino acid substitutions only at positions 34 and 35, said amino acid substitutions being F34A or F34D, and V35S or V35T, and said PTH is not PTH(1-34).

2. 2. The PTH-Fc fusion protein of claim 1, wherein the PTH is selected from mutant PTH(1-84), PTH(1-74), PTH(1-64), PTH(1-54), and PTH(1-44).

3. The PTH is a mutant PTH(1-84), PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH(1-73), or PTH(1-7 2), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH(1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1-61), or PTH(1-60), or PTH(1-59) , or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH(1-48), or PTH(1-47), or PTH(1-46), or or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH(1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1-36), or PTH(1-35).

4. The PTH-Fc fusion protein according to any one of claims 1 to 3, wherein the PTH is PTH(1-74).

5. The PTH-Fc fusion protein of any one of claims 1 to 4, wherein the Fc region or derivative thereof is hFcLALA, which contains L234A and L235A substitutions.

6. The PTH-Fc fusion protein of any one of claims 1 to 5, wherein the PTH is glycosylated.

7. The PTH-Fc fusion protein of any one of claims 1 to 6, which is PTH-66 having the amino acid sequence of SEQ ID NO:8, PTH-67 having the amino acid sequence of SEQ ID NO:9, PTH-68 having the amino acid sequence of SEQ ID NO:10, or PTH-69 having the amino acid sequence of SEQ ID NO:

11.

8. A nucleic acid encoding the PTH-Fc fusion protein of any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid of claim 8.

10. A host cell comprising the nucleic acid of claim 8 or the expression vector of claim 9.

11. The host cell of claim 10 , wherein the host cell is a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell.

12. The host cell of claim 10 or 11, wherein the host cell is a CHO cell.

13. The PTH-Fc fusion protein of any one of claims 1 to 7, which has a serum half-life longer than the serum half-life of wild-type PTH (1-84).

14. The PTH-Fc fusion protein of any one of claims 1 to 7, which has a serum half-life at least two times longer than the serum half-life of wild-type PTH (1-84).

15. The PTH-Fc fusion protein of any one of claims 1 to 7, which has a serum half-life at least 10 times longer than the serum half-life of wild-type PTH (1-84).

16. The PTH-Fc fusion protein of any one of claims 1 to 7, which has a serum half-life at least 20 times longer than the serum half-life of wild-type PTH (1-84).

17. A PTH-Fc fusion protein according to any one of claims 1 to 7 and 13 to 16, comprising one copy of PTH and one copy of Fc, or comprising two copies of PTH and one copy of Fc.

18. A pharmaceutical composition comprising the PTH-Fc fusion protein of any one of claims 1 to 7 and 13 to 17 and a pharma- ceutically acceptable carrier.

19. A pharmaceutical formulation comprising a PTH-Fc fusion protein according to any one of claims 1 to 7 and 13 to 17, or a pharmaceutical composition according to claim 18.

20. 20. The pharmaceutical formulation of claim 19, which is in a liquid dosage form suitable for administration by injection or infusion.

21. A composition for controlling serum calcium levels in a subject in need thereof, comprising a PTH-Fc fusion protein according to any one of claims 1 to 7 and 13 to 17.

22. 22. The composition of claim 21, wherein the subject has hypoparathyroidism.

23. A composition for treating a subject having hypoparathyroidism, comprising a PTH-Fc fusion protein according to any one of claims 1 to 7 and 13 to 17.

24. 20. A composition for treating a subject suffering from hypocalcemia due to hypoparathyroidism, comprising a PTH-Fc fusion protein according to any one of claims 1 to 7 and 13 to 17.

25. A composition according to any one of claims 18 and 21 to 24, or a pharmaceutical formulation according to claim 19 or 20, which is administered subcutaneously.

26. A composition according to any one of claims 18 and 21 to 25, or a pharmaceutical formulation according to claim 19 or 20, which is administered once daily or once weekly.

27. The composition of any one of claims 18 and 21 to 26, or the pharmaceutical formulation of claim 19 or 20, provided to a subject in an amount of 20 μg / day to 100 μg / day of PTH-Fc fusion protein.

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