Growth hormone receptor antagonists, fusion proteins, and uses thereof

Abstract

Growth hormone receptor antagonists (GHAs), including polypeptides and fusion proteins, as well as targeting and protease-activated domains, comprising one or more mutations at site 2 and additional modifications at site 1, of a growth hormone. The GHAs, including compositions and formulations thereof, can be administered by various routes, alone or in combination with other agents, in targeted therapies directed to oncological, metabolic, vascular, endocrine, and other disorders, as well as in other therapeutic and diagnostic applications.

Description

[0001] Attorney Docket No. 1362.0003-1WO

[0002] GROWTH HORMONE RECEPTOR ANTAGONISTS, FUSION PROTEINS, AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority under 35 U. S. C. § 119(e) and Article 8 of the Patent Cooperation Treaty (PCT) to U. S. Provisional Application No. 63 / 776,713, filed March 24, 2025, the entire contents of which are hereby incorporated by reference in their entirety.

[0005] FIELD

[0006] Tire present disclosure relates to growth hormone receptor antagonists, including modified growth hormone polypeptides and fusion proteins; targeted compositions and fonnulations comprising such antagonists; and their use in therapies directed to endocrine, oncological, vascular, and metabolic disorders, as well other in other medical, veterinary, and diagnostic applications.

[0007] BACKGROUND

[0008] Growth hormone (GH), also called somatotropin, is a 22 kilodalton protein produced and released into the bloodstream by the anterior pituitary gland. A key endocrine regulator, GH exerts systemic effects on distant targets such as the liver, w here it stimulates release of insulin-like growth factor-1 (IGF-1), which further promotes growth throughout the body. Okada et al. 2001, Trends Mol. Med. 7, 126-132. In addition to its endocrine role, GH can play autocrine and paracrine roles via local expression in

[0009] extra-pituitary tissues such as immune, neural, reproductive, muscular, alimentary, skeletal, respiratory, visual, and cardiovascular cells. Perry et al. 2013, Curr. Opin. Endocrinol. Diabetes Obes. 20. 307-313.

[0010] GH exerts its effects via the growth hormone receptor (GHR), which shows w ide expression in body tissues, including liver, immune, nerve, reproductive, bone, kidney, muscle, adipose, and lung cells. The GHR is a type I cytokine receptor that lacks intrinsic kinase activity and is activated via recruitment of cytosolic domain-associated kinases such as Janus kinase 2 (JAK2) to transduce signals into tire cell. See, e.g., Dehkhoda et al. 2018, Front Endocrinol (Lausanne) 9, 35; Strous et al. 2020, Front Endocrinol (Lausanne) 11, 597573. Recruitment depends on GH binding, via site 1 and site 2, to the GHR, triggering confirmational changes that allow JAK2 activation. Brown, et al. 2005, Nat. Struct. Mol. Biol. 12. 814-821. Activated JAK2 kinase can then activate downstream signaling cascades, including JAK-STAT, Ras-ERK, and PI3K-Akt pathways. Waters et al. 2006, J. Mol. Endocrinol. 36, 1-7.

[0011] The biological effects of GH are tissue specific and variable, depending on the targeted region. For example, human GH (hGH) stimulates anabolic effects in muscle and bone tissues and catabolic effectsAttorney Docket No. 1362.0003-1WO

[0012] in adipose tissue. Healy et al. 2003, J. Clin. Endocrinol. Metab. 88, 5521-5526; Moller and Jorgensen, 2009, Endocr Rev. 30, 152-177. Excessive or unregulated GH expression is implicated in disease pathology. See Lu et al. 2019, Signal Transduct. Target Ther. 4, 3. Chronic excessive secretion of GH, arising for example from a GH-secreting pituitary adenoma, leads to acromegaly - a serious condition marked by abnormal skeletal growth, soft tissue expansion, and increased risk of cardiovascular and gastrointestinal problems. Abnormal GH levels have also been linked to cancer. See Wang et al. 2023. Endocr. Relat. Cancer 30, e230033. High GH levels can accelerate tumorigenesis through mechanisms such as cell proliferation, transformation, angiogenesis, and lymphangiogenesis in multiple tumor types, including breast, endometrial, and hepatocellular carcinomas, and lung, prostate, and colon cancers. In addition, elevated GH levels can contribute to diabetes mellitus and associated microvascular complications such as retinopathy and nephropathy. Strous et al. 2020, Front Endocrinol (Lausanne) 11, 597573. Consistent with these observations, individuals with GH resistance, such as in Laron syndrome - a rare genetic condition resulting in GHR inactivation - show protection from cancer and diabetes. Laron et al. 2017, Mutat. Res. Rev. Mutat. Res. 772, 123-133.

[0013] These observations highlight therapeutic strategies focused on blocking GH release or inhibiting GHR activation. Current treatment options are limited to pegvisomant, a PEGylated analog of GH and the only clinically approved GHR antagonist, which is marketed worldwide to treat patients with acromegaly and, in some cases, gigantism. See, e.g., Parkinson et al. 2003, Adv. Drug Deliv. Res. 55, 1303-1314; Freda et al. 2015, Endocr. Pract. 21. 264-274. However, pegvisomant has several limitations, such as the requirement for daily subcutaneous injections, reduced GHR affinity, manufacturing complexities necessitated by PEGylation, potential immune responses, hypersensitivity, and long-term PEG accumulation in tissues such as the liver and kidneys. More generally, the sole therapeutic focus of pegvisomant on acromegaly may limit its suitability to other diseases and conditions, where complicated endocrine, autocrine, and paracrine functions of GH may necessitate more tailored therapeutics.

[0014] Accordingly, there is a need for improved GHR antagonists (GHAs) to address therapeutic limitations. The present disclosure addresses these and other needs by providing mutated GH variants and fusion proteins thereof, including targeting and protease activation domains, as well as tailored formulations, offering improved pharmacokinetics, stability, and therapeutic versatility to provide enhanced efficacy, reduced dosing frequency, and broader applicability to GH- and GHR-related disorders.

[0015] SUMMARY

[0016] Provided are growth hormone antagonists (GHAs), including polypeptides and fusion proteins thereof, compositions comprising or encoding GHAs, and their use in numerous methods, including therapeutic, veterinary, and diagnostic applications.

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[0018] In embodiments, GHAs correspond to Set One GHAs, which comprise mutations at one or more positions in a growth hormone, specifically targeting residues interacting at an interface (site 2) between the GH and growth hormone receptor (GHR), wherein the targeted mutations disrupt GH binding. In embodiments, the site 2 mutations include a modification to one or more residues at positions 8, 12, 16, 103, and 119 of a human GH, or at corresponding positions of other GHs, as described further herein. A GHA with one or more site 2 mutations can further comprise mutations that specifically target residues interacting at an interface (site 1) between the GH and grow th hormone receptor (GHR), wherein the targeted mutations enhance or stabilize GH binding. In embodiments, the site 1 mutations include modifications to one or more residues at positions 18, 46, 62, 175, and 190 of a human GH, or at corresponding positions of other GHs, as described further herein.

[0019] In embodiments, GHAs correspond to Set Two GHAs, which comprise mutations at one or more site 2 residues at positions 10. 14, 21, and 120; and mutations at one or more site 1 residues at positions 18, 167, 168, 171, 172, 174, 176, and 179 of a human GH, or at corresponding positions of other GHs, as described further herein.

[0020] In embodiments, a GHA can comprise one or more Set One and Set Two mutations, at site 1 and site 2.

[0021] In embodiments, GHAs can comprise a carrier protein, such as a serum albumin, a transferrin, an immunoglobulin Fc domain, or a growth hormone binding protein, which can be joined to the N terminus or C terminus of the GHA directly or indirectly via a peptide linker, as described further herein. Furthermore, GHAs, including fusion proteins, can be provided as compositions, including ones formulated for specific routes of administration or for targeted delivery of GHA proteins and nucleic acids, as described further herein.

[0022] In embodiments, GHAs can comprise a targeting (or homing domain) or protease-cleavable domain, and comprise various modular formats, as disclosed further herein.

[0023] In embodiments, a GHA, or composition thereof, is used in methods to treat conditions associated with excessive GH activity or GHR activation as disclosed further herein. These conditions include endocrine disorders such as acromegaly or gigantism, oncological disorders such as hepatocellular carcinoma, breast cancer, endometrial cancer, and melanoma, and metabolic disorders such as insulin resistance, type 2 diabetes, related diabetic complications, and vascular disorders, as described further herein. In embodiments, a GHA, or composition thereof, is administered to a subject to reduce STAT3 or STA5 phosphorylation. In embodiments, methods are directed to veterinary indications in non -human mammals, such as dogs, cats, and horses. In embodiments, a GHA, or composition thereof, is used for diagnostic applications directed to endocrine, oncological, metabolic, vascular, and other conditions, and may be provided as a kit, as described further herein. In embodiments, a GHA, or composition thereof, may also be administered to a subject in combination with other active agents or therapies.

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[0025] BRIEF DESCRIPTION OF FIGURES

[0026] For a more complete understanding of the invention, reference is made to the Detailed Description and Examples in conjunction with the accompanying figures. This patent application contains at least one drawing executed in color. Copies of this parent application or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fee.

[0027] FIG. 1. Co-cry stal structure (3HHR) of tire complex between human GH and the extracellular domain of the human GHR, as previously reported by De Vos et al. 1992, Science 255, 306-312. Chain A corresponds to GH (as a four-helix bundle), and chains B and chain C correspond to the extracellular GHR domains. The solved structure has revealed that one GH molecule binds to 2 GHR molecules via two distinct and asymmetric binding sites: site 1 between chain A and chain B, and site 2 between chain A and chain C.

[0028] FIG. 2. PISA interface list, describing the interactions between A, B, and C chains of the human growth hormone-receptor complex in the 3HHR co-crystal structure. For each interaction between Structure 1 and Structure 2, as denoted by the left and middle panels, the right panel shows the interface area, the change in free energy (AG), number of hydrogen bonds (NHB), number of salt bridges (NSB). and tire absence of disulfide bonds (NDS).

[0029] FIG. 3. Interacting amino acid residues at: (A) the site 2 interface between chain A (GH) and chain C of the GHR categorized into hydrogen bonds (left panel) and salt bridges (right panel): and (B) the site 1 interface between chain A (GH) and chain B of the GHR, categorized into hydrogen bonds (left panel) and salt bridges (right panel).

[0030] FIG. 4. Comparison of interaction energies between BC, AB, and AC chain pairs for wild-type (3HHR) and mutated GH molecules. (A) Individual interaction energies in kilocalories per mole (kcal / mol) for the BC (orange: left bar in each molecule grouping), AB (red: middle bar in each molecule grouping), and AC (pink: right bar in each molecule grouping) pairs. (B) Total interaction energies (kcal / mol) of all chain pairs for each molecule.

[0031] FIG. 5. Structural model of an albumin-GHAl fusion protein, showing the human albumin domain joined to the GHA1 domain by a peptide linker.

[0032] FIG. 6. (A) Structural model of a GHAl-IgG Fc fusion protein, showing the human IgG Fc domain joined to the GHA1 domain by a peptide linker. (B) Schematic depiction of a dimerized IgG Fc-GHA fusion (as described further in Example 12).

[0033] FIG. 7. Effects of a GHA1 -Albumin fusion protein on HepG2 Cell Proliferation. The boxplot shows cell metabolic activity, an indicator of cell proliferation, across six groups: no treatment (control), sorafenib alone (3 pM, suboptimal dose), and sorafenib (3 pM) combined with GHA1 -Albumin fusion

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[0035] protein at concentrations of 40 pg / mL, 80 pg / mL. 120 pg / tnL, and 240 pg / mL. The metabolic activity was measured using a colorimetric assay and is shown on the y-axis. Each box represents the interquartile range (IQR) of metabolic activity for each treatment group, with the median show n as a line inside each box. Whiskers denote data within 1.5 times the IQR, and any outliers are displayed as individual points. Tire trend shows a dose-dependent decrease in cell metabolic activity when GHAl-Albumin is combined with sorafenib, indicating enhanced inhibition of cell proliferation.

[0036] FIG. 8. Effects of a GHA1 -Transferrin fusion protein on HepG2 Cell Proliferation. Hie boxplot shows cell metabolic activity, an indicator of cell proliferation, across five groups: no treatment, sorafenib (3 pM, suboptimal dose), and sorafenib (3 pM) combined with GHA1 -Transferrin fusion protein at concentrations of 40 pg / mL, 80 pg / mL, and 120 pg / mL. The metabolic activity was measured using a colorimetric assay and is shown on the y-axis. Each box represents the interquartile range (IQR) of metabolic activity for each treatment, with the line inside each box showing the median value. Whiskers denote data within 1.5 times the IQR, and circles represent outliers. All GHA1 -Transferrin combinations reduced proliferation compared to sorafenib alone, indicating enhanced anti-proliferative effects.

[0037] FIG. 9. Effects of a GHAl-IgG Fc fusion protein on HepG2 Cell Proliferation. The box plot shows the percentage change in absorbance values for different treatment groups compared to the "no treatment" group. Each box illustrates the distribution of percentage changes in a group, with the median marked by a horizontal line. The whiskers indicate the variability within the group, displaying potential outliers (circles) or spread. The treatment groups include sorafenib alone (3 pM, suboptimal dose) and sorafenib (3 pM) combined with GHAl-IgG Fc fusion protein at concentrations of 120 pg / mL, 80 pg / mL, and 40 pg / mL. The analysis highlights the relative efficacy of each treatment in reducing absorbance, with greater negative percentages indicating higher efficacy.

[0038] FIG. 10. Detection of STAT3 phosphorylation in HepG2 cells treated with varying concentrations of GHAl-Albumin in the presence of GH. Left Panel: Western blot analysis of phospho-STAT3 bands, lane 1: 160 pg / mL GHA-1 + 1 pg / mL GH; lane 2: 80 pg / mL GHA-1 + 1 pg / mL GH; lane 3: 40 pg / mL GHA-1 + 1 pg / mL GH; lane 4: 1 pg / mL GH (positive control); lane 5: untreated cells (negative control). Right Panel: GAPDH bands serve as a loading control, confirming similar protein loading across all samples. The highest p-STAT3 signal is observed in the positive control (lane 4), indicating maximal STAT3 phosphorylation with GH alone. GHA1 treatment reduces p-STAT3 levels in a dosedependent manner when combined with GH (Lanes 1 to 3).

[0039] FIG. 11. Line graph depicting tumor volume over time (in days) in a mouse liver cancer model. The line on top (solid circles; blue) represents the control group, and the line on bottom (solid squares; red) represents the treatment group with GHAl-Albumin. The tumor volume is plotted on the y-axis, while the

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[0041] time in days is plotted on the x-axis. The control group shows a rapid increase in tumor volume over time, whereas the treated group shows a significantly reduced rate of tumor growth. The asterisks (*) denote statistically significant suppression of tumor growth compared to the control at days 16, 20, 24, 28, and 32.

[0042] FIG. 12. Tumor growth inhibition graph (based on data in Figure 11), depicting relative tumor inhibition in tire treatment group compared to the control group over the 30 day period.

[0043] FIG. 13. Reduced STAT5 phosphorylation in Hepal-6 cells treated with GHAs in the presence of GH. Hepal-6 cells were serum-starved (24 h), treated with inhibitors (160 pg / mL; 2 h), and stimulated with GH (2 pg / mL; 20 min). Samples receiving no further treatment (NT) or GH alone (GH) served as the unstimulated and stimulated controls. (A) Western blot analysis of phospho-STAT5 and GAPDH. (B). Quantification of pSTAT5 levels (normalized to GAPDH).

[0044] FIG. 14. Reduced proliferation of Hepal-6 cells treated with GHAs in the presence of GH. Hepal-6 cells were serum-starved (24 h), treated with inhibitors (160 pg / mL; 2 h), and stimulated with GH (2 pg / mL; 20 min). The sample receiving GH alone (GH) served as the stimulated control. ATP-luminescence values were measured 48 h after GH addition and correspond to tire percent of GH-only control (control mean = 100).

[0045] FIG. 15. Reduced STAT5 phosphory lation in Hepal-6 cells treated with GHA5 (or GHA5-Fc) in the presence of GH. Hepal-6 cells were serum-starved, pre-treated with inhibitors (2 h) at the indicated concentrations, and stimulated with GH. Samples receiving no further treatment (NT) or GH alone (GH) served as the unstimulated and stimulated controls. Levels of pSTAT5 and GAPDH (as the loading control) were detected by Western blot analysis.

[0046] FIG. 16. Reduced STAT5 phosphorylation in Hepal-6 cells treated with GHA5 (or GHA5-TBP) in the presence of GH. Hepal-6 cells were serum-starved, pre-treated with inhibitors (2 h) at the indicated concentrations, and stimulated with GH. Samples receiving no further treatment (NT) or GH alone (GH) served as the unstimulated and stimulated controls. (A) Western blot analysis of phospho-STAT5 and GAPDH. (B). Quantification of pSTAT5 levels (normalized to GAPDH).

[0047] FIG. 17. Clustal alignment of human, cattle, dog, and cat GH protein sequences, highlighting residues modified in GHA-1 to GHA-4 (from Set One). Residues at tire site 1 interface are highlighted in gray, and residues at site 2 interface are underlined. A star (*) indicates a fully conserved residue where all sequences in the alignment have the same amino acid at that position; two dots (:) signify a position where the residues are strongly similar; a single dot (.) indicates a position where the residues are weakly similar; and no symbol denotes a position where the residues show' no similarity'. All five highlighted residues at the A-B (Site 1) interface show7homology across all sequences. Four of the five residues at the A-C (Site 2) interface show homology across all sequences.

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[0049] FIG. 18. Clustal alignment of human, cattle, dog, and cat GH protein sequences, highlighting residues modified in GHA-5 to GHA-9 (from Set Two). Residues at the site 1 interface are highlighted in gray, and residues at site 2 interface are underlined. A star (*) indicates a fully conserved residue where all sequences in the alignment have tire same amino acid at that position; two dots (:) signify a position where the residues are strongly similar; a single dot (.) indicates a position where the residues are weakly similar; and no symbol denotes a position where the residues show no similarity. All eight highlighted residues at the A-B (Site 1) interface show homology across all sequences. All four underlined residues at the A-C (Site 2) interface show identity (or strong similarity) across all sequences.

[0050] DETAILED DESCRIPTION

[0051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0052] For the sake of brevity, all publications, including patent applications, patents, and other citations mentioned herein, are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference. Citation of any such publication, however, shall not be construed as an admission that it is prior art to the present invention.

[0053] Terms And Definitions

[0054] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms are defined herein for clarity or ready reference so that the present disclosure may be more readily understood, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0055] As used herein, the terms '‘a,” “an,” and “the” are to be understood as meaning both singular and plural, unless explicitly stated otherwise. Thus, “a,” “an,” and “the” (and grammatical variations thereof where appropriate) refer to one or more. For example, tire term “a capsule” includes a plurality of capsules, and mixtures thereof. The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent. As will become apparent to one of ordinary skill in the art after reading this document, the illustrated embodiments and their various alternatives may be implemented without confinement to the illustrated examples.

[0056] A group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and / or” unless expressly

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[0058] stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and / or” unless expressly stated otherwise. Furthermore, although items, elements, or components of the invention may be described or claimed in tire singular, the plural is contemplated to be within the scope thereof, unless limitation to the singular is explicitly stated.

[0059] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range fonnat is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1. 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0060] Moreover, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0061] As used herein, the term “about” or “approximately” means within an acceptable range for a particular value as determined by one skilled in the art, and may depend in part on how the value is measured or determined, e.g., the limitations of the measurement system or technique. In embodiments, “about” mean a range of up to 10%, up to 5%, or up to 1% or less on either side of a given value. For example, the phrase “about 50 mg” includes ±10% of 50, or from 45 to 55 mg. To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about.” However, is understood that whether the term “about” is used explicitly or not, even’ quantity given herein is meant to refer to both the actual given value and the approximation of such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and / or measurement conditions for such given value. Accordingly, for any embodiment of the disclosure in which a numerical value is prefaced by “about” or “approximately,” the disclosure includes an embodiment in which the exact value is recited. Conversely, for any embodiment of the disclosure in which a numerical value is not prefaced by “about” or “approximately,” the disclosure includes an embodiment in which the value is preceded by “about” or approximately.”

[0062] The terms “comprising” and “including” are used herein in their open, non-limiting sense. Other terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended, as opposed to limiting. Thus, the term “example” is used to provide exemplary

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[0064] instances of the item in discussion, not an exhaustive or limiting list thereof. Similarly, adjectives such as ‘'conventional,” '‘traditional,” “normal,” “criterion,” “known,” and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but they should be read to encompass conventional, traditional, nonnal, or criterion technologies that may be available or known now or at any time in the future. Likewise, where this document refers to technologies that would be apparent or known to one of ordinary skill in the art, such technologies encompass those apparent or known to the skilled artisan now or at any time in the future.

[0065] The terms “polypeptide” and “protein” refer to a polymer of amino acid residues. Tire terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog). The terms encompass amino acid chains of any length, including foil length proteins (i.e., a GHA polypeptide or GHA protein fusion), wherein the amino acid residues are linked by covalent peptide bonds. Where an amino acid sequence is provided herein. L-, D-, or beta amino acid versions of tire sequence are also contemplated as well as retro, inversion, and retro-inversion isoforms. Peptides also include amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acids. In addition, the term applies to amino acids joined by a peptide linkage or by other modified linkages (e.g., where the peptide bond is replaced by an a-ester, a 0-ester, a thioamide, phosphonamide, carbamate, hydroxylate, and the like. See, e.g., Spatola 1983, in Chemistry and Biochemistry of Amino Acids Peptides and Proteins (Weinstein, ed.) Marcel Dekker. New York, 267-357; Morley 1980, Trends Pharm. Sci. 1, 463-468; Hudson et al. 1979, Int. J. Pept. Prot. Res. 14, 177-185; Spatola et al. 1986, Life Sci. 38, 1243-1249.

[0066] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that arc later modified, e.g., hydroxyprolinc, y-carboxyglutamatc, and O-phosphoscrinc. An amino acid may be an L- or D-amino acid. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from foe general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

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[0068] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUP AC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0069] One of skill in the art will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence represent a ‘‘conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, substitutions may be made wherein an aliphatic amino acid (e.g., G, A, I, L, or V) is substituted with another member of the group. Similarly, an aliphatic polar-uncharged group such as C, S, T, M, N, or Q, may be substituted with another member of the group; and basic residues, e.g., K, R, or H, may be substituted for one another. See, e.g., Creighton 1984, Proteins. Structures and molecular properties, W. H. Freeman and Company, New York). Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the present disclosure. Moreover, one of skill in the art will also recognize that in certain protein regions, such as the site 1 and site 2 interacting regions of a GH described herein, even conservative substitutions may have functional consequences, for example, by reducing hydrogen bonding potential, attractive electrostatic interactions, or hydrophobic interactions with the GHR. On tire other hand, conservative substitutions at other protein regions may be expected to have minimal functional effects.

[0070] " Sequence identity” (or “identity”) as used herein, refers to the percentage of amino acid residues in a single given sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve tire maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. “Sequence homology” (or “homology”), as used herein, refers to the percentage of amino acid residues in a single given sequence that arc identical or have similar chemical properties to the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. Thus, in contrast to sequence identity, sequence homology considers any conservative substitutions (as described herein) as part of the sequence alignment. Sequence identity or sequence homology can be determined using well known methods, including publicly available computer programs such as the NCBI Basic Local Alignment Search Tool (BLAST) software. These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity or sequence homology between the given and reference sequences.

[0071] “Variants or variant” refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar,

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[0073] and, in many regions, identical to the reference nucleic acid or polypeptide. As used herein, 'variant” refers to a GHA polypeptide, GHA fusion, or carrier protein, differing in sequence from the native protein but retaining at least one functional or therapeutic property thereof as described elsewhere herein or otherwise known in the art.

[0074] '‘Administering” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and deliver}’ systems known to those skilled in the art. Preferred routes of administration for agents of the invention, such as GHAs, include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase '‘parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. Alternatively, a GHA of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. In embodiments, administering refers to parenteral administration in the case of GHAs used herein.

[0075] Tire tenns “individual,” “subject,” and “patient” are used interchangeably herein and can be a vertebrate, in particular, a mammal, more particularly, a primate (including non-human primates and humans) and include a laboratory animal in the context of a clinical trial or screening or activity experiment. Thus, as can be readily understood by one of ordinary skill in the art, the formulations of the present disclosure are particularly suited to administration to any vertebrate, particularly a mammal, and more particularly, a human.

[0076] As used in the present disclosure, the term “effective amount” is interchangeable with “therapeutically effective amount” and means an amount or dose of a GHA, or other active components disclosed herein, effective in treating the particular disease, condition, or disorder disclosed herein. Tirus “treating” includes producing a desired preventative, inhibitory, relieving, or ameliorative effect, as defined further herein. In methods of treatment according to the disclosure, “an effective amount” of any one of the presently described formulations is administered to a subject (e.g., a mammal). The “effective amount” will vary, depending on numerous factors, such as the active ingredient, the disease (and its severity), the treatment desired, and age and weight of the subject.

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[0078] A '‘therapeutically effective amount’’ or “therapeutically effective dosage” of a drug or therapeutic agent, such as a GHA of the disclosure, is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impainnent or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro or in vivo assays.

[0079] By way of example, an anti-cancer agent promotes cancer regression in a subject. “Promote cancer regression” means that administering an effective amount of the drug, alone or in combination with an anti -neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. In embodiments, a therapeutically effective amount of the drug promotes cancer regression to tire point of significantly reducing or eliminating the cancer. In addition, the terms '‘effective” and '‘effectiveness” with regard to a treatment includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the drug to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and / or organism level (adverse effects) resulting from administration of the drug. An anti -cancer agent promotes cancer regression, can also halt cancer progression on imaging, improve quality of life, and improve survival outcomes such as time to tumor progression (TTP), progression-free survival (PFS), and overall survival (OS).

[0080] By way of example for the treatment of tumors, a therapeutically effective amount of the drug preferably inhibits cell growth or tumor growth by at least about 20%, by at least about 40%, by at least about 60%, and by at least about 80% relative to untreated subjects. In other preferred embodiments of the invention, tumor regression may be observed and continue for a period of at least about 20 days, at least about 40 days, or at least about 60 days. Notwithstanding these ultimate measurements of therapeutic effectiveness, evaluation of immunotherapeutic drugs, which can be used in combination with a GHA described herein, must also make allowance for "immune -related” response patterns.

[0081] The term “fonnulation,” as used herein, is the form in which the drug is to be administered to the subject or patient. GHAs can be administered as part of a formulation that includes non-active agents, such as biodegradable polymers or hydrogels in controlled or sustained release formulations, phosphate or citrate salt buffers in liquid formulations, matrix components in depot formulations, or cryoprotectants in lyophilized formulations.

[0082] -12- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0083] The term ‘'pharmaceutically acceptable,’’ as used in connection with formulations of the present disclosure, refers to molecular entities and other ingredients of such formulations that are physiologically tolerable and do not typically produce untoward reactions when administered to an animal (e.g., human) according to their intended mode of administration (e.g., intravenous, oral, topical, ocular, transdermal, intramuscular, intranasal, pulmonary, etc.).

[0084] A “pharmaceutically acceptable excipient” refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological formulation or otherwise used as a vehicle, carrier, or diluent to facilitate administration of an agent and that is compatible therewith. Suitable pharmaceutical excipients include those described in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005). In certain embodiments, the excipient is a solid component.

[0085] As used herein, the term “inert” refers to any inactive ingredient of a described formulation. The definition of “inactive ingredient” as used herein follows that of the U. S. Food and Drug Administration, as defined in 21 C. F. R. 201.3(b)(8), which is any component of a drug product other than tire active ingredient.

[0086] As used herein, “suitable for oral administration” refers to a sterile, pharmaceutical product, such as a product produced under good manufacturing practices (GMP), as understood in the art, suitable for administration to a subject (e.g.. a human subject) orally.

[0087] As used herein, the term “disorder” is used interchangeably with “disease” or “condition.” For example, an endocrine (or growth) disorder also means an endocrine (or growth) disease or an endocrine (or growth) condition.

[0088] The terms “treat,” “treating.” and “treatment” cover therapeutic methods directed to a diseasestate in a subject and include: (i) preventing the disease-state from occurring, in particular, when the subject is predisposed to the disease-state but has not yet been diagnosed as having it; (ii) inhibiting the diseasestate, e.g., arresting its development (progression) or delaying its onset; and (iii) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. These terms also include ameliorating a symptom of a disease (e.g.. reducing the pain, discomfort, or deficit), wherein such amelioration may be directly affecting the disease (e.g., affecting the disease’s cause, transmission, or expression) or not directly affecting the disease.

[0089] An “adverse event” (AE) as used herein is any unfavorable and generally unintended or undesirable sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system or expansion of immune system cells (e.g., T cells) in response to a treatment. A medical treatment may have one or more associated AEs and each AE may have the same or different level of severity. Reference to -13- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0090] methods capable of ‘'altering adverse events’’ means a treatment regime that decreases the incidence or severity of one or more AEs associated with the use of a different treatment regime.

[0091] An ‘'antibody” (Ab) shall include, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each El chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI. CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy -tenninus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e g., effector cells) and the first component (C Iq) of the classical complement system.

[0092] An ‘'antibody” may be of any immunoglobulin isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE. The term “antibody” may include, for instance, monoclonal, chimeric, recombinant, deimmunized, affinity matured, humanized and human antibodies, as well as antibodies from other species such as rodents, rabbits, mice, rats, hamsters, goats, and llamas. Antibodies may be derived solely from a single source, or may be “chimeric,” that is. different portions of the antibody (such as CDRs, framework regions, variable region, constant region) may be derived from two different antibodies. The definition of “antibody” according to the invention comprises full-length antibodies, also including camelid antibodies, and other immunoglobulins generated by biotechnological or protein engineering methods or processes. An antibody may also be produced in hybridomas. Where not expressly stated, and unless the context indicates otherwise, the term “antibody” also includes an antigen-binding fragment (such as an Fab fragment, scFv, or nanobody) or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab.

[0093] The terms “cancer,” “cancerous,” or “malignant” refer to or describe various diseases typically characterized by unregulated cell growth, and more particularly, a cancer characterized by overexpression of GH or GHR. Examples of cancers include liver cancer, prostate cancer, colorectal cancer, breast cancer, endometrial cancer, lung cancer, glioblastoma, and melanoma. Even more particular forms of cancer include

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[0095] hepatocellular cancer (HCC). With respect to HCCs, the term can encompass any stage, including early stage, intermediate stage, and advanced stage.

[0096] An ‘'immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. The term “immunotherapy” refers to tire treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing, or otherwise modifying an immune response. In embodiments, immunotherapy refers to cancer immunotherapy, and more particularly, immune checkpoint inhibitor (“ICI” or “CPI”) therapy, as described further herein. In embodiments, immunotherapy comprises administering a therapeutically effective amount of a PD-1 ICI, which can include an anti-PD-1 antibody such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), cemiplimab (Libtayo®), dostarlimab (Jemperli®), retifanlimab (Zynx®), as well as other suitable antibodies. In embodiments, immunotherapy comprises administering a therapeutically effective amount of a CTLA-4 ICI, which can include an anti-CTLA-4 antibody such as ipilimumab (Y ervoy®) or tremelimumab (Imjudo®). In embodiments, immunotherapy comprises administering a therapeutically effective amount of a PD-L1 ICI, which can include an anti-PD-Ll antibody such as atezolizumab (Tecentriq®), avelumab (Bavencio®), ordurvalumab (Imfinzi®). Immunotherapies include both monotherapies based on administering a single ICI, as well as combination therapies based on administering two (or more) ICIs.

[0097] A “signal transduction pathway” or '‘signaling pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of the cell. A “cell surface receptor” includes, for example, molecules and complexes of molecules that are located on tire surface of a cell and are capable of receiving a signal and transmitting such a signal across the plasma membrane of a cell. An example of a cell surface receptor of the present invention is the growth hormone receptor, which transmits a signal that drives downstream signaling cascades, including the JAK-STAT, Ras-ERK, and PI3K-Akt pathways. An “inhibitor” of signaling refers to a compound or agent, such as a GHA polypeptide or fusion protein, which antagonizes or reduces the initiation, reception, or transmission of a signal, be that signal stimulatory or inhibitory, by any component of a signaling pathway such as a receptor or its ligand.

[0098] Reference will now be made to the embodiments of the present invention, examples of which are illustrated by and described in conjunction with the accompanying examples and enumerated embodiments -15- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0099] and claims. While certain embodiments are described herein, it is understood that the embodiments described are not intended to limit the scope of the invention. On the contrary, the present disclosure is intended to cover alternatives, modifications, and equivalents that can be included within the invention as defined by the appended enumerated embodiments and claims.

[0100] Growth Hormone Receptor Antagonists

[0101] Tire present disclosure encompasses rowth hormone receptor antagonists (GHAs) for use in therapeutic, diagnostic, and other applications. A GHA includes a “GHA polypeptide,” comprising the mutated GH by itself. A GHA also includes a “GHA fusion protein,” (“GHA fusion” or “fusion protein”) wherein the mutated GH is joined, directly or indirectly via a linker, to a carrier protein, such as albumin, transferrin, an Fc domain, or growth hormone binding protein (GHBP). A GHA fusion or fusion protein can also include other domains, in addition to carrier (half-life enhancing) domains, such as flexible peptide linkers, targeting domains, spacers, and protease cleavable domains, as described further herein.

[0102] Accordingly, as used herein, tire tenn “GHA” refers to both a GHA polypeptide itself, as well as a GHA fusion protein with one or more additional domains. Hie subject disclosure also covers variants of such GHAs.

[0103] In addition to covering the amino acid sequences of GHAs disclosed herein, e.g., GHA polypeptides and GHA fusion proteins, the present disclosure also provides polynucleotides (nucleic acids) encoding the GHAs, including RNA, DNA, and modified nucleic acids, as well as viral vectors containing these nucleic acids, and cells encoding the GHAs.

[0104] GHA Polypeptides

[0105] In embodiments, the present disclosure provides GHA polypeptides, comprising targeted mutations that competitively inhibit endogenous GH binding to the GHR. GH is a monomeric protein and previous structural studies, as depicted in Figure 1 and described further in the Examples, reveal that GH (A chain) binds HGR in a 1:2 ratio via two non-identical motifs, sites 1 and 2. Site 1 mediates the interaction of GH (A chain) with the first HGR (chain B), and site 2 mediates the interaction of GH (A chain) with the second HGR (chain C).

[0106] As described further herein, including in the Examples, the interaction at the interface between the A and C chains is secondary to tire primary high affinity interaction at the interface between the A and B chains. In turn, as described in Examples 1-3, a complex and detailed analysis of the A-C and A-B interfaces supports design strategies for new and improved GHAs that can inhibit downstream growth hormone receptor (GHR) signaling.

[0107] -16- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0108] Accordingly, in some embodiments, the present disclosure provides a GHA that comprises one or more mutations (modifications, substitutions) targeting the Site 2 interface between the A chain (GH) and a C chain of a GHR. In embodiments, the GHA comprises, in addition to one or more mutations targeting the Site 2 interface, mutations targeting the Site 1 interface between the A chain (GH) and a B chain of a GHR. In embodiments, tire present disclosure provide Set One and Set Two GHAs, as described herein, which may also comprise additional domains in the case of fusions, including carrier (half-life enhancing) proteins, flexible peptide linkers, targeting domains, spacers, and protease cleavable domains.

[0109] Set One GHAs: Mutations Targeting the Site 2 Interface.

[0110] The present disclosure provides GHAs, which may also be referred to as "‘Site 2 GHAs.” comprising mutations at one or more positions within an A chain of a GH, specifically modifying GH residues interacting at a Site 2 interface between the A chain and a C chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations selectively disrupt GH binding at the A-C interface.

[0111] Examples 1 and 2 provide details from structural and bioinformatics studies that highlight functional properties of the site 2 interface, as well as key interacting human GH residues at the interface that are amenable to targeted mutations at site 2.

[0112] Set One (encompassing modifications in GHA1 to GHA4)

[0113] In embodiments, the site 2 targeting mutations comprise a modification to one or more residues at positions 8, 12, 16, 103, and 119 of GH, and more particularly, human GH (hGH), or at corresponding homologous residues in non-human GH proteins.

[0114] In embodiments, the modifications to one or more residues at positions 8, 12, 16, 103, and 119 of the GH are selected from:

[0115] a substitution of arginine (Arg; R) at position 8 with alanine (Ala; A);

[0116] a substitution of asparagine (Asn; N) at position 12 with glutamine (Gin; Q);

[0117] a substitution of arginine (Arg; R) at position 16 with lysine (Lys; K);

[0118] a substitution of tyrosine (Tyr; Y) at position 103 with phenylalanine (Phe; F): and

[0119] a substitution of glutamate (Glu; E) at position 119 with aspartate (Asp; D).

[0120] In embodiments, the site 2 targeting mutations in the GHA can comprise modifications to one residue, two residues, three residues, four residues, or five residues selected from positions 8, 12, 16, 103, and 119 of tire GH. In embodiments, the GHA comprises a combination of one or more mutations, two or

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[0122] more mutations, three or more mutations, or four or more mutations, selected from the group consisting of Arg8Ala, Asnl2Gln, Argl6Lys, TyrlO3Phe, and Glul 19Asp.

[0123] In embodiments, the modifications to one or more residues at positions 8, 12, 16, 103, and 119 of the GH are selected from:

[0124] a substitution of arginine (Arg; R) at position 8 with glutamate (Glu; E), leucine (Leu; L), glycine (Gly; G), or tryptophan (Trp; W);

[0125] a substitution of asparagine (Asn; N) at position 12 with alanine (Ala; A), isoleucine (He; I), serine (Ser; S), or glutamine (Gin; Q);

[0126] a substitution of Arginine (Arg; R) at position 16 with glutamate (Glu; E), leucine (Leu; L); alanine (Ala; A), or tryptophan (Trp; W);

[0127] a substitution of tyrosine (Tyr; Y) at position 103 with phenylalanine (Phe; F), Leucine (Leu; L), or Serine (Ser; S);

[0128] and a substitution of glutamate (Glu; E) at position 119, with arginine (Arg; R), valine (Vai; V), or alanine (Ala; A).

[0129] The site 2 targeting mutations in the GELA can therefore comprise a modification to one residue, two residues, three residues, four residues, or five residues selected from positions 8, 12, 16, 103, and 119 of the GH. More particularly, the GHA comprises modification to five residues and corresponds to the amino acid sequence of GHA1 (also referred to herein as NT-GHA-1, NT-GHA1, or GHA-1), GHA2 (also referred to herein as NT-GHA-2. NT-GHA2, or GHA-2), GHA3 (also referred to herein as NT-GHA-3, NT-GHA3, or GHA-3), or GHA4 (also referred to herein as NT-GHA-4, NT-GHA4, or GHA -4). More particularly, the GHA comprises the amino acid sequence of GHA 1.

[0130] In embodiments, the modifications to one or more residues at positions 8, 12, 16, 103, and 119 of the GH are selected from:

[0131] a substitution of arginine (Arg; R) at position 8 with alanine (Ala; A), glutamate (Glu; E), leucine (Leu; L), glycine (Gly; G), or tryptophan (Trp; W);

[0132] a substitution of asparagine (Asn; N) at position 12 with glutamine (Gin; Q), alanine (Ala; A), isoleucine (He; I), or serine (Ser; S);

[0133] a substitution of arginine (Arg; R) at position 16 with lysine (Lys; K), glutamate (Glu; E), leucine (Leu; L); alanine (Ala; A), or tryptophan (Trp; W);

[0134] a substitution of tyrosine (Tyr; Y) at position 103 with phenylalanine (Phe; F), leucine (Leu; L), or serine (Ser; S); and

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[0136] and a substitution of glutamate (Glu; E) at position 119 with aspartate (Asp; D), arginine (Arg; R), valine (Vai; V), or alanine (Ala; A).

[0137] The site 2 targeting mutations in the GHA can therefore comprise a modification to one residue, two residues, three residues, four residues, or five residues selected from positions 8, 12, 16, 103, and 119 of the GH.

[0138] In embodiments, the GHA comprises a modification to five residues and comprises the amino acid sequence of GHA1, GHA2, GHA3, or GHA4.

[0139] In one aspect, the GHA comprises the amino acid sequence of GHA1 (with the 5 amino acid substitutions shown in bold and underlined):

[0140] FPTIPLSELF DAAMLEAHRL HQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVFGASDSNV YDLLKDLERG IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFRKDM DKVETFLRIV QCRSVEGSCG F

[0141] (GHA k SEQ ID NO:1)

[0142] In one aspect, the GHA comprises the amino acid sequence of GHA2 (with the 5 amino acid substitutions shown in bold and underlined):

[0143] FPTIPLSLLF DIAMLLAHRL HQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVLGASDSNV YDLLKDLEVG IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFRKDM DKVETFLRIV QCRSVEGSCG F

[0144] (GHA2; SEQ ID NO: 2)

[0145] In one aspect, the GHA comprises the amino acid sequence of GHA3 (with the 5 amino acid substitutions shown in bold and underlined):

[0146] FPTIPLSGLF DSAMLAAHRL HQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVSGASDSNV YDLLKDLEAG IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFRKDM DKVETFLRIV QCRSVEGSCG F

[0147] (GHA3: SEQ ID NO:3)

[0148] In one aspect, the GHA comprises the amino acid sequence of GHA4 (with the 5 amino acid substitutions shown in bold and underlined):

[0149] FPTIPLSWLF DQAMLWAHRL HQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS

[0150] -19- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0151] LVFGASDSNV YDLLKDLERG IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFRKDM DKVETFLRIV QCRSVEGSCG F

[0152] (GHA4; SEQ ID NO:4)

[0153] Without being limited to a particular mechanism, different mutations in a GHA may disrupt GH binding at the A-C interface by altering hydrogen bonding, electrostatic interactions (such as salt bridges), hydrophobic potentials, or by exerting other effects.

[0154] At position 8, for example, an Arg8Ala or Arg8Leu substitution removes the positive charge and introduces a hydrophobic side group that could disrupt hydrogen bonding potential and polar interactions; an Arg8Glu substitution creates a local charge reversal that could repel other negatively charged regions or attract positively charged areas; an Arg8Gly substitution introduces a small non-charged group that could weaken structure and charge-dependent interaction; and an Arg8Trp substitution introduces a bulky and hydrophobic aromatic group that could create steric hindrance, while still allowing for stacking interactions with other aromatic residues.

[0155] At position 12, for example, an Asnl2Ala substitution introduces a small non-hydrophobic side group that could weaken potential hydrogen bonding interactions and promote nonpolar interactions and compactness; an Asnl2Ile substitution introduces a larger bulkier hydrophobic side group that could potentially reduce flexibility and adaptability in polar environments and stabilize certain nonpolar interactions: an Asnl2Ser substitution introduces a smaller side group that is more flexible and may weaken interactions dependent on particular structural and spatial features: and an Asnl2Gln substitution that introduces a longer side group that introduce steric effects if bulkiness interferes with the interaction.

[0156] At position 16, for example, an Arg 16Lys substitution maintains charge of the side group but alters its spatial geometry; an Argl6Glu substitution introduces a negative charge that could repel other negatively charged regions or attract positively charged areas, potentially altering binding specificity; an Argl6Leu substitution introduces a hydrophobic side group that could disrupt polar interactions at the site 2 interface; and Argl6Ala substitution introduce a smaller side group, potentially weakening interactions at the site 2 interface that are dependent on specific structural and positional features; and an Argl6Trp substitution that introduce a larger side group, potentially weakening interactions at the site 2 interface by steric hindrance.

[0157] At position 103, for example, a Tyrl03Phe substitution retains the aromatic ring but removes the hydroxyl group, eliminating hydrogen-bonding potential while preserving hydrophobic stacking, which may maintain stability in nonpolar regions but alter binding dynamics where polar interactions were involved; a Tyrl03Leu substitution removes a hydroxyl group and aromatic stacking interactions, creating a purely hydrophobic side chain that may enhance stability in hydrophobic environments but reduce versatility for

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[0159] binding, especially where aromatic interactions are beneficial; and a TyrlO3Ser substitution replaces an aromatic group with a smaller hydrophobic group, potentially weakening interactions dependent on specific structural and positional features or on aromatic ring interactions.

[0160] At position 119, for example, a Glut 19Asp substitution may alter electrostatic interactions, such as salt bridge formation; a Glut 19Arg substitution introduces a positive charge, which could alter electrostatic attractions and introduce stronger hydrogen bonding and ionic interactions due to arginine’s flexible, nitrogen-rich side chain; a Glul 19Val substitution introduces a hydrophobic side group that could disrupt electrostatic interactions at the site 2 interface, such as hydrogen bonding or salt bridges; and a Glul 19Ala substitution introduces a smaller side group, potentially weakening interactions at the site 2 interface that are dependent on specific structural and positional features.

[0161] Accordingly, in embodiments, certain targeted GH mutations in GHA can reduce hydrogen bonding potential, attractive electrostatic interactions, or hydrophobic interactions between the A chain and C chain in the GHR complex, and more particularly at the site 2 interface.

[0162] In embodiments, the GHA significantly affects GH binding at an interface between an A chain and a C chain of the GHR in tire GH: GHR complex, without significantly affecting GH binding at the interface between an A chain and a B chain of the GHR in the GH-GHR complex.

[0163] In embodiments, the GHA significantly affects GH binding at an interface between the A chain and a C chain of the GHR in the GH: GHR complex, thereby inhibiting downstream growth hormone receptor (GHR) signaling.

[0164] In embodiments, the targeted GH mutations enhance specificity for growth hormone receptor blockade, resulting in selective inhibition of downstream signaling pathways such as STAT3 or STAT5 phosphorylation.

[0165] Set One GHAs: Additional Modifications Targeting the Site 1 Interface.

[0166] In embodiments, a Site 2 GHA as described above is further modified with additional mutations that selectively enhance or stabilize GH binding at the Site 1 interface between the A chain and a B chain of a GHR.

[0167] Examples I and 3 provide details from structural and bioinformatics studies that highlight functional properties of the site 1 interface, as well as key interacting GH residue at the interface that are amenable to targeted mutations at site 1.

[0168] GHAs comprising both disrupting Site 2 mutations and enhancing / stabilizing Site 1 mutations can have a greater capacity for competitive inhibition with endogenous GH, thereby enhancing their potential as effective therapeutic candidates. Accordingly, the present disclosure includes GHAs that comprise, in -21- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0169] addition to one or more targeted mutations that selectively disrupt the A-C interface at Site 2, one or more targeted mutations that selectively enhance or stabilize the A-B interface at Site 1.

[0170] In embodiments, the present disclosure provides a GHA, comprising (i) mutations at one or more positions within an A chain of a growth hormone (GH), specifically targeting residues interacting at an interface (site 2) between the A chain and a C chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations disrupt GH binding at the A-C interface; and (ii) mutations at one or more positions within an A chain of a growth hormone (GH), specifically targeting residues interacting at an interface (site 1) between the A chain and a B chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations enhance GH binding at the A-B interface.

[0171] In embodiments of such GHAs. the site 2 targeting mutations include those in any GHA described above and herein, and therefore can include modifications to one or more residues at positions 8, 12, 16, 103, and 119 of the GH. In particular embodiments, the site 2 targeting mutations comprise any one or more of the five mutations found in GHA1, GHA2, GHA3, or GHA4.

[0172] In embodiments of such GHAs, the additional site 1 targeted mutations comprise a modification to one or more residues at positions 18, 46, 62, 175, and 190 of the GH. In embodiments, the site 1 targeted modifications to one or more residues at positions 18, 46, 62, 175, and 190 of the GH are selected from: a substitution of histidine (His; H) at position 18 with asparagine (Asn; N) or glutamine (Gin; Q); a substitution of glutamine (Gin; Q) at position 46 with asparagine (Asn; N);

[0173] a substitution of serine (Ser; S) at position 62 with asparagine (Asn; N);

[0174] a substitution of threonine (Thr; T) at position 175 with aspartate (Asp; D) or glutamate (Glu; E); and

[0175] a substitution of glycine (Gly; G) at position 190 with asparagine (Asn; N) or glutamine (Gin; Q).

[0176] Hie site I targeting mutations in the GHA can therefore comprise modifications to one residue, two residues, three residues, four residues, or five residues selected from positions 18, 46, 62, 175, and 190 of the GH.

[0177] In embodiments, the GHA comprises: one or more site 2 targeting mutations selected from the group consisting of Arg8Ala, Asnl2Gln, Argl6Lys, Tyrl03Phe, and Glul 19Asp; and one or more site 1 targeting mutations selected from the group consisting of His 18 Asn (or Hisl8Gln), Gln46Asn, Scr62Asn, Thrl75Asp (or Thrl75Glu), and Gly 190Asn (or Gly 190Gln).

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[0179] In embodiments, the GHA comprises: the five site 2 targeting mutations in GHA1, GHA2, GHA3, or GHA4; and one or more site 1 targeting mutations selected from Hisl8Asn (or Hisl8Gln), Gln46Asn, Ser62Asn, Thrl75Asp (or Thrl75Glu), and Glyl90Asn (or Glyl90Gln).

[0180] In embodiments, the GHA comprises:

[0181] a modification to one or more residues at positions 8, 12, 16, 103, and 119 of the GH selected from:

[0182] a substitution of arginine (Arg; R) at position 8 with alanine (Ala; A), glutamate (Glu; E), leucine (Leu; L), glycine (Gly; G), or tryptophan (Trp; W);

[0183] a substitution of asparagine (Asn; N) at position 12 with glutamine (Gin; Q), alanine (Ala; A), isoleucine (He; I), or serine (Ser; S);

[0184] a substitution of arginine (Arg; R) at position 16 with lysine (Lys; K), glutamate (Glu; E), leucine (Leu; L); alanine (Ala; A), or tryptophan (Trp; W); a

[0185] a substitution of tyrosine (Tyr; Y) at position 103 with phenylalanine (Phe; F), leucine (Leu; L), or serine (Ser; S); and

[0186] a substitution of glutamate (Glu; E) at position 119 with aspartate (Asp; D), arginine (Arg; R), valine (Vai; V), or alanine (Ala; A); and

[0187] a modification to one or more residues at positions 8, 12, 16, 103, and 119 of the GH selected from:

[0188] a substitution of histidine (His; H) at position 18 with asparagine (Asn; N) or glutamine (Gin; Q); a substitution of glutamine (Gin; Q) at position 46 with asparagine (Asn; N);

[0189] a substitution of serine (Ser; S) at position 62 with asparagine (Asn; N);

[0190] a substitution of threonine (Thr; T) at position 175 with aspartate (Asp; D) or glutamate (Glu; E); and

[0191] a substitution of glycine (Gly; G) at position 190 with asparagine (Asn; N) or glutamine (Gin; Q).

[0192] In any of the preceding embodiments, a GHA can comprise a modification to one residue, two residues, three residues, four residues, or five residues at positions 18. 46, 62, 175, and 190 of the GH. Additionally, a GHA can comprise: a modification to one residue, two residues, three residues, four residues, or five residues at positions 18, 46, 62, 175, and 190 of the GH; and a modification to one residue, two residues, three residues, four residues, or five residues at positions 8, 12, 16, 103, and 119 of the GH Without being bound to a particular mechanism, different site 1 targeting mutations in a GHA may enhance or stabilize GH binding at the A-B interface (site 1) by altering hydrogen bonding, electrostatic interactions (such as salt bridges), hydrophobic potentials, or by exerting other effects.

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[0194] At position 18, for example, a Hisl8Asn or Hisl8Gln substitution may enhance hydrogen bonding potential at the A-B interface.

[0195] At position 46, for example, a Gln46Asn substitution may enhance hydrogen bonding potential at the A-B interface.

[0196] At position 62, for example, a Ser62Asn substitution may enhance hydrogen bonding potential at the A-B interface.

[0197] At position 175, for example, a Thrl75Asp or Thrl75Glu substitution may enhance salt bridge potential at the A-B interface.

[0198] At position 190, for example, a Gly 190Asn or Glyl90Gln substitution may enhance salt bridge potential at the A-B interface.

[0199] In embodiments, a GHA with targeted mutations at site 1 and site 2 inhibit GHR signaling. In embodiments, a GHA with targeted mutations at site 1 and site 1 enhance specificity for growth hormone receptor blockade, resulting in selective inhibition of downstream signaling pathways such as STAT3 or STAT5 phosphory lation.

[0200] Also provided are GHAs comprising a modification:

[0201] to two or more residues at positions 8, 12, 16, 103, and 119 of a (human) GH selected from: a substitution of arginine (Arg; R) at position 8 with alanine (Ala; A), glutamate (Glu; E), leucine (Leu; L), glycine (Gly; G), or tryptophan (Trp; W); a substitution of asparagine (Asn; N) at position 12 with glutamine (Gin; Q), alanine (Ala; A), isoleucine (He: I), or serine (Ser: S);a substitution of arginine (Arg; R) at position 16 with lysine (Lys: K), glutamate (Glu; E), leucine (Leu: L): alanine (Ala; A), or try ptophan (Trp; W): a substitution of tyrosine (Tyr; Y) at position 103 with pheny lalanine (Phe; F), leucine (Leu; L), or serine (Ser; S); and a substitution of glutamate (Glu; E) at position 119 with aspartate (Asp; D), arginine (Arg; R), valine (Vai; V), or alanine (Ala; A);

[0202] and to two or more residues at positions 18, 46, 62, 175. and 190 of the (human) GH selected from: a substitution of histidine (His; H) at position 18 with asparagine (Asn; N) or glutamine (Gin: Q): a substitution of glutamine (Gin; Q) at position 46 with asparagine (Asn; N); a substitution of serine (Ser; S) at position 62 with asparagine (Asn; N); a substitution of threonine (Thr; T) at position 175 with aspartate (Asp; D) or glutamate (Glu; E); and a substitution of gly cine (Gly; G) at position 190 with asparagine (Asn; N) or glutamine (Gin; Q).

[0203] In embodiments, a GHA comprises one or more, two or more, three or more, or four or more mutations at positions 8, 12, 16, 103, and 119, selected from the group consisting of Arg8Ala, Arg8Glu, Arg8Leu, Arg8Ala, Arg8Trp; Asnl2Gln, Asnl2Ala, Asnl2Ile, Asnl2Ser: Argl6Lys, Argl6Glu, Argl6Leu,

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[0205] Argl6Ala, Argl6Trp; Tyrl03Phe, TyrlO3Leu; TyrlO3Ser; and Glul 19Asp, Glul 19Arg, Glul 19Val, and Glu 119Ala; and one or more, two or more, three or more, or four or more mutations at positions 18, 46, 62, 175, and 190, selected from the group consisting of Hisl8Asn (or Hisl8Gln), Gln46Asn, Ser62Asn, Thrl75Asp (or Thrl75Glu), and Glyl90Asn (or Glyl90Gln).

[0206] In embodiments, the GHA comprises the amino acid sequence of GHA1 (SEQ ID NO: 1), GHA2 (SEQ ID NO:2), GHA3 (SEQ ID NO:3), or GHA4 (SEQ ID NO:4). In embodiments, the GHA comprises: the five mutations in GHA1, GHA2. GHA3. or GHA4; and one or more mutations selected from Hisl8Asn (or Hisl8Gln), Gln46Asn. Ser62Asn, Thrl75Asp (or Thrl75Glu). and Glyl90Asn (or Glyl90Gln).

[0207] In embodiments, the modifications at positions 8, 12, 16, 103. and 119 are within an A chain of the GH, targeting residues interacting at an interface (site 2) between the A chain and a C chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations disrupt GH binding at the A-C interface; and wherein the modifications at positions 18, 46, 62, 175, and 190 are within an A chain of the GH, targeting residues interacting at an interface (site 1) between the A chain and a B chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations enhance or stabilize GH binding at the A-B interface.

[0208] Set Two GHAs: Mutations Targeting the Site 2 and Site 1 Interfaces.

[0209] In embodiments, the GHA comprises: a site 2 modification at one or more residues at positions 10, 14, 21, and 120 of GH; and a site 1 modification at one or more residues at positions 18, 167, 168. 171, 172. 174, 176. and 179 of GH. and more particularly, human GH (hGH), or at corresponding homologous residues in non-human GH proteins.

[0210] In embodiments, the modifications to one or more residues at positions 10, 14, 21, and 120 of the GH are selected from:

[0211] a substitution of phenylalanine (Phe; F) at position 10 with alanine (Ala; A) or histidine (His; H); a substitution of methionine (Met: M) at position 14 with tryptophan (Trp; W) or glycine (Gly; G) a substitution of histidine (His; H) at position 21 with asparagine (Asn; N); and

[0212] a substitution of glycine (Gly; G) at position 120 with a lysine (Lys; K) or arginine (Arg; R).

[0213] In embodiments, the modifications to one or more residues at positions 18, 167, 168, 171, 172, 174, 176, and 179 of the GH arc selected from:

[0214] a substitution of histidine (His; H) at position 18 with aspartate (Asp; D) or asparagine (Asn; N); a substitution of arginine at position 167 with asparagine (Asn; N) or glutamate (Glu; E) a substitution of lysine (Lys: K) at position 168 with alanine (Ala; A);

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[0216] a substitution of aspartate (Asp; D) at position 171 with a serine (Ser; S);

[0217] a substitution of lysine (Lys; K) at position 172 with an arginine (Arg; R);

[0218] a substitution of glutamate (Glu; E) at position 174 with a serine (Ser: S) or threonine (Thr; T); a substitution of phenylalanine (Phe; F) at position 176 with a tyrosine (Tyr; Y); and

[0219] a substitution of isoleucine (lie, I) at position 179 with a threonine (Thr; T).

[0220] In embodiments, a GHA comprises a modification:

[0221] to two or more residues at positions 10, 14, 21, and 120 of a human growth hormone (GH), selected from:

[0222] a substitution (mutation) of phenylalanine (Phe; F) at position 10 with alanine (Ala; A) or histidine (His; H),

[0223] a substitution of methionine (Met; M) at position 14 with tryptophan (Trp; W) or glycine (Gly; G), a substitution of histidine (His; H) at position 21 with asparagine (Asn; N), and

[0224] a substitution of glycine (Gly; G) at position 120 with a lysine (Lys; K) or arginine (Arg; R); and

[0225] to two or more residues at positions 18, 167, 168, 171, 172, 174, 176, and 179 of the human GH, wherein the modification is selected from:

[0226] a substitution of histidine at position 18 with aspartate (Asp; D,) or asparagine (Asn; N), a substitution of arginine at position 167 with asparagine (Asn; N) or glutamate (Glu; E), a substitution of lysine (Lys: K) at position 168 with alanine (Ala; A);

[0227] a substitution of aspartate (Asp: D) at position 171 with a serine (Ser; S);

[0228] a substitution of lysine (Lys; K) at position 172 with an arginine (Arg; R);

[0229] a substitution of glutamate (Glu; E) at position 174 with a serine (Ser; S) or threonine (Thr; T); a substitution of phenylalanine (Phe; F) at position 176 with a tyrosine (Tyr; Y); and

[0230] a substitution of isoleucine (He, I) at position 179 with a threonine (Thr; T).

[0231] In embodiments, the modification comprises two or more mutations, or three or more mutations selected from the group consisting of PhelOAla (or PhelOHis), Metl4Trp (or Metl4Gly), His21Asn, and Glyl20Lys (or Glyl20Arg); and three or more mutations, four or more mutations, five or more mutations, six or more mutations, or seven or more mutations selected from the group consisting of Hisl8Asp (or Hisl8Asn), Argl67Asn (or Argl67Glu), Lysl68Ala, Asp 171 Ser. Lysl72Arg, Glul74Ser (or Glul74Thr), Phel76Tyr, and Ilel79Thr.

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[0233] In embodiments, the GHA comprises a combination of one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of PhelOAla (or PhelOHis), Metl4Trp (or Metl4Gly), His21Asn, and Glyl20Lys (or Glyl20Arg).

[0234] In embodiments, the GHA comprises a combination of one or more mutations, two or more mutations, three or more mutations, four or more mutations, five or more mutations, six or more mutations, or seven or more mutations selected from the group consisting of His 18 Asp (or Hisl8Asn), Argl67Asn (or Argl67Glu), Lysl68Ala, Aspl71Ser, Lysl72Arg. Glul74Ser (or Glul74Thr), Phel76Tyr, and Ilel79Thr.

[0235] In embodiments, the GHA comprises a combination of one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of Phe 1 OAla (or Phe 1 OHis), Metl4Trp (or Metl4Gly). His21Asn, and Glyl20Lys (or Glyl20Arg); and a combination of one or more mutations, two or more mutations, three or more mutations, four or more mutations, five or more mutations, six or more mutations, or seven or more mutations selected from the group consisting of His 18Asp (or Hisl8Asn), Argl67Asn (or Argl67Glu), Lysl68Ala, Asp 171 Ser, Lysl72Arg, Glul74Ser (or Glul74Thr), Phel76Tyr, and Ilel79Thr.

[0236] In embodiments, the GHA comprises a mutation at each of residues 18, 21, 120, 167, 171, and 174. More particularly, tire six mutations at residues 18, 21, 120, 167, 171, and 174 are selected from Hisl8Asp (or Hisl8Asn), His21Asn, Glyl20Lys (or Glyl20Arg, Argl67Asn (or Argl67Glu), Aspl71Ser, and Glul74Ser (or Glul74Thr). In embodiments, the GHA further comprises one or more mutations at residues 10, 14, 168, 172, 176, and 179, and more particularly, the one or more mutations at residues 10, 14, 168, 172, 176, and 179 are selected from PhelOAla (or PhelOHis), Metl4Trp (or Metl4Gly), Lysl68Ala, Lysl72Arg, Phel76Tyr, and Ilel79Thr.

[0237] In embodiments, the GHA comprises the amino acid sequence of GHA5, GHA6, GHA7, GH8, orGHA9.

[0238] In one aspect, the GHA comprises the amino acid sequence of GHA5 (with the amino acid substitutions shown in bold and underlined):

[0239] FPTIPLSRLF DNAMLRADRL NQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVYGASDSNV YDLLKDLEEK IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFNADM SRVSTFLRTV QCRSVEGSCG F

[0240] (GHA5; SEQ ID NO 21)

[0241] In one aspect, the GHA comprises the amino acid sequence of GHA6 (with the amino acid substitutions shown in bold and underlined):

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[0243] FPTIPLSRLA DNAWLRADRL NQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEP VQFLRSVFANS LVYGASDSNV YDLLKDLEER IQTLMGRLEDG SPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFNKDM SKVSTYLRTV QCRSVEGSCG F

[0244] (GHA6: SEQ ID NO:22).

[0245] In one aspect, the GHA comprises the amino acid sequence of GHA7 (with the amino acid substitutions shown in bold and underlined):

[0246] FPTIPLSRLH DNAGLRANRL NQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVYGASDSNV YDLLKDLEER IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFNKDM SKVSTYLRTV QCRSVEGSCG F

[0247] (GHA7; SEQ ID NO:23).

[0248] In one aspect, the GHA comprises the amino acid sequence of GHA8 (with the amino acid substitutions shown in bold and underlined):

[0249] FPTIPLSRLF DNAMLRADRL NQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVYGASDSNV YDLLKDLEER IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFEKDM SKVTTFLRIV QCRSVEGSCG F

[0250] (GHA8; SEQ ID NO: 24).

[0251] In one aspect, the GHA comprises the amino acid sequence of GHA9 (with the amino acid substitutions shown in bold and underlined):

[0252] FPTIPLSRLF DNAMLRANRL NQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVYGASDSNV YDLLKDLEER IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDALLKNY GLLYCFNKDM SKVSTFLRTV QCRSVEGSCG F

[0253] (GHA9; SEQ ID NO:25).

[0254] Without being limited to a particular mechanism, different mutations in a GHA may disrupt GH binding at the site 1 interface (A-B) or site 2 interface (A-C) by altering hydrogen bonding, electrostatic interactions (such as salt bridges), hydrophobic potentials, or by exerting other effects.

[0255] For example, regarding proposed site 2 disruptions (between A and C): PhelO is flanked by identified A-C contacts Arg8 and Asnl2, and a PhelOAla or PhelOHis substitution is predicted to be

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[0257] destabilizing, for example by disrupting hydrophobic packing or polarity at the A-C interface. Met 14 is flanked by identified A-C contacts Asnl2 and Arg 16, and a Metl4Trp or Metl4Gly substitution is predicted to be destabilizing, for example, by disrupting packing, folding, or helical properties at the A-C interface. His21 can contribute to site 2 binding, and a His21Asn substitution is predicted to be destabilizing, for example, by eliminating aromatic or hydrogen bond interactions at the A-C interface. Gly 120 is flanked by identified A-C contact Glut 19, and a Gly 120Lys or Gly 120 Arg substitution is predicted to be destabilizing, for example, by introducing steric hindrance or disrupting folding at the A-C interface.

[0258] For example, regarding proposed site 1 enhancements (between A and B): His 18 is an identified A-B contact, and a Hisl 8Asp or Hisl8Asn substitution is predicted to be stabilizing, for example, by helping to ensure a strong, reliable hydrogen bond network at the A-B interface. Arg 167, Lysl68, and Asp 171 are identified A-B contacts (as part of helix 4 of hGH) and the disclosed substitutions (Argl67Asn (or Argl67Glu), Lysl68Ala, and Aspl71Ser) are predicted to be stabilizing, for example, by enhancing electrostatic interactions (or reducing electrostatic repulsion) at the A-B interface. Lys 172, Glu 174, Phe 176, and He 179 are also part of helix 4 of hGH. and the disclosed substitutions (Lysl72Arg, Glul74Ser (or Glul74Thr), Phel76Tyr, and Ilel79Thr) are predicted to be stabilizing, for example, by enhancing electrostatic interactions (or reducing electrostatic repulsion) or hydrogen bonding at the A-B interface.

[0259] In embodiments, a GHA with targeted mutations at site 1 and site 2 inhibits GHR signaling. In embodiments, a GHA with targeted mutations at site 1 and site 1 enhances specificity for growth hormone receptor blockade, resulting in selective inhibition of downstream signaling pathways such as STAT3 or STAT5 phosphory lation.

[0260] Also provided are GHAs comprising a modification:

[0261] to two or more residues at positions 10, 14, 21, and 120 of the (human) GH selected from: a substitution (mutation) of phenylalanine (Phe; F) at position 10 with alanine (Ala; A) or histidine (His; H), a substitution of methionine (Met; M) at position 14 with tryptophan (Trp; W) or glycine (Gly; G), a substitution of histidine (His; H) at position 21 with asparagine (Asn; N). and a substitution of glycine (Gly; G) at position 120 with a lysine (Lys; K) or arginine (Arg; R);

[0262] and to two or more residues at positions 18, 167, 168, 171, 172, 174, 176, and 179 of the (human) GH selected from: a substitution of histidine at position 18 with aspartate (Asp; D,) or asparagine (Asn; N), a substitution of arginine at position 167 with asparagine (Asn; N) or glutamate (Glu; E), a substitution of lysine (Lys; K) at position 168 with alanine (Ala; A); a substitution of aspartate (Asp; D) at position 171 with a serine (Ser; S); a substitution of lysine (Lys; K) at position 172 with an arginine (Arg; R); a substitution of glutamate (Glu; E) at position 174 with a serine (Ser; S) or threonine (Thr; T); a substitution

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[0264] of phenylalanine (Phe; F) at position 176 with a tyrosine (Tyr; Y); and a substitution of isoleucine (He, I) at position 179 with a threonine (Thr; T).

[0265] In embodiments, the GHA comprises: two or more mutations, or three or more mutations at positions 10, 14, 21, and 120 selected from the group consisting of PhelOAla (or PhelOHis), Metl4Trp (or Metl4Gly), His21Asn, and Glyl20Lys (or Glyl20Arg); and three or more mutations, four or more mutations, five or more mutations, six or more mutations, or seven or more mutations selected from the group consisting ofHisl8Asp (or Hisl8Asn), Argl67Asn (or Argl67Glu), Lysl68Ala, Aspl71Ser, Lysl72Arg, Glul74Ser (or Glul74Thr). Phel76Tyr, and Ilel79Thr.

[0266] In embodiments, the GHA comprises a substitution at each of residues 18, 21, 120, 167, 171, and 174. selected from HisI8Asp (or Hisl8Asn), His21Asn. GlyI20Lys (or Gly 120Arg), Argl67Asn (or Argl67Glu), Asp 171 Ser, and Glul74Ser (or Glul74Thr), and can further comprise one or more mutations at residues 10, 14, 168, 172, 176, and 179, selected from PhelOAla (or PhelOHis), Metl4Trp (or Metl4Gly), Lysl68Ala, Lysl72Arg, Phel76Tyr, and Ilel79Thr.

[0267] In embodiments, the GHA comprises the amino acid sequence of GHA5 (SEQ ID NO:21), GHA6 (SEQ ID NO:22), GHA7 (SEQ ID NO:23), GH8 (SEQ ID NO:24), or GHA9 (SEQ ID NO:25).

[0268] In embodiments, the modifications at positions 10, 14, 21, and 120 are within an A chain of tire GH, targeting residues interacting at an interface (site 2) between the A chain and a C chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations disrupt GH binding at the A-C interface: and wherein the modifications at positions 18, 167. 168, 171. 172, 174. 176, and 179 are within an A chain of the GH, targeting residues interacting at an interface (site 1) between the A chain and a B chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations enhance or stabilize GH binding at the A-B interface.

[0269] Combined Set One and Set Two GHAs

[0270] In embodiments, a GHA comprises one or more site 1 mutations from Set One or Set Two. In embodiments, a GHA comprises one or more site 2 mutations from Set One or Set Two. In embodiments, a GHA comprises one or one or more site 1 mutations from Set One or Set Two; and one or one or more site 2 mutations from Set One or Set Two.

[0271] Variants

[0272] In embodiments, the present disclosure encompasses polypeptide variants that consist of or comprises an amino acid sequence sharing at least 80% homology with any one of the GHAs disclosed herein (including polypeptides and fusion proteins), or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 85% homology with any one of the mutated

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[0274] GHAs disclosed herein, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 90% homology with any one of the mutated GHAs disclosed herein, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 95% homology with any one of the mutated GHAs disclosed herein, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 97% homology with any one of the mutated GHAs disclosed herein, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 99% homology with any one of the mutated GHAs disclosed herein, or a fragment thereof.

[0275] GHA Fusion Proteins

[0276] In embodiments, the GHA is a GHA fusion protein - also referred to as a " GHA fusion7’ or ‘■fusion protein,” in which the GHA further comprises a carrier protein. The carrier protein may be joined (or fused) directly to the N-terminus of the GHA, the C-terminus of a GHA, or to both the N-terminus and C-terminus of a GHA. The carrier protein may also be joined indirectly to either tenninus or both termini of a GHA via a linker, as discussed further herein.

[0277] While GHAs arc useful in treating conditions associated with excessive grow th hormone (GH) activity, a limitation in their application - particularly where long-term action is desirable - is their typical short half-life, which can require frequent dosing to maintain effective plasma levels, potentially reducing patient compliance and therapeutic efficacy. Extending the half-life of GHAs may therefore enhance their clinical utility and reduce the frequency of administration.

[0278] Fusing GHAs to larger, long -circulating carrier proteins is one approach to increase their halflife. This strategy leverages the pharmacokinetic properties of these carrier proteins, which resist rapid degradation and clearance, thereby prolonging the presence of the GHA in circulation. By increasing molecular size, protein fusions can also reduce renal filtration, w hich is a common elimination pathway for smaller therapeutic proteins and peptides. Additionally, such fusions can provide steric hindrance against enzymatic degradation, further extending GHA stability in tire bloodstream.

[0279] The choice betw een an N and C-terminus fusion (or both) depends on numerous factors, such as the protein’s structure and function, the expression system used, purification requirements, necessary post-translational modifications, and the intended biological activity.

[0280] Carrier Proteins

[0281] Accordingly, in embodiments, the GHA, including any one of GHA1-GHA9 and variants thereof, is a GHA fusion comprising a carrier protein (or carrier). More particularly, the carrier protein is selected from albumin, transferrin, an immunoglobulin Fc region (also referred to as an “Fc domain”), and growth hormone binding protein (GHBP).

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[0283] Albumin

[0284] Carrier protein options include serum albumin (Albumin or HSA) proteins. Human serum albumin is a 585 amino acid protein synthesized by hepatocytes and is the most abundant protein in blood plasma, having a long half-life and numerous biological roles, including a carrier of endogenous and exogenous ligands, such as fatty acids, nucleic acids, hormones, metals, toxins, and drugs. De Simone et al.

[0285] 2021, Int. J. Mol. Sci. 18,10086.

[0286] HSA's extended half-life appears to reflect binding to the neonatal Fc receptor (FcRn) and subsequent recycling back into circulation. Chaudhury et al. 2006. Biochemistry. 45, 4983-4990 (Recycling by FcRn can also account for the extended half-life of Fc domain-containing proteins, as discussed below). The neonatal Fc receptor (FcRn) is expressed in a variety of cell types in adults, including vascular endothelium and antigen-presenting cells (APCs).

[0287] Tire HSA protein, as well as HSA -binding motifs, have found use as fusion partners to extend plasma half-lives of protein drugs. Rogers et al. 2015, Curr. Pharm. Des. 21, 1899-1907; Nilvebrant and Hober, 2013, Comput. Struct. Biotechnol. J. 6, e201303009. HSA also tends to accumulate in tumors and in inflamed tissues, suggesting that albumin fusions may help to target therapeutic agents to those sites. Kratz 2008, J. Control. Release 132:171-183

[0288] Accordingly, in embodiments, a carrier is a serum album protein and, more specifically, is human serum album (HSA). In embodiments, human albumin comprises the 585 amino acid sequence of SEQ ID NO: 5:

[0289] 1 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE 61 NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV 121 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP 181 KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK 241 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA 301 DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC 361 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST 421 PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES 481 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT 541 KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGL

[0290] (hALB; SEQ ID NO:5).

[0291] Transferrin

[0292] In embodiments, a carrier protein is a transferrin (TF) protein. Human TF is a 698 amino acid protein synthesized almost exclusively in the liver and secreted into the blood, where it serves as the

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[0294] principal transport protein for iron throughout the body, delivering iron to cells expressing the transferrin receptor (TfR) on the plasma membranes. The transferrin protein is composed of alpha helices and beta sheets that form two homologous domains, each capable of binding a single ferric iron (Fe3+) ion.

[0295] Human transferrin has a long-half-life in serum, reported to be at least 7 days, reflecting a clathrin-dependent transferrin receptor-mediated mechanism that recycles TfR-bound transferrin back into circulation. Gomme et al. 2005, Drug Discov. Today 10, 267-273. Transferrin has been used to deliver small molecule drugs, peptides, proteins, and genes to various target tissues. Li and Qian, 2002, Med. Res. Rev. 22, 225-250.

[0296] Accordingly, in embodiments, a carrier protein is transferrin, and more specifically, is human transferrin (hTRANSF). In one aspect, human transferrin can act as a targeting moiety, directing a fusion protein to liver cells (which have elevated levels of TfR). This may result in improved homing of the therapeutic protein to the liver, which can increase its efficacy and reduce side effects. In embodiments, human transferrin comprises the 698 amino acid sequence of SEQ ID NO:6:

[0297] 1 MRIAVGALLV CAYLGLCLAV PDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVK 61 KASYLDCIRA IAANEADAVT LDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVV 121 KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPE PRKPLEKAVA NFFSGSCAPC 181 ADGTDFPQLC QLCPGCGCST LNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRD 241 QYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKED LIWELLNQAQ EHFGKDKSKE 301 FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA KMYLGYEYVT AIRNLREGTC PEAPTDECKP 361 VKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKI MNGEADAMSL DGGFVYIAGK 421 CGLVPVLAEN YNKSDNCEDT PEAGYFAVAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWN 481 IPMGLLYNKI NHCRFDEFFS EGCAPGSKKD SSLCKLCMGS GLNLCEPNNK EGYYGYTGAF 541 RCLVEKGDVA FVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKSV EEYANCHLAR 601 APNHAVVTRK DKEACVHKIL RQQQHLFGSN VTDCSGNFCL FRSETKDLLF RDDTVCLAKL 661 HDRNTYEKYL GEEYVKAVGN LRKCSTSSLL EACTFRRP

[0298] (hTRANSF; SEQ ID NO: 6)

[0299] As annotated, SEQ ID NO:6 includes an N-terminal signal sequence (spanning residues 1 to 19) highlighted in bold and underlined text, followed by the mature 679 amino transferrin protein (spanning residues 20 to 698). See, e.g., Yang et al. 1984, Proc. Natl. Acad. Set. USA 81, 2752-2756. In embodiments, a transferrin carrier protein comprises the entire sequence of SEQ ID NO:6 and therefore includes the signal peptide. In other embodiments, a transferrin carrier protein comprises the mature transferrin protein, spanning residues 20 to 698 of SEQ ID NO:6.

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[0301] In some embodiments, the transferrin moiety may be modified so it does not exhibit N-linked or O-linked glycosylation. For example, the modified Tf moiety may contain a mutation within or adjacent to the N-linked glycosylation site comprising the sequence N-X-S / T. In embodiments, the transferrin carrier contains a mutation at N432 or N630 or an adjacent S / T residue of SEQ ID NO:6.

[0302] Fc Domain

[0303] Carrier protein options also include immunoglobulin-derived fragment crystal I izablc region (Fc) regions, and more particularly, IgG Fc regions. The Fc domain is the tail portion of an antibody, derived from constant regions, that interacts with cell surface receptors called Fc receptors, as well as with proteins in the complement system.

[0304] Fc-containing IgG immunoglobulins are among the most abundant proteins in human blood, with circulation half-lives that can reach several weeks. Like HSA, the long half-life of Fc-containing proteins reflects FcRn-binding and recycling back into circulation. Roopenian and Akilesh 2007, Nature Rev. Immunol. I, 715-725. This attribute has spurred efforts to develop both C-tenninal and N-terminal IgG fusions to therapeutic agents, as discussed below.

[0305] A prototype Fc region comprises an N-terminal hinge region, CH2 domain, and CH3 domain of an IgG, such as IgGl, IgG2, and IgG4. When the IgG Fc region is joined to a therapeutic protein of interest, the resulting fusion protein is typically a homodimer, comprising two identical Fc-fiision monomers linked through cysteine residues in the hinge region of IgG Fc. Such an Fc fusion protein is therefore structurally similar to an IgG molecule without the CHI domains and light chains. Due to this structural homology, IgG Fc fusion proteins may exhibit pharmacokinetic profiles similar to human IgG, an approach that has been applied to several biologic drugs, including cytokines and soluble receptors. See, e.g., U. S. Pat. No.

[0306] 5,349,053; Capon et al. 1989, Nature 337, 525-531; Harris et al. 1990, Eur. J. Biochem. 194, 611-620;

[0307] Chamow et al. 1996, Trends. Blotechnol. 14, 52-60; Hecht et al. 2012. PloS One 7, e4935.

[0308] Accordingly, in embodiments, a carrier protein is Fc, and more specifically, is human IgG Fc (IgG Fc). In one aspect, the IgG Fc fusion protein is specifically targeted to specific cells, tissues, or organs, and less likely to produce off-target effects, reducing side effects and toxicity.

[0309] In embodiments, the Fc domain is a human IgGl Fc domain, and more particularly, comprises an amino acid sequence of SEQ ID NO:7:

[0310] 1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG

[0311] (hlgG Fc; SEQ ID NO: 7).

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[0313] In embodiments, the Fc domain is a human IgGl Fc domain, comprising one or more effector-reducing mutations selected from the group consisting of L14A, L15A, N77A, and P109G (referring to SEQ ID NO: 7).

[0314] In embodiments, the Fc domain is a human IgGl Fc domain, comprising one or more mutations that enhance binding affinity to neonatal Fc receptor (FcRn) relative to wild-type Fc, selected from the group consisting of M32Y / S34T / T36E (YTE), M208L / 224S (LS), and combinations thereof (referring to SEQ ID NO:7).

[0315] In embodiments, the Fc domain is a human Fc domain, such as an IgG4 Fc domain or modified IgGl Fc domain, comprising a stabilizing proline residue at position 228 (based on EU numbering).

[0316] In embodiments, tire Fc domain is an engineered human IgG Fc domain, and more particularly, comprises an amino acid sequence of SEQ ID NO:34:

[0317] 1 DKTHTCPPCP APEAAAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY 61 VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL GAPIEKTISK 121 AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVL HEALHSHYTQ KSLSLSPGK

[0318] (hlgG Fc (mod); SEQ ID NO:34).

[0319] Because an Fc domain naturally dimerizes, a GHA-containing fusion protein comprising any of the Fc domains described herein can be bivalent for the GHR. Accordingly, in embodiments, the disclosure provides a fusion protein, in which the fusion protein is a homodimer comprising two identical polypeptide chains, each chain comprising the GH antagonist polypeptide linked to the Fc domain.

[0320] Growth Hormone Binding Protein

[0321] Carrier protein options also include a growth hormone binding protein (GHBP) or an extracellular domain of a GHR. In some embodiments, the GBHP corresponds to residues 1 1-256 of the human GHR (Accession No. AAA52555), comprising tire 246 amino acid sequence of SEQ ID NO:8:

[0322] 1 ALAGSSDAFS GSEATAAILS RAPWSLQSVN PGLKTNSSKE PKFTKCRSPE RETFSCHWTD 61 EVHHGTKNLG PIQLFYTRRN TQEWTQEWKE CPDYVSAGEN SCYFNSSFTS IWIPYCIKLT 121 SNGGTVDEKC FSVDEIVQPD PPIALNWTLL NVSLTGIHAD IQVRWEAPRN ADIQKGWMVL 181 EYELQYKEVN ETKWKMMDPI LTTSVPVYSL KVDKEYEVRV RSKQRNSGNY GEFSEVLYVT 241 LPQMSQ

[0323] (hGHBP 246; SEQ ID NO: 8).

[0324] In some embodiments, tire GBHP corresponds to residues 19-256 of the human GHR (Accession No. AAA52555), comprising the 238 amino acid sequence of SEQ ID NO:9:

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[0326] 1 FSGSEATAAI LSRAPWSLQS VNPGLKTNSS KEPKFTKCRS PERETFSCHW TDEVHHGTKN 61 LGPIQLFYTR RNTQEWTQEW KECPDYVSAG ENSCYFNSSF TSIWIPYCIK LTNGGTVDES 121 KCFSVDEIVQ PDPPIALNWT LLNVSLTGIH ADIQVRWEAP RNADIQKGWM VLEYELQYKE 181 VNETKWKMMD PILTTSVPVY SLKVDKEYEV RVRSKQRNSG NYGEFSEVLY VTLPQMSQ (hGHBP_238; SEQ ID NO: 9).

[0327] In embodiments, a GHA polypeptide disclosed herein can be fused at its N-terminus, C-tenninus (or both termini) to any carrier protein disclosed herein, with or without a linker. In embodiments, the GHA domain in the fusion protein is GHA1, GHA-2, GHA-3. or GHA-4. In embodiments, the GHA domain in the fusion protein is GHA5, GHA-6, GHA-7, GHA-8. or GHA-9.

[0328] Linkers

[0329] In embodiments, tire carrier protein is joined to the GHA molecule by a flexible linker consisting of one or more amino acids. The linker can join the carrier protein to the N-terminus of a GHA, the C-terminus of a GHA, or to both the N-terminus and C-tenninus of a GHA.

[0330] Generally, the peptide linker will have no (or minimal) biological activity other than joining the carrier molecule to the GHA molecule or preserving a minimal distance or spatial relationship between them. However, in embodiments, the constituent amino acids of the linker may be selected to influence properties of the fusion protein, GHA molecule, or carrier molecule, such as folding or net charge.

[0331] In embodiments, the linker is relatively short, and in particular embodiments, is less than about 10 amino acids, less than about 8 amino acids, or less than 6 amino acids.

[0332] Non-limiting examples include glycine, serine, and glycine-serine linkers. Glycine-rich linkers, such as (G)n or (GGG)n, may offer flexibility but reduced solubility. Serine-rich linkers such as (S)n or (SSS)n may offer rigidity but greater solubility. Glycine-serine linkers may confer flexibility and solubility. In embodiments, the linker is a glycine-serine mixture, such as (GGSGG)n, (GGSGGS)n or (GGGS)n, where n varies from 1 to 10, 1 to 8. 1 to 6, or 1 to 4. More particularly, a glycine-serine linker (also referred to as a glycine rich linker) corresponds to (GGGGS)n, where n varies from 1 to 10. In embodiments, a glycine-serine linker corresponds to (GGGGS)3or (GGGGS)4, i.e., GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS, or corresponds to a combination, such as (GGGGS)2GGGG, i.e., GGGGSGGGGSGGGG.

[0333] In embodiments, a linker between a GHA and carrier can also comprise non-natural amino acids or a chemical spacer not based on amino acids.

[0334] Selecting a linker reflects numerous considerations that can allow tire constituent protein domains to operate synergistically while retaining their individual functions. Such considerations include

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[0336] reducing steric hindrance, attaining appropriate flexibility to properly orient the domains, increasing overall stability, improving solubility, and protecting against undesired proteolytic cleavage.

[0337] In embodiments, the present disclosure provides a fusion protein comprising any GHA and any albumin carrier protein disclosed herein. In embodiments, the fusion protein comprises an N-terminal albumin domain, a linker sequence, and a C-terminal GHA domain. In embodiments, the fusion protein comprises an N-terminal GHA domain, a linker domain, and a C-terminal albumin domain. In embodiments, the fusion protein comprises an N-terminal albumin domain, a GHA domain flanked on each side by a linker sequence, and a C-terminal albumin domain. In specific embodiments, the albumin domain is HSA. more particularly, an amino acid sequence of SEQ ID NO:5; and the GHA domain can be GHA1, GHA2, GHA3, GHA4, GHA5, GHA6, GHA7, GHA8, or GHA9. In specific embodiments, the linker domain is GGGGSGGGGSGGGGS.

[0338] In embodiments, the present disclosure provides a fusion protein comprising any GHA and any transferrin carrier protein disclosed herein. In embodiments, tire fusion protein comprises an N-terminal transferrin domain, a linker sequence, and a C-terminal GHA domain. In embodiments, the fusion protein comprises an N-terminal GHA domain, a linker domain, and a C-terminal transferrin domain. In embodiments, the fusion protein comprises an N-terminal transferrin domain, a GHA domain flanked on each side by a linker domain, and a C-terminal transferrin domain. In specific embodiments, the transferrin domain is human transferrin, more particularly, an amino acid sequence of SEQ ID NO:6; and the GHA domain is GHA1, GHA2, GHA3, GHA4, GHA5, GHA6, GHA7, GHA8, or GHA9. In specific embodiments, the linker domain is GGGGSGGGGSGGGGS.

[0339] In embodiments, the present disclosure provides a fusion protein comprising any GHA and any Fc carrier protein described herein. In embodiments, the fusion protein comprises an N-terminal Fc domain, a linker sequence, and a C-terminal GHA domain. In embodiments, the fusion protein comprises an N-terminal GHA domain, a linker domain, and a C-terminal Fc domain. In some embodiments, the fusion protein comprises an N-terminal Fc domain, a GHA domain flanked on each side by a linker sequence, and a C-terminal Fc domain. In specific embodiments, the Fc domain is human IgG Fc, more particularly, an amino acid sequence of SEQ ID NO:7; and the GHA domain is In specific embodiments, the transferrin domain is human transferrin, more particularly, an amino acid sequence of SEQ ID NO:6; and tire GHA domain is GHA1, GHA2, GHA3, GHA4, GHA5, GHA6, GHA7, GHA8, or GHA9. In specific embodiments, the linker domain is GGGGSGGGGSGGGGS. In specific embodiments, the linker domain is GGGGSGGGGSGGGGS.

[0340] In embodiments, the present disclosure provides a fusion protein comprising any GHA and any GHBP carrier domain described herein. In embodiments, the fusion protein comprises an N-terminal GHBP

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[0342] domain, a linker sequence, and a C-terminal GHA domain. In embodiments, the fusion protein comprises an N-terminal GHA domain, a linker domain, and a C-terminal GHBP domain. In embodiments, the fusion protein comprises an N-terminal GHBP domain, a GHA domain flanked on each side by a linker sequence, and a C-terminal GHBP domain. In specific embodiments, the GHBP domain is human GHBP, more particularly, an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9: and the GHA domain is GHA1, GHA2, GHA3, or GHA4. In embodiments, the linker domain is GGGGSGGGGSGGGGS.

[0343] Targeting Domains

[0344] In embodiments, a GHA (including a fusion protein) comprises a targeting (or homing) domain. A targeting domain is a functional module within a therapeutic protein that directs (homes) the molecule to a specific cell type, tissue, organ, or subcellular location. It acts as an "address label," ensuring the therapeutic payload is delivered where it is needed, minimizing off-target effects and improving efficacy.

[0345] Targeting domains are commonly used in fusion proteins, antibody-drug conjugates (ADCs), bispecific antibodies, and engineered biologies, where one domain targets and another domain exerts the therapeutic effect.

[0346] Targeting domains can include a receptor ligand or a peptide-based domain. They can also include a domain based on an antibody; an antibody fragment, such as an Fab fragment (typically comprising one light chain and the Vnand CHI domains of one heavy chain, held together by disulfide bonds); an scFv (single-chain variable fragment, which typically comprises tire VH and VL domains connected by a flexible linker peptide: or a nanobody (VHH): a single-domain antibody fragment derived from camelids.

[0347] In embodiments, the targeting domain is a transferrin receptor (TfR) binding domain. In embodiments, the TfR binding domain comprises an antibody or antibody fragment that binds TfR, such as an Fab, scFv, nanobody, or antigen-binding fragment thereof.

[0348] In embodiments, the targeting domain is the transferrin receptor-binding peptide T12 (also designated THRor TBP), a 12-amino acid synthetic peptide with tire sequence THRPPMWSPVWP, which binds the human transferrin receptor (TfRl / CD71). See, e.g., U. S US 6,743,893. Notably, TBP binds to a site of TfRl that is distinct from that of native transferrin. Consequently, TBP does not interfere with transferrin binding, iron homeostasis is maintained, and circulating holo-transferrin does not compete for receptor occupancy — a limitation that has historically plagued transferrin-conjugated drug delivery' approaches.

[0349] Protease-Cleavable Domains

[0350] In embodiment, a GHA (including a fusion protein) comprises a protease-cleavable linker (also referred to as a protease-cleavable sequence or a cleavable peptide sequence). A protease -cleavable linker is a peptide sequence engineered into a therapeutic protein construct that is selectively cut by a specific protease enzyme. This cleavage releases or activates a functional domain — enabling spatially and

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[0352] temporally controlled drug activity, such as at the intended disease site (e.g., a tumor or inflamed tissue). See, e.g., Poreba 2020, FEBSJ. 287, 1936-1969.

[0353] Design Considerations include selectivity: the linker must not be cleaved by off-target proteases in circulation; kinetics: the rate of cleavage must be fast enough for therapeutic effect; stability: the cleavage domain must resist serum proteases during systemic transit; and length and flexibility: the linker must not preclude steric accessibility of the cleavage site.

[0354] Examples of cleavable peptide sequences include Val-Cit (VC), which is cleaved by Cathepsin-B; GFLG, which is cleaved by Cathepsin-A; PLGLAG / PLGVR, which is cleaved by MMP-2 / 9; Asn-Ala-Ala-Leu, which is cleaved by Legumain; and DEVD, which is cleaved by Caspase-3.

[0355] In embodiments, the protease cleavable sequence is cleaved by one or more matrix metalloproteinases (MMPs). which are zinc-dependent endopeptidases. Notably, MMPs can be overexpressed in the tumor microenvironment (TME), making MMP-specific cleavage sites useful for cancer-targeted drug delivery. See, e.g., Geiger et al. 2020, Nat. Commun. 11, 3196. This enables stimulus-responsive (enzyme-triggered) release of therapeutics or imaging agents specifically at disease sites.

[0356] In embodiments, the cleavable linker corresponds to “PLGLAG,” which is selectively cleaved by MMPs, particularly MMP-2 (gelatinase A) and MMP-9 (gelatinase B). MMP-2 and MMP-9 share highly- overlapping substrate specificities, so most " MMP-2 cleavable" linkers are in practice MMP-2 / 9 cleavable (but this is generally acceptable as MMP-2 and MMP-9 are typically co-overexpressed in tumors).

[0357] In embodiments, a cleavage motif, such as PLGLAG, is flanked on either side by spacers to ensure steric accessibility. Such spacers can comprise one or more copies of glycine rich sequences, as disclosed herein, including (GGG)n or (GGGS)n, when n varies from 1 to 10, such as (GGGS)3and (GGGS)4.

[0358] GHA Modular Designs

[0359] As described herein, a GHA, such as a GHA fusion, can include a carrier protein, as well as other domains, such as linkers, targeting domains, protease -cleavable sequences, and spacers. In embodiments, such GHAs (fusions) have a modular design.

[0360] In embodiments, a GHA fusion has a modular design exemplified by: [Transferrin (Tf) or TfR binding domain] — [Protease-cleavable linker] — [Flexible Linker] — [GHA] — [Carrier Protein]. In other embodiments, the order of the domains within the fusion design can be arranged in any functional permutation. For example, in embodiments, a GHA fusion can have a modular design exemplified by: [Carrier Protein] — [Flexible Linker] — [GHA] — [Protease-cleavable linker] — [Transferrin (Tf) or TfR binding domain].

[0361] In embodiments, the Tf domain comprises a transferrin polypeptide, such as human transferrin, which can function as a targeting domain, as described herein,.

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[0363] In embodiments, the TfR binding domain comprises an antibody or antibody fragment that binds TfR, optionally an Fab, scFv, nanobody, or antigen-binding fragment thereof.

[0364] In embodiments, the TfR binding domain is a Tf-mimetic peptide or engineered protein domain that binds TfR, such as transferrin receptor-binding peptide T12 (THRPPMWSPVWP), as described further in Example 13.

[0365] In embodiments, the protease-cleavable linker comprises a peptide substrate peptide cleavable by a protease selected from matrix metalloproteinases (MMPs), cathepsins, urokinase-type plasminogen activator (uPA), plasmin, legumain, and furin. In embodiments, the protease-cleavable linker comprises a peptide substrate cleavable by cathepsin B or cathepsin L or both. In embodiments, the protease -cleavable linker comprises a peptide substrate cleavable by MMP-2 or MMP-9, or both.

[0366] In embodiments, the protease-cleavable linker comprises a cleavage motif selected from PLGLAG (cleaved primarily by MMPs. especially MMP-2 (gelatinase A) and MMP-9 (gelatinase B).

[0367] GPLGVRG (also an MP-cleavable sequence, with selectivity toward MMP-2 and MMP-9, GFLG (recognized by cathepsins, particularly cathepsin B), RVRR (substrate for furin, which is also known as PACE), and AAN (cleaved by legumain - a lysosomal cysteine protease). In embodiments, the protease cleavable linker is PLGLAG, as in SEQ ID NO:33 (see Example 13. In embodiments, the protease cleavable linker is flanked by a spacer domain, such as a glycine rich sequences, as disclosed herein, including (GGG)n or (GGGS)n, when n varies from 1 to 10, such as (GGGS)3and (GGGS)4. In embodiments, the protease-cleavable linker is cleavable by a protease that is upregulated or enriched in a target tissue microenvironment relative to healthy tissue, such as MMPs in tumor microenvironments.

[0368] Tire flexible linker can include any of tire linkers disclosed herein, including glycine and serine linkers, as well as glycine-serine linkers (glycine rich linkers) such as (GGSGG)n or (GGSGGS)n or (GGGS)n. where n varies from 1 to 10.

[0369] In embodiments, the GHA can be any of the growth hormone antagonist disclosed herein. Thus, a GHA can comprise any combination of site 1 and site 2 mutations from Set One or Set Two. More particularly, a GHA comprises GHA1, GHA2, GHA3, GHA4, GHA5. GHA6, GHA7, GHA8, or GHA9. In embodiments, the GHA comprises pegvisomant or a pegvisomant-like antagonist variant that blocks GHR activation without agonism.

[0370] In embodiments, the carrier domain includes any of the carrier proteins disclosed herein for extending half-life of the GHA fusion. In embodiments, the carrier protein (half-life extension moiety) is selected from selected from albumin, an albumin-binding domain, an immunoglobulin (IgG), or an IgG Fc domain. In embodiments, the half-life extension moiety comprises an Fc domain configured to form a homodimer or heterodimer.

[0371] In embodiments, these GHAs (or variants thereof) can be used in any of the formulations, compositions, and methods disclosed herein. In embodiments, these GHAs (or variants thereof) can be used -40- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0372] in the manufacture of a medicament for treating a disease associated with excessive GH / GHR signaling, disclosed further herein.

[0373] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 10: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLGGGGS'GGGGSGGGGS'FPT I PL SEL FDAAMLEAHRLHQLAFDT YQE FEEAY I PKEQKY S FLQNP QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERG IQTLMGRLEDGSPRTGQI FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCF

[0374] (hALB-GHAl; SEQ ID NO: 10).

[0375] SEQ ID NO: 10 includes an N-temrinal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGGS ), which is followed by the mutant human GHA1 sequence ( FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSL CFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTL MGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) contain ing site 2 interaction mutations highlighted in bold and underlined text.

[0376] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 11: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI R RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH

[0377] -41- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0378] CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLGGGGS'GGGGSGGGGS'FPT I PL SLL EDI AMLLAHRLHQLAFDT YQE FEEAY I PKEQKY S FLQNP QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVLGASDSNVYDLLKDLEVG IQTLMGRLEDGSPRTGQI FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCF

[0379] (hALB-GHA2; SEQ ID NO: 11).

[0380] SEQ ID NO: 11 includes an N-terminal human serum albumin domain ( DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGGS ), which is followed by the mutant human GHA2 sequence ( FPTIPLSLLFDIAMLLAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCF SESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVLGASDSNVYDLLKDLEVGIQTLMG RLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations shown in bold and underlined text.

[0381] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 12: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLGGGGSGGGGSGGGGSFPT I PL SGL FDSAMLAAHRLHQLAFDT YQE FEEAY I PKEQKY S FLQNP QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVSGASDSNVYDLLKDLEAG IQTLMGRLEDGSPRTGQI FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCF

[0382] -42- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0383] (hALB-GHA3; SEQ ID NO: 12).

[0384] SEQ ID NO: 12 includes an N-terminal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGGS ), which is followed by the mutant human GHA3 sequence ( FPTIPLSGLFDSAMLAAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSL CFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVSGASDSNVYDLLKDLEAGIQTL MGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) contain ing site 2 interaction mutations shown in bold and underlined text.

[0385] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 13: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI R RHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLK VMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLGGGGSGGGGSGGGGSFPT I PL SWL FDQAMLWAHRLHQLAFDT YQE FEE Y I PKEQKY S FLQNP QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERG IQTLMGRLEDGSPRTGQI FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCF

[0386] (hALB-GHA4; SEQ ID NO: 13).

[0387] SEQ ID NO: 13 includes an N-terminal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK

[0388] -43- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0389] CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGGS ), which is followed by the mutant human GHA4 sequence ( FPTIPLSWLFDQAMLWAHRLHQLAFDTYQEFEEAYIPKE QKYSFLQNPQTSL CFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTL MGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations shown in bold and underlined text.

[0390] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 14: FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCFGGGGSGGGGSGGGGSDAHKSEVAHRF KDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELL FFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICT LSEKERQIKKQT AL VELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL

[0391] (GHAl-hALB; SEQ ID NO: 14).

[0392] SEQ ID NO: 14 includes an N-temiinal mutant human GHA1 sequence

[0393] ( FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations highlighted in bold and underlined text, followed by a peptide linker ( GGGGSGGGGSGGGGS ), which is followed by a human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSL HTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKY LYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERA FKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKP LLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYE TTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV

[0394] -44- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0395] PKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAE EGKKLVAASQAALGL).

[0396] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 15: FPTIPLSLLFDIAMLLAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVLGASDSNVYDLLKDLEVGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCFGGGGSGGGGSGGGGSDAHKSEVAHRF KDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELL FFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICT LSEKERQIKKQT LVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0397] (GHA2-hALB; SEQ ID NO: 15).

[0398] SEQ ID NO: 15 includes an N-terminal mutant human GHA2 sequence

[0399] ( FPTIPLSLLFDIAMLLAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVLGASDSNVYDLLKDLEVGIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations shown in bold and underlined text, followed by a peptide linker (GGGGSGGGGSGGGGS ), which is followed by a human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKS LHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGER AFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEK PLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTY ETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVE VSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETY VPKE FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA EEGKKLVAASQAALGL).

[0400] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 16: FPTIPLSGLFDSAMLAAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVSGASDSNVYDLLKDLEAGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCFGGGGSGGGGSGGGGSDAHKSEVAHRF

[0401] -45- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0402] KDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELL FFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICT LSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0403] (GHA3-hALB: SEQ ID NO: 16).

[0404] SEQ ID NO: 16 includes an N-terminal mutant human GHA3 sequence

[0405] ( FPTIPLSGLFDSAMLAAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVSGASDSNVYDLLKDLEAGIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations shown in bold and underlined text, followed by a peptide linker (GGGGSGGGGSGGGGS ), which is followed by a human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENC DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETF LKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKF GERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKEC CEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPT LVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVD ETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCKADDKETC FAEEGKKLVAASQAALGL ).

[0406] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 17: FPTIPLSWLFDQAMLWAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCFGGGGSGGGGSGGGGSDAHKSEVAHRF KDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELL FFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA

[0407] -46- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0408] KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICT LSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0409] (GHA4-hALB; SEQ ID NO: 17).

[0410] SEQ ID NO: 17 includes an N-terminal mutant human GHA4 sequence

[0411] ( FPTIPLSWLFDQAMLWAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations shown in bold and underlined text, followed by a peptide linker (GGGGSGGGGSGGGGS ), which is followed by a human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCD KSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFG ERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECC EKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAK TYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTL VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETC FAEEGKKLVAASQAALGL).

[0412] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 18: FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCFGGGGSGGGGSGGGGSMRLAVGALLVC AVLGLCLAVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDA GLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLY CDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHS TIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHER LKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEA GYFAVAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCK LCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTR KSVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAK LHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP

[0413] (GHAl-hTRANSF; SEQ ID NO: 18).

[0414] -47- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0415] SEQ ID NO: 18 includes an N-terminal mutant human GHA1 sequence ( FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations highlighted in bold and underlined text, followed by a peptide linker ( GGGGSGGGGSGGGGS), which is followed by a human transferrin

[0416] domain (MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVK KASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAE FYGSKEDPQTFYYAVAVVKKDSGFQMNQLR GKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQY FGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMG GKEDLIWELLNQAQEHFGKDKSKE FQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREG TCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGK CGLVPVLAENYNKSDNCEDTPEAGYFAVAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINH CRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGK NPDPWAKNLNEKDYELLCLDGTRKSVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSG NFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP ).

[0417] In embodiments, a GHA1 -Transferrin fusion protein does not include the underlined signal sequence (MRL VGALLVCAVLGLCL ) of the transferrin domain.

[0418] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 19: FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCFGGGGSGGGGSGGGGSDAHKSEVAHRF KDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELL FFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICT LSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV7YASQAALGL (GHAl-hlgGl Fc; SEQ ID NO: 19).

[0419] SEQ ID NO: 19 includes an N-terminal mutant human GHA1 sequence

[0420] ( FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTY

[0421] -48- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0422] SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations shown in bold and underlined text, followed by a peptide linker ( GGGGSGGGGSGGGGS ), which is followed by a human IgG 1-Fc binding

[0423] domain ( DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG).

[0424] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 19: FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCFGGGGSGGGGSGGGGSDAHKSEVAHRF KDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELL FFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICT LSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0425] (GHAl-hlgGl Fc: SEQ ID NO: 19).

[0426] SEQ ID NO: 19 includes an N-temiinal mutant human GHA1 sequence

[0427] ( FPTIPLSELFDAAMLEAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVFGASDSNVYDLLKDLERGIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF) containing site 2 interaction mutations shown in bold and underlined text, followed by a peptide linker ( GGGGSGGGGSGGGGS, which is followed by a human IgGl-Fc binding domain (DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ).

[0428] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:26: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI R RHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH

[0429] -49- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0430] CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLGGGGSGGGGSGGGGFPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQ TSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKI QTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF

[0431] (hALB-GHA5; SEQ ID NO:26).

[0432] SEQ ID NO:26 includes an N-terminal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGG ), which is followed by the mutant human GHA5 sequence ( FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQT SLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQ TLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF).

[0433] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:27: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNC ELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH EKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHK PKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGFPTIPLSRL ADNAWLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLL LIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHND DALLKNYGLLYCFNKDMSKVSTYLRTVQCRSVEGSCGF

[0434] -50- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0435] (hALB-GHA6; SEQ ID NO:27).

[0436] SEQ ID NO:27 includes an N-terminal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGG ), which is followed by the mutant human GHA6 sequence ( FPTIPLSRLADNAWLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQ TSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERI QTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRTVQCRSVEGSCGF).

[0437] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:28: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGLGGGGSGGGGSGGGGFPTIPLSRLHDNAGLRANRLNQLAFDTYQEFEEAYIPKEQKYSFLQNP QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEER IQTLMGRLEDGSPRTGQI FKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRTVQCRSVEGSCGF

[0438] (hALB-GHA7; SEQ IDNO:28).

[0439] SEQ ID NO:28 includes an N-terminal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG

[0440] -51- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0441] KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGG ), which is followed by the mutant human GHA7 sequence ( FPTIPLSRLHDNAGLRANRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPT PSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERIQTLMGRLEDGS PRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRTVQCRSVEGSCGF).

[0442] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 29: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI R RHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLGGGGSGGGGSGGGGFPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQ TSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERI QTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFEKDMSKVTTFLRIVQCRSVEGSCGF

[0443] (hALB-GHA8; SEQ ID NO:29).

[0444] SEQ ID NO:29 includes an N-terminal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGG ), which is followed by the mutant human GHA8 sequence ( FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLC FSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERIQTLM GRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFEKDMSKVTTFLRIVQCRSVEGSCGF).

[0445] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:30:

[0446] -52- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0447] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLGGGGSGGGGSGGGGFPTIPLSRLFDNAMLRANRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQ TSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERI QTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTFLRTVQCRSVEGSCGF

[0448] (hALB-GHA9; SEQ ID NO:30).

[0449] SEQ ID NO: 30 includes an N-terminal human serum albumin domain (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIA RRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKS HCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL ) followed by a peptide linker ( GGGGSGGGGSGGGG ), which is followed by the mutant human GHA9 sequence ( FPTIPLSRLFDNAMLRANRLNQLAFDTYQEFEEAYIPKE QKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVY DLLKDLEERIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTFLRTVQCR SVEGSCGF).

[0450] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:31: FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGFGGGGSGGGGSGGGGSGGGGSDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG

[0451] -53- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0452] (GHA5-hIgGl Fc; SEQ ID NO: 31).

[0453] SEQ ID NO: 31 includes an N-terminal mutant GHA5 sequence ( FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF) followed by a peptide

[0454] linker ( GGGGSGGGGSGGGGSGGGGS ), which is followed by a human IgGl Fc domain ( DKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG).

[0455] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:32: FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGFGGGGSGGGGSGGGGSGGGGSDKTHT CPPCPAPEAAAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQK SLSLSPGK

[0456] (GHA5-hIgG Fc (mod); SEQ ID NO: 32).

[0457] SEQ ID NO: 32 includes an N-terminal mutant GHA5 sequence

[0458] ( FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTY SKFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF) followed by a peptide

[0459] linker (GGGGSGGGGSGGGGSGGGGS ), which is followed by an engineered human IgGl Fc domain of SEQ ID NO:34 ( DKTHTCPPCPAPEAAAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VLHEALHSHYTQKSLSLSPGK).

[0460] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:33:

[0461] THRPPMWSPVWPGGGSGGGSGGGSPLGLAGGGGSGGGSGGGSGGGSDAHKSEVAHRFKDLGEENFKALVLIA FAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPER NECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQ AADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH

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[0463] TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKD VCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLN QLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALV ELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGFPT IPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLEL LRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFD TNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF

[0464] (TBP-MMP-hALB-GHA5; SEQ ID NO:33).

[0465] SEQ ID NO:33 includes anN-terminal mutant transferrin-binding peptide T12 (THRPPMWSPVWP ), followed by a spacer ( GGGSGGGSGGGS ), which is followed by an MMP2 / MMP9 cleavage domain ( PLGLAG), which is followed by a spacer ( GGGSGGGSGGGSGGGS ), which is followed by a human serum albumin domain ( DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTE FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEV DVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKAS SAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALL VRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA AFVEKCCKADDKETCFAEEGKKLVAASQAALGL ), which is followed by a peptide linker ( GGGGSGGG GSGGGG ), which is followed by the mutant GHA5 sequence ( FPTIPLSRLFDNAMLRADRLNQLA FDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSV FANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNA DMSRVSTFLRTVQCRSVEGSCGF).

[0466] Variants

[0467] In embodiments, the present disclosure encompasses polypeptide variants that consist of or comprises an amino acid sequence sharing at least 80% homology with any one of the carrier proteins disclosed herein, including SEQ ID NOS:5-9, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 85% homology with any one of the carrier proteins disclosed herein, including SEQ ID NOS:5-9, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 90% homology with any one of the carrier proteins disclosed herein, including SEQ ID NOS:5-9, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 95% homology with any one of the carrier proteins -55- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0468] disclosed herein, including SEQ ID NOS:5-9, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 97% homology with any one of the carrier proteins disclosed herein, including SEQ ID NOS:5-9, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 99% homology with any one of the carrier proteins disclosed herein, including SEQ ID NOS:5-9, or a fragment thereof.

[0469] In embodiments, the present disclosure encompasses protein variants that consist of or comprises an amino acid sequence sharing at least 80% homology with any one of the fusion proteins disclosed herein, including SEQ ID NOS: 10-19, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 85% homology with any one of the fusion proteins disclosed herein, including SEQ ID NOS: 10- 19, or a fragment thereof including SEQ ID NOS: 10-19. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 90% homology with any one of the fusion proteins disclosed herein, including SEQ ID NOS: 10-19, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 95% homology with any one of the fusion proteins disclosed herein, including SEQ ID NOS: 10-19, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 97% homology with any one of the fusion proteins disclosed herein, including SEQ ID NOS: 10-19, or a fragment thereof. In embodiments, a variant consists of or comprises an amino acid sequence sharing at least 99% homology with any one of the fusion proteins disclosed herein, including SEQ ID NOS: 10-19, or a fragment thereof.

[0470] Compositions and Formulations

[0471] While one or more GHAs, such as GHA polypeptides and GHA fusion proteins, of the current disclosure may be administered alone, they may also be present as part of a pharmaceutical composition as a mixture with a conventional excipient, i.e., a pharmaceutically acceptable organic or inorganic substance, vehicle, or carrier suitable for administration. Any of the GHAs can also be used in the manufacture of a medicament for treating any of the diseases, as described herein, as well as used in any of the diagnostic methods herein.

[0472] Pharmaceutical compositions of the invention in general comprise one or more GHA proteins or GHA polynucleotides (such as mRNA or DNA encoding such GHAs), together with one or more acceptable excipients.

[0473] Excipient are “acceptable” in the sense of being compatible with other ingredients of the formulation and not deleterious to the recipient thereof. Suitable pharmaceutically acceptable excipients include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, fatty acid esters, hydroxymethyl-cellulose, and polyvinylpyrrolidone. The

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[0475] pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously react with the active compounds.' Examples of suitable pharmaceutical excipients are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

[0476] The particular vehicle, excipient or carrier used may vary, depending on the patient and condition, therapeutic agent (e.g., a GHA polypeptide versus a GHA polynucleotide), and mode of administration.

[0477] In a preferred embodiment, the composition is fonnulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0478] GHA Proteins

[0479] In embodiments, a GHA composition is formulated for delivery of a GHA protein, i.e., any GHA polypeptide or GHA fusion protein disclosed herein.

[0480] Formulations for GHA proteins can comprise liquid based formulations suitable for immediate and direct use. for example, as an injectable. Such formulations can comprise a solution with additional excipients, such as a buffer, e.g., a phosphate or citrate salt to provide a suitable pH. typically about 6 to 7.5; a stabilizer, e.g., trehalose or mannitol; and a preservative, e.g., benzyl alcohol.

[0481] Formulations for GHA proteins can comprise a lyophilized formulation, e.g., a freeze-dried powder, offering enhanced stability and a longer shelf life. Such lyophilized formulations may include excipients such as cryoprotectants, e.g., sucrose or glycine; and bulking agents, e.g., mannitol. Such freeze-dried formulations can be reconstituted before use, e.g., in sterile water or saline.

[0482] Formulations for GHA proteins can also comprise microparticle or nanoparticle formulations, allowing encapsulation of GHA for controlled and sustained release. Such formulations may be based on biodegradable polymers such as PLGA or chitosan, allowing nanoparticle-encapsulation of a GHA protein; or be based on lipid-based carriers, allowing liposome-encapsulation of a GHA protein. The particle size can be tailored, e.g., from 50 nm - 200 pm, in accordance with the desired release kinetics.

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[0484] Formulations for GHA proteins can also comprise a long-acting depot formulation, allowing direct injection into a target organ to deliver a high concentration of drug locally and to minimize the risk of systemic toxicity. Such depot injectables create a storage (or depot) of tire drug in the injected target, allowing slow sustained release into the body over an extended period of time, e.g., weeks, or even months. Such depot preparations can include an injectable gel or microsphere-based depot, with excipients that may include matrix components such as biocompatible polymers, e.g., hyaluronic acid or a poloxamer: and oils, e.g., sesame or castor oil. They may also include bioavailability enhancers, such as lipid-based components that provide a self-emulsifying delivery system. Depot formulations can be administered by intramuscular or subcutaneous injections, as well as by direct injections into a desired targeted organ.

[0485] In any of these protein formulations, the GHA protein may optionally be conjugated to high molecular weight molecules to increase its multivalency and avidity of binding. In embodiments, the high molecular weight molecules are water soluble molecules, including, but not limited to, peptides, saccharides, poly(vinyls), poly(ethers), poly(amines), poly(carboxylic acids) and the like. In embodiments, the water-soluble polymer is dextran, polyethylene glycol (PEG), polyoxyalkylene, polysialic acid, starch, or hydroxyethyl starch. More specifically, the water soluble polymer is PEG. Any suitable method may be used to conjugate the GHA protein or fusion to water-soluble polymers such as PEG. See, e.g., Hermanson, Bioconjugate Techniques 2ndEd., Academic Press, Inc. 2008.

[0486] GHA Polynucleotides

[0487] In embodiments, a GHA composition is formulated for delivery' of a GHA polynucleotide (nucleic acid), such as a polynucleotide encoding any GHA polypeptide or GHA fusion protein disclosed herein. Polynucleotides include, but arc not limited to, RNA molecules and DNA molecules.

[0488] In embodiments, the polynucleotide encodes a GHA polypeptide, and in specific embodiments, encodes an amino acid sequence of GHA1 (SEQ ID NO: 1), GHA2 (SEQ ID NO:2), GHA3 (SEQ ID NO:3), or GHA4 (SEQ ID NO:4). GHA5 (SEQ ID NO:21), GHA6 (SEQ ID NO:22), GHA 7 (SEQ ID NO:23), GHA8 (SEQ ID NO:24), or GHA9 (SEQ ID NO:25).

[0489] In embodiments, the polynucleotide encodes any GHA fusion disclosed herein, such as a fusion encoding a GHA joined directly (or indirectly via a linker) at its amino terminus (or carboxy terminus) to an albumin, transferrin, immunoglobulin Fc domain, or growth hormone binding protein (GHBP), as well as a fusion comprising additional domains, such as targeting sequence, protease cleavable linker, and a spacer sequence. In embodiments, the GHA domain encoded by the polynucleotides comprises an amino acid sequence of GHA1 (SEQ ID NO: 1), GHA2 (SEQ ID NO:2), GHA3 (SEQ ID NO:3), or GHA4 (SEQ ID NO:4), GHA5 (SEQ ID NO 21), GHA6 (SEQ ID NO:22). GHA7 (SEQ ID NO:23), GHA8 (SEQ ID NO:24). or GHA9 (SEQ ID NO:25).

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[0491] In embodiments, the polynucleotide is delivered by a viral vector. As disclosed herein, viral delivery systems can comprise lipid nanoparticles (LNPs), hydrogel matrices, and polymer coatings, and in particular embodiments, viral vector based formulations are tailored to protect the viral vector, enhance transduction efficiency, and reduce immunogenicity.

[0492] RNA

[0493] In embodiments, the nucleic acid is an RNA molecule, including an mRNA or a synthetic RNA, encoding a GHA disclosed here.

[0494] Delivery systems for RNA agents include lipid nanoparticles (LNPs), polymeric nanoparticles, and hydrogel matrices.

[0495] In embodiments, formulations for GHA RNAs can include lipid nanoparticles (LNPs) for IV or IM delivery’, wherein the RNA in encapsulated in LNPs.

[0496] In embodiments, formulations of GHA RNAs can include polymeric nanoparticles, such as PLGA polymer-based nanoparticles.

[0497] In embodiments, the lipid nanoparticles or polymeric nanoparticles are coated with a mucoadhesive polymer, a specific polymer designed to stick finnly to mucosal surfaces, like the lining of the mouth or intestine, allowing for prolonged drug delivery to a specific area of tire body by enhancing its residence time at that site: essentially, the lipid or polymeric nanoparticle acts as a drug carrier while the mucoadhesive polymer ensures that the nanoparticle stays in place for extended periods.

[0498] Formulations of GHA RNAs can also include a hydrogel-encapsulated mRNA for SC or IM injection, in which the mRNA is embedded in a biocompatible hydrogel matrix for controlled release and a depot-like sustained release profile.

[0499] Formulations of GHA RNAs can also include dry powder formulations, in which the mRNA is stabilized in a dry powder form suitable for non-invasive routes of administration, such as pulmonary delivery by inhalation, or for reconstitution.

[0500] Formulations of GHA RNAs can also be based on direct administration without a carrier, for example, based on chemically modified mRNAs (e.g., with pseudouridine or 5 -methylcytidine) to ensure stability, for IM or intratumoral injection. Such modifications can also enhance translation efficiency and reduce an innate immune response.

[0501] In embodiments, an RNA molecule encoding a GHA is delivered by an RNA vector, and more particularly, an RNA viral vector. Exemplary RNA viral vectors are derived from lentiviruses and retroviruses. Lentiviral vectors can transduce both dividing and non-diving cells, making them suitable for

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[0503] applications requiring durable expression. Retroviral vectors can stably integrate into the host genome, providing a means for long-term expression in dividing cells.

[0504] DNA

[0505] In embodiments, the nucleic acid is a DNA molecule.

[0506] In embodiments, the DNA molecule is delivered by a DNA vector, and more particularly, a DNA viral vector. Exemplary DNA viral vectors include those based on adeno-associated virus (AAV), which is a single -stranded DNA virus; and adenovirus, which is a double-stranded DNA virus. DNA can also be integrated into a cell by a lentivirus, which is an RNA virus belonging to the family Retroviridae - a group of enveloped viruses that contain a reverse transcriptase enzyme that converts RNA into DNA before becoming integrated into tire genome of the host. AAV vectors are known for high safety profdes and longterm expression in non-dividing cells, making them suitable for treating chronic conditions. Adenoviral vectors offer high transduction efficiency and robust expression, making them suitable for acute or shortterm therapies. Lentiviral vectors are suitable for delivery to dividing, as well as non-diving cells, making them desirable for applications requiring durable expression.

[0507] Formulations for GHA viral DNA can include optimized polyethylene glycol (PEG) - such as in a coated adenoviral vector for IV administration. Such coated viral particles can enhance stability and circulation time and reduce immune detection.

[0508] Formulations for GHA viral DNA can include lipid-coated viral particles, in which the viral vector is encapsulated in lipid nanoparticles (LNPs), such as an LNP-encapsulated AAV vector encoding GHA for IM injection.

[0509] Formulations for GHA viral DNA can also include hydrogel encapsulated vectors, in which the viral vector is embedded in a biocompatible hydrogel matrix for sustained releases. Such formulations can allow for prolonged expression and localized delivery and include, for example, a hydrogel-encapsulated lentiviral vector encoding GHA for SC administration.

[0510] Formulations for GHA viral DNA also include viral vectors coated with mucoadhesive polymers for enhance nasal or mucosal delivery. Such formulations can increase retention time, improve absorption, and include, for example, a mucoadhesive adenoviral vector encoding GHA for intranasal delivery.

[0511] Formulations for GHA viral DNA also include viral vectors without an additional carrier or modifications, such as a lentiviral vector encoding GHA for direct intratumoral injection.

[0512] Formulations for GHA viral DNA also include viral vectors encapsulated in biodegradable microspheres for controlled release. Such formulation can allow sustained release, reducing the frequency of dosing, and include microsphere-encapsulated AAV vector for long-term delivery of GHAs.

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[0514] Administration Routes

[0515] Suitable route of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, intravenous, oral, topical, ocular, transdermal, intrathecal / epidural, rectal / vaginal, subcutaneous, intramuscular, intranasal, intraperitoneal, and intradermal administration.

[0516] Intravenous: For intravenous (IV) administration, GHAs can be formulated as sterile stable solutions ready for infusion. As described further herein, such IV formulations are useful in cancer applications, such as in acute HCC patients, allowing rapid systemic effects and controlled dosing.

[0517] Oral: In some embodiments, the GHA composition is formulated for oral administration in a solid dosage formulation comprising the drug. As used herein, a “solid dosage formulation” or “solid dosage form,” as intended for oral administration, includes such solid dosage formulations, as disclosed, e.g. in Remington: The Science and Practice of Pharmacy, 21st Edition (2020) or Dilip M. Parikh, Handbook of Pharmaceutical Granulation Technology, 4thed (2021) CRC Press, and comprises, but is not limited to, tablets, including chewable tablets, capsules, pills, lozenges, troches, cachets, capsules, and pellets.

[0518] To overcome GI degradation issues, GHAs can be formulated as oral formulations with enteric coatings, such as Eudragit®, nanoemulsions, inhibitors of proteases and other degradative enzymes, as well as mucoadhesive polymers. Examples of enteric coatings that can find use according to the subject disclosure include enteric components known in the art, including acid-insoluble polymers and film-forming polymers. In exemplary embodiments, the acid-insoluble polymer can also be selected from the group consisting of acrylic and methacrylic acid copolymers, cellulose acetate esters such as phthalate, butyrate, hydroxypropyl methylcellulose phthalate, and salts thereof. In exemplary embodiments, the film-forming polymer is selected from the group consisting of cellulose acetate phthalate, cellulose acetate trimellitate, HPMCP (hypromellose phthalate), HPMCAS (hydroxy propyl methyl cellulose acetate succinate), polyvinyl acetate phthalate (PVAP), and methacrylic acid copolymers.

[0519] Topical: For topical administration, GHAs can be formulated as an ointment, cream, gel, lotion, or drops. The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol. In particular embodiments, GH agonists can be formulated as topical eye drops enabling penetration to the retina. As described further herein, such topical formulations are usefid in non-invasive deliver}' methods particularly suitable for early intervention in non-proliferative diabetic retinopathy.

[0520] Ocular: For ocular administration, GHAs can be formulated as sterile drops, gels, or intraocular injectable solutions. As described further herein, such ocular formulations are particularly useful in reducing pathological angiogenesis in diabetic retinopathy.

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[0522] Transdermal: For transdermal administration, GHAs can be formulated in the form of a transdermal patch. The formulation may contain conventional components, such as permeation enhancers and may be in the form of a microneedle patch. As described further herein, transdermal formulations may be particularly suitable for slow, continuous release, potentially useful for chronic suppression in acromegaly or other growth disorders. Transdermal formulations may include absorption enhancers, such as surfactants, e.g.. polysorbates, or bile salts.

[0523] Intrathecal / Epidural: For intrathecal or epidural administration, GHAs can be formulated as sterile pyrogen free solutions ready for injection, allowing direct CNS dosing. Such formulations are particularly useful in neurological indications, including those arising by metastatic cancer in the CNS.

[0524] Intra-articular / Intralesional: For intra-articular or intralesional administration. GHAs can be formulated as injectable solutions or suspensions, allowing targeted delivery at high concentrations. As described further herein, such formulations can be particularly useful for treating GH-sensitive tumors.

[0525] Rectal / Vaginal: For rectal or vaginal administration, GHAs can be formulated as suppositories, gels, or other stable suitable formulations. As described further herein, such formulations advantageously avoid first-pass metabolism, and may be particularly useful for patients unable to receive injections, ensuring systemic absorption without injection-related complications.

[0526] Intramuscular: For intramuscular (IM) administration, GHAs, and more particularly, long-lasting variants such as, pegylated or carrier-fused GHAs can be formulated for intermittent administration - for example, as a monthly depot injectable. As described further herein, such formulations can allow sustained release, which may be particularly usefill for long-term cancer treatment requiring chronic suppression of GH activity.

[0527] Subcutaneous: For subcutaneous administration, GHAs, and more particularly, long-lasting variants such as pegylated or carrier-fused GHAs, can be formulated for intermittent administration - for example, weekly injectable forms. As described further herein, such formulations can allow convenient selfadministration and sustained release, which may be particularly useful for chronically managing diseases such as acromegaly through steady suppression of GH activity.

[0528] Intranasal: For intranasal administration, GHAs can be formulated as a metered nasal spray, drop formulation, or mucoadhesive nasal powder. As described further herein, such fonnulations allow convenient delivery through the nasal cavity, providing a rapid systemic effect. In embodiments, they are particularly useful for rapid GH suppression in patients with acute exacerbations acromegaly symptoms.

[0529] Pulmonary: For pulmonary (inhalable) administration, GHAs can be formulated, at a desired particle size, as pressurized metered-dose inhaler, a drag powder inhaler (DPI) or as a nebulized solution. As

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[0531] described further herein, such formulations provide a non-invasive means for rapid systemic absorption through the respiratory system. Pulmonary formulations may include excipients, such as stabilizers, e.g., lactose, and surfactants, e.g., lecithin.

[0532] Doses

[0533] Useful doses, including therapeutically effective doses, of the active agents of the present disclosure can be determined by various methods, as recognized by one skilled in the art.

[0534] In embodiments, useful doses of the active agents can be determined by modeling, for example by comparing their in vitro activity and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0535] In embodiments, useful amounts or doses of the active agents may be ascertained by routine methods such dose escalation studies or clinical trials, and by taking into consideration numerous factors, e.g., the mode or route of administration or drug delivery, the phannacokinetics of the agent, tire severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, concomitant medications, and the judgment of the treating physician

[0536] For example, an exemplary dose can be in tire range from 0.1 mg to 200 gm per day, from 0.1 mg to 50 gm per day, from.1 mg to 10 gm per day, from 0.1 mg to 1000 mg per day, from 1 mg to 1000 mg per day, from 1 mg to 200 mg per day, or from 1 mg to 50 mg per day.

[0537] Similarly, an exemplary' dose can be in the range from 0.001 mg / kg to 2 gm / kg per day, from 0.001 mg / kg to 0.5 gm / kg per day, from.001 mg / kg to 0.1 gm / kg per day, from 0.001 mg / kg to 10 mg / kg per day, from.01 mg / kg to 10 mg / kg per day, from.01 mg / kg to 2 mg / kg per day, or from.01 mg / kg to 0.5 mg / kg per day

[0538] In embodiments, the desired dose may be presented in a unit dosage form; for example, a composition containing from 0.1 to 1000 mg, from 0.1 to 200 mg, from 0.5 to 100 mg, or from 1 to 50 mg, of active agent per unit dosage fonn.

[0539] In other embodiments, the desired dose may be presented in divided doses administered at appropriate intervals, for example, as two. three, four, or more sub-doses per day. (e.g., BID, TID, QID). The sub-dose itself may be further divided, e.g., into multiple temporally distinct administrations, used according to the compositions and methods of the present disclosure.

[0540] In embodiments, a formulation comprising an active agent is prepared for systemic administration (e g., intravenous, intramuscular, or subcutaneous) at a dose of 0.01 mg / kg to 1000 mg / kg, more specifically 0.02 mg / kg to 10 mg / kg, administered from once a day to once a month.

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[0542] In embodiments, a formulation comprising an active agent is prepared for localized administration (e.g., as an intratumoral, intravitreal, or intra-articular injection) at a dose of 0.1 mg to 1000 mg, administered from once a week to once every three months.

[0543] In embodiments, a formulation comprising an active agent is prepared for oral administration (e.g., as a nano-emulsion or enteric coated solid tablet) at a dose of 1 mg to 2000 mg, administered from once a day to once a week.

[0544] In embodiments, a formulation comprising an active agent is prepared for intranasal administration (e.g., as a metered spray) at a dose of 0.1 mg to 100 mg, administered from 1-6 times a day.

[0545] In embodiments, a formulation comprising an active agent is prepared for pulmonary’ (inhalable) administration (e.g., as a metered spray) at a dose of 0.1 mg to 500 mg, administered, for example, once a day or twice a day.

[0546] In embodiments, a formulation comprising an active agent is prepared for topical or transdermal administration at a dose of 0.5 mg to 5000 mg, administered on a daily to a weekly basis.

[0547] In embodiments, a fonnulation comprising an mRNA-encoded agent is prepared for systemic administration (e.g.. intravenous, intramuscular, or subcutaneous) at an equivalent protein expression dose of 0.1 mg / kg to 1000 mg / kg, administered by injection about every11 to 4 weeks, depending on the duration of expression and therapeutic needs.

[0548] In embodiments, a viral vector formulation comprising a DNA-encoded agent is prepared for systemic administration (e.g., intravenous, intramuscular, or subcutaneous) at an equivalent protein expression dose of 0.1 mg / kg to 2000 mg / kg, administered by a single injection or repeated injections about every 3 to 12 months, depending on tire duration of expression and therapeutic needs.

[0549] Combinations

[0550] In embodiments, GHA compositions and formulations disclosed herein can be administered to a subject in combination with other agents or treatments, the combination depending on the condition and therapeutic intervention.

[0551] In exemplary’ embodiments involving treating an endocrine disorder such as acromegaly or gigantism, a GHA disclosed herein can be administered in combination with a somatostatin analog or dopamine agonist, as well as in conjunction with radiotherapy or surgical intervention, as described further herein.

[0552] In exemplary embodiments involving treatment of a cancer, including hepatocellular carcinoma (HCC), a GHA disclosed herein can be administered in combination with a kinase inhibitor, immune

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[0554] checkpoint inhibitor, or anti -angiogenic agent, as well as in conjunction with radiotherapy or a surgical intervention, as described further herein.

[0555] In exemplary embodiments involving treatment of diabetes, a GHA disclosed herein can be administered in combination with insulin, an insulin analog, an insulin sensitizer, a GLP-1 receptor agonist, or metformin, as described further herein.

[0556] In exemplary embodiments involving treatment of a diabetic complication, such as a vascular disorder, a GHA disclosed herein can be administered in combination with an anti-VEGF agent, a corticosteroid, an angiotensin-converting enzyme (ACE) inhibitor, or a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, as described further herein.

[0557] Moreover, reference to administering a GHA composition or formulation in combination with a second agent (such as an ICI) or other treatment modality (such as radiotherapy) does not mean that the GHA composition or formulations must be administered at the same time or with some dosing regimen as the additional agent (or treatment). Unless explicitly stated otherwise, parameters for administering a composition are independent of the parameters for administering a second agent or treatment to a subject, and will depend on such factors as the particular indication, subject needs, therapeutic objectives, and other considerations.

[0558] Therapeutic Methods

[0559] As described in further detail herein, GHAs disclosed herein, including polypeptides and fusion proteins, as well as compositions thereof, are usefid in numerous therapeutic methods.

[0560] Accordingly, in embodiments, the present disclosure provides method of treating a condition associated with excessive (or aberrant) growth hormone activity or excessive (or aberrant) growth hormone receptor activation, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA disclosed herein.

[0561] Accordingly, in embodiment the present disclosure provides the use of any GHA in the manufacture of a medicament for treating a disease associated with excessive (or aberrant) growth honnone activity or excessive (or aberrant) growth hormone receptor activation, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA disclosed herein.

[0562] Acromegaly and Gigantism

[0563] In embodiments, tire condition associated with excessive growth hormone activity or growth hormone receptor activation, as targeted by the therapeutic methods herein, is an endocrine disorder, and more particularly, is acromegaly or gigantism.

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[0565] Gigantism and acromegaly are both caused by excess growth hormone (GH) but they affect different age groups. Gigantism occurs in children and adolescents before their growth plates fuse, whereas acromegaly occurs in adults after their growth plates have fused.

[0566] Acromegaly (or gigantism) is a chronic disease primarily caused by benign pituitary adenomas, although it can occasionally arise from tumors in other locations. This condition is characterized by excessive growth of the hands, feet, and facial features, organ enlargement, and may also include vertebral deformities, abnonnal calcium levels, increased cardiovascular risk, respiratory issues, and glucose intolerance. Elevated GH levels can lead to high circulating IGF-1, influencing various metabolic processes and tissue responses.

[0567] Accordingly, in embodiments, the present disclosure provides methods of treating acromegaly or gigantism, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA disclosed herein.

[0568] In embodiments, the GHA is administered in combination with somatostatin analogs such as octreotide or lanreotide, to achieve synergistic suppression of growth hormone and insulin-like growth factor 1 (IGF-1) levels in acromegaly (or gigantism) patients.

[0569] In embodiments, the GHA is co-administered with dopamine agonists, such as cabergoline or bromocriptine, to provide an enhanced treatment regimen for acromegaly, particularly in patients with partial resistance to somatostatin analogs.

[0570] In embodiments, the GHA is administered in conjunction with a radiotherapy or a surgical intervention to improve long-term management of acromegaly (or gigantism) by mitigating residual growth hormone receptor activity post-treatment.

[0571] In embodiments, the GHA is administered intravenously. In embodiments, the GHA is administered orally. In embodiments, the GHA is administered subcutaneously. In embodiments, the GHA is administered at a frequency ranging from once daily to once monthly. In embodiments, the GHA is administered at a dose of 0.1 mg / kg to 1000 mg / kg.

[0572] Cancer

[0573] In embodiments, the condition associated with excessive growth hormone activity or growth hormone receptor activation, as targeted by the therapeutic methods herein, is cancer.

[0574] Increasing evidence links GH to several cancers, including liver, breast, colon, and endometrial malignancies. Supporting this view, individuals with GH resistance, such as those with Laron syndrome, show protection against cancers. Conversely, high GH levels can accelerate tumor progression through mechanisms such as cell proliferation, transformation, angiogenesis, and lymphangiogcncsis, affecting -66- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0575] various cancers including breast, endometrial, and colon cancers. See Kaulsay 1999, Exp. Cell Res. 250, 35-50.

[0576] Disrupted GH signaling is associated with elevated p53 in colon tissues, a role for GH in tumor promotion by suppressing tumor suppressor genes. Genetic variants in GH-related genes are also linked to increased cancer risks, notably lung cancer due to prolonged GH signaling. See Steuerman et al. 2011, Eur. J. Endocrinol. 164, 485-489.

[0577] Preclinical studies with pegvisomant support the efficacy of GHR antagonism as a monotherapy in some tumor types, although responses can vary and predictive biomarkers are lacking. Combining GHR antagonism with radiotherapy and chemotherapy may enhance anticancer effects, as shown by the ability of pegvisomant to reduce tumor regrowth in a xenograft model. See Minoia et al. 2012, J. Clin. Endocrinol. Metab. 97, E907-E916; Evans et al. 2016, Cancer Lett. 379, 117-123. Preclinical studies suggest GH antagonism could enhance tire efficacy of existing cancer therapies and reduce chemoresistance, particularly when used in combination with other treatments.

[0578] These and other observations underscore a role of GH in cancer progression and as a therapeutic target in oncology, along with the potential for growth hormone antagonists in cancer treatment. However, current antagonists like pegvisomant were designed for acromegaly and are not optimized for cancer-specific indications. In this regard, certain cancers overexpress HGR and produce excess hGH levels, underscoring autocrine and paracrine influences on growing tumors. See, e.g., Basu et al. 2018, Eur. J. Endocrinol. 178, R155-R181; Lu et al. 2019, Signal Transduct. Target. Then 4, 3. More generally, GH has differential effects depending on whether it originates by pulsatile secretion from tire pituitary or by secretion from cancer cells or other cells in the tumor microenvironment. See Lu et al. 2019, Signal Transduct. Target Then 4, 3.

[0579] Autocrine expression of GH in breast cancer cells, for example, leads to a more aggressive cellular phenotype compared to the effects seen by exogenously adding GH to mimic endocrine secretion. Kaulsay, et al. 1999, Exp. Cell Res. 250, 35-50; Mukhina et al. 2004, roc. Natl Acad. Sci. USA 101, 15166-15171; Perry et al. 2008, J. Mammary Gland Biol. Neoplasia 13, 131-145; Mertani et al. 2001, J. Biol. Chem. 276, 21464-21475.

[0580] In summary, while pegvisomant has proven to be a treatment for acromegaly, its limitations — both in acromegaly and broader contexts — highlight the need for next-generation GH antagonists.

[0581] Specifically, there is a need to develop cancer-specific GH antagonists that leverage preclinical insights, offering improved therapeutic outcomes and addressing the shortcomings of current therapies. The development of antagonists tailored specifically for cancer remains a significant unmet need. Such drugs -for example, long lasting forms suitable for targeted delivery - could address limitations in efficacy, reduce side effects, improve patient compliance, and provide cost-effective solutions.

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[0583] The present disclosure addresses these challenges by introducing novel growth hormone antagonist (GHA) molecules with targeted mutations that enhance receptor binding, improve stability, and extend half-life. These mutations nay modulate interaction profiles without compromising efficacy, achieving reduced immunogenicity and prolonged activity. Tire present disclosure also incorporates fusion proteins, linking GHAs to stabilizing carriers such as albumin, transferrin, or IgG, coupled with optimized linker sequences (as well as targeting domains and protease cleavage domains) to maintain independent and directed functionality and further extend pharmacokinetics.

[0584] Additionally, the present disclosure supports diverse administration routes, including intravenous, subcutaneous, intralesional, and innovative non-invasive methods such as transdermal, oral, and inhalable formulations. Advanced delivery techniques, including nanoparticle encapsulation, hydrogels, and biodegradable microspheres, enhance bioavailability and therapeutic convenience.

[0585] In summary, the present disclosure presents a class of mutant-based GHAs, including those comprising GHA1-GHA9, to improve pharmacokinetics, stability, and therapeutic versatility. These innovations address limitations in current treatments, offering enhanced efficacy, reduced dosing frequency, and broader applicability to GH-related disorders and cancer.

[0586] Accordingly, in embodiments, the present disclosure provides methods of treating a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA disclosed herein.

[0587] In embodiments, the cancer is characterized by overexpression of growth hormone and or its receptors, including GHR. In embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma, breast cancer, endometrial cancer, and melanoma.

[0588] In embodiments, the GHA is administered with a kinase inhibitor, including but not limited to sorafenib or lenvatinib, for treatment of growth hormone receptor-positive hepatocellular carcinoma, enhancing anti-proliferative and pro-apoptotic effects.

[0589] In embodiments, the GHA is co-administered with an immune checkpoint inhibitor, such as pembrolizumab or nivolumab, to enhance the immune-mediated suppression of tumor growth in cancers expressing a growth hormone receptor.

[0590] In embodiments, the GHA is administered in combination with an anti-angiogenic agent, such as bevacizumab, to suppress growth homione-mediated vascular proliferation in a solid tumor.

[0591] In embodiments, the GHA is administered in conjunction with a radiation therapy to enhance radiosensitivity in a cancer characterized by an overactive growth hormone signal transduction pathway.

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[0593] In embodiments, the GHA is administered intravenously. In embodiments, the GHA is administered orally.

[0594] In embodiments, the GHA is administered by intratumoral injection. In embodiments, the GHA is administered at a frequency ranging from once a week to once every three months. In embodiments, the GHA is administered at a dose of 1 mg to 2000 mg.

[0595] In embodiments, a method of treating cancer with a GHA, alone or in combination with other agents or therapies, can promote cancer regression. A method of treating cancer with a GHA can also halt cancer progression as assessed by imaging, improved quality of life, and improved survival outcomes such as time to tumor progression (TTP), progression-free survival (PFS), and overall survival (OS).

[0596] Hepatocellular Carcinoma

[0597] In particular embodiments, GHAs of the present disclosure are useful in cancer treatment methods directed to hepatocellular carcinoma (HCC).

[0598] HCC is the most common primary liver cancer and third major cause of cancer mortality’ worldwide. Llovet et al. 2021, Nat. Rev. Dis. Primers 7, 6. HCC presents most frequently in connection with liver cirrhosis, less frequently in chronic liver disease, and rarely in patients with a healthy liver. HCC prevalence has risen in recent years, particularly in western nations, due to the increasing frequency of non-alcoholic fatty liver disease and obesity. Leowattanaet al. 2023, World J. Gastroenterol. 29, 1551-1568. HCC remains among the most challenging malignancies to treat, with significant variation in clinical presentation and outcomes based on disease stage, and a low overall survival rate. Yilma et al. 2024, JAMA New. Open 7, e2435066.

[0599] HCC can be divided into three stages, each presenting different therapeutic and strategic considerations.

[0600] Early- Stage:

[0601] Early-stage HCC is typically defined by low tumor burden confined to the liver without major vascular invasion (e g., portal vein or hepatic artery) or distant metastasis. Early stage patients with preserved liver function are amenable to curative treatments such as local radiofrequency ablation or surgical resection, with liver transplantation as an additional consideration. Most HCC patients present beyond the early stage, however, due to a lack of symptoms. Moreover, relapse is common even in early HCC patients receiving potentially curative approaches, reaching a relapse rate of 50% within two years and 70% within five years after resection. These observations highlight the need for perioperative therapies to improve long-tenn outcomes. See Tan et al. 2015, Ann. Surg. 261, 947-955; Roayaie et al. 2013, Hepatology 57, 1426- 1435.

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[0603] Intermediate-Stage:

[0604] Intermediate-Stage HCC is typically defined by locally advanced tumors that are confined to the liver without distant metastasis. These cases are generally not suitable for curative treatments such as resection or transplantation. Instead, they are typically treated using locoregional modalities such as trans-arterial chemoembolization (TACE) or radioembolization via hepatic artery’ catheterization. Despite these approaches, tumor control remains suboptimal, underscoring the need for additional interventions.

[0605] Advanced-Stage:

[0606] Advanced-Stage HCC represents the majority (70%-80%) of diagnosed cases and is typically defined by major vascular invasion or distant metastatic disease. These cases are not amenable to resection or other surgical treatments and are treated systemically. While the terms “advanced HCC" (“aHCC") and “unresectable HCC” (“uHCC”) are often used interchangeably, they differ in degree: “Advanced HCC" refers to a broader category of hepatocellular carcinoma (HCC) that that include unresectable tumors, but also tumors with significant vascular invasion, extrahepatic spread, or poor liver function, whereas "unresectable HCC" specifically indicates a tumor that cannot be surgically removed due to its size, location, or involvement with major blood vessels within the liver, usually considered a part of advanced HCC. In essence, "unresectable HCC" is a subset of "advanced HCC" that highlights the inability to surgically resect the tumor as a key factor in its advanced stage.

[0607] For years, the only approved first line systemic treatment for advanced HCC was the tyrosine kinase inhibitor (TKI) sorafenib, which has activity against numerous protein kinases, including VEGFR, PDGFR and RAF kinases. More recently, therapeutic options have expanded with the approval of first-line and second-line treatments, encompassing both monotherapies and combination therapies.

[0608] Recently approved first-line therapies for advanced HCC include the oral multiple kinase inhibitor lenvatinib (Lenvima®; Kisplyx®), which targets VEGF1, VEGF2, and VEGF3 kinases and has shown superior efficacy to sorafenib with respect to clinically significant secondary’ endpoints. Other recent approvals include doublet therapies: One doublet therapy comprises the immune checkpoint inhibitor atezolizumab (Tecentriq®), a monoclonal antibody targeting PD-L1, in combination with the angiogenic inhibitor bevacizumab (targeting VEGF). A second doublet therapy comprises two immune checkpoint inhibitors: durvalumab (targeting PD-L1) and tremelimumab (targeting CTLA-4).

[0609] Recently approved second-line therapies have been used in aHCC patients who have progressed on sorafenib treatment. These include two TKI monotherapies: regorafenib (Stivarga®), which targets tumor angiogenesis (e.g., VEGFRl, VEGFR2, VEGFR3, TIE2), tumor survival (e.g., KIT, RAFI, BRAF), and tumor microenvironment (e.g., PDGFR); and cabozantinib (Cabometyx®), which targets tumor angiogenesis and other activities through inhibition of targets such as MET, RET, AXL, VEGFR2, FLT3, and KIT.

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[0611] Another second-line monotherapy is ramucirumab (Cyramza®), an anti -angiogenic monoclonal antibody targeting the vascular endothelial growth factor receptor 2 (VEGFR-2) protein.

[0612] Other second-line options for uHCC are based on immunotherapy. These include two ICI monotherapies: nivolumab (Opdivo®), a monoclonal antibody targeting PD-1; and pembrolizumab (Keytruda®), a monoclonal antibody targeting PD-1, These also include an ICI doublet, comprising ipilimumab (Yervoy®), a monoclonal antibody targeting CTLA-4, in combination with nivolumab (Opdivo®), a monoclonal antibody targeting PD-1.

[0613] While systemic therapies have demonstrated significant improvements in overall survival (OS) and progression-free survival (PFS) in patients with uHCC, response rates remain modest, with no approved therapies in third-line settings for patients who have progressed on first- and second-line treatments See. e.g, Leowattana et al. 2023, World J. Gastroenterol. 14, 1551-1568; Abou-Alfaet al. 2023, Future Oncol. 19, 2505-2516; Finn et al. 2020, N. Engl. J. Med. 382, 1894-1905; Yau et al. 2020, JAMA Oncol. 6, e204564. More generally, available systemic therapies are largely palliative, offering only a modest improvement in survival outcomes, and are not based on hypothesis-driven criteria for personalized patient selection based on clinical or biomarker-based strategies. Indeed, even in resectable HCC. there are no approved neoadjuvant or adjuvant therapies to lower recurrence and improve survival.

[0614] Given the limitations of existing treatments, there is an urgent need for novel therapeutic strategies to address the unique challenges presented by each stage of HCC. The best outcomes generally are observed after surgical resection - which is potentially curative. Thus, new, and novel treatment approaches are needed to improve outcomes in HCC, particularly unresectable HCC (uHCC). See, e.g., Falette-Puisieux ct al. 2022, Cancers (Basel) 14, 2357.

[0615] As described herein, GHAs offer a promising approach for treating HCC at multiple stages. The liver is a major target of GHR signaling, and clinical and preclinical evidence suggest that GHR signaling pathways plays a role in HCC pathogenesis. In a clinical study examining a large cohort of 767 HCC patients and 200 healthy controls, GH levels in peripheral blood were significantly elevated in half (49.5%) of HCC patients, and these patients had significantly more aggressive clinicopathological features and poorer clinical outcomes. Moreover, pegvisomant inhibited HCC tumor progression in two patients with advanced staged HCC and high GH who developed sorafenib resistance. Kaseb et al. 2022, Front Oncol. 12, 986305.

[0616] Similarly, in a study of uHCC patients receiving doublet therapy with durvalumab and tremelimumab, higher pre-treatment GH levels were correlated with poorer OS. Chamseddine et al. 2024, J. Hepatocell.

[0617] Carcinoma. 11, 455-461.

[0618] Supporting a role for GHRs in HCC pathogenesis is genetic evidence from mouse models of HCC induced by diethylnitrosamine (DEN). Following DEN-injections. HCC developed in 93.5% of wild-

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[0620] type mice but only in 5.6% of mutant mice with a global genetic disruption of GHR expression. In mice with liver-restricted GHR disruption, significantly fewer tumors developed compared to control, suggesting that GHR in the liver cells might increase tumor burden. Haque et al. 2022, J. Hepatocell. Carcinoma. 9, 823-837. In nude mice, sorafenib alone failed to significantly suppress the growth of Hep2 xenograft tumors in nude mice; however, the GHR antagonist pegvisomant alone inhibited tumor grow th, and sorafenib and pegvisomant led to greater suppression of tumor growth. Moreover, in nude mice with sorafenib-resistant HCC xenografts, pegvisomant potentiated the effects of sorafenib and overcame sorafenib resistance.

[0621] In vitro results from experiments in HCC cell-line support these in vivo observations: Compared to controls, blocking GHR signaling in HCC cells with pegvisomant or GHR siRNA induced substantial inhibitory cellular effects, including decreases in cellular proliferation, adhesion, and migration.

[0622] Mechanistically, pegvisomant decreased phosphorylation of downstream signaling and survival proteins, including JAK2, STAT3, STAT5. IRS-1, AKT, ERK, and IGF-IR. Extending these observations, in vitro studies in two different HCC cell lines (Huh7 and Hep3B) confirmed that forced GH expression promoted cell proliferation, cell survival, anchorage-independent grow th, cell migration, and invasion and further showed that forced GH expression promoted cancer stem cell (CSC)-like properties by inhibiting expression of the tight junction protein, CLAUDIN-1. Chen et al. 2017, Int. J. Mol. Sci. 15, 1274.

[0623] Taken together, results from clinical and preclinical studies support a role of the GHR pathway in HCC pathogenesis, initiation, and progression. Elevated levels of circulating GH are associated with advanced stages of HCC and may serve as a predictive and prognostic biomarker in HCC.

[0624] Accordingly, in embodiments, a GHA disclosed herein can be used as a monotherapy or in a combination therapy to treat HCC at distinct stages in HCC progression.

[0625] In early stage HCC, GHAs may be utilized perioperatively, either as neoadjuvant therapy to reduce tumor size before surgical resection or as adjuvant therapy to target residual microscopic disease postsurgery, thereby reducing recurrence rates. GHAs could also enhance the efficacy of ablative therapies in this setting. Such perioperative use of GHAs may address high relapse rates in surgically treated patients, providing a new avenue for adjuvant therapy development.

[0626] In intermediate stage HCC, GHAs can be combined with locoregional therapies such as TACE. radioembolization, or ablative procedures to improve tumor control. In patients awaiting liver transplantation, GHAs administered via intra-arterial delivery through the hepatic artery may serve as effective bridging therapies. Locoregional delivery of GHAs could amplify their antitumor activity, especially in combination with established liver-directed therapies.

[0627] In advanced stage HCC, a GHA can be used in a third line setting, where no approved options currently exists. In addition, a GHA can be combined with ICIs (CPIs) or TKIs in first or second line settings -72- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0628] to enhance response rates and survival outcomes in advanced HCC. By targeting the GH axis, GHAs may overcome resistance to existing therapies, including CPIs and TKIs. In addition, localized delivery minimizes systemic side effects, improving tolerability and expanding treatment options for patients with comorbidities.

[0629] In embodiments, a GHA disclosed herein, including a fusion protein comprising transferrin (or other carrier) is locally administered to treat HCC. Local drug administration is a particularly attractive approach for HCC, given the liver's unique vascular supply and the potential to deliver high drug concentrations to the tumor while minimizing systemic toxicity. In one aspect, a GHA is administered by intratumoral delivery: a GHA can be injected directly into the tumor, delivering therapeutic concentrations precisely at the site of disease. In one aspect, a GHA is administered by intra-arterial delivery: a GHA can be administered via hepatic artery catheterization, in combination with locoregional therapies such as TACE or radioembolization, to enhance drug retention within tire tumor. In particular embodiments, a GHA can be administered in a sustained release fonnulation, such as in drug-eluting beads or hydrogels loaded with a GHA to prolonged release of the drug at the tumor site, enabling continuous therapeutic effects with minimal systemic exposure.

[0630] Diabetes and Diabetic Complications

[0631] In embodiments, the condition associated with excessive (or aberrant) growth honnone activity or growth hormone receptor activation, as targeted by the therapeutic methods herein, is diabetes, including diabetic complications, such as diabetic retinopathy and diabetic nephropathy.

[0632] Diabetes mellitus

[0633] Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels due to insulin deficiency or resistance. Insulin is essential for maintaining glucose homeostasis by promoting glucose uptake into cells, enhancing its utilization, and storing it as fat and glycogen in peripheral tissues. It also reduces gluconeogenesis (the synthesis of glucose from non-carbohydrate sources) and glycogenolysis (the breakdown of glycogen). Elevated GH levels can also contribute to diabetic complications, such as diabetic retinopathy and nephropathy.

[0634] GH has actions that counteract those of insulin, contributing to diabetes by increasing glucose production via gluconeogenesis and glycogenolysis, particularly in the liver and kidneys, and by inhibiting glucose uptake in adipose tissues. GH also promotes lipolysis. In healthy adults and adolescents, elevated GH levels can impair glucose tolerance and promote insulin resistance. In contrast, individuals with Laron syndrome, who have reduced circulating IGF-1, exhibit increased insulin sensitivity.

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[0636] Patients with poorly controlled diabetes often exhibit elevated levels of growth hormone, which can contribute to poor metabolic control. GH antagonists disclosed herein can help improve diabetic control by countering the effects of elevated GH.

[0637] Accordingly, in embodiments, the present disclosure provides a method of treating insulin resistance or diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA disclosed herein. In embodiments, the diabetes is type 2 diabetes mellitus.

[0638] In embodiments, the GHA is administered with insulin or insulin analogs to improve glycemic control in patients with growth hormone-mediated insulin resistance.

[0639] In embodiments, the GHA is administered with a GLP-1 receptor agonist, such as semaglutide or dulaglutide, to address both hyperglycemia and growth hormone-related metabolic complications in diabetes.

[0640] In embodiments, the GHA is administered with an insulin sensitizer selected from the group consisting of metformin and pioglitazone.

[0641] In embodiments, the GHA is administered with metformin to enhance insulin sensitivity and mitigate the effects of growth hormone-induced hepatic glucose production in patients with type 2 diabetes.

[0642] In embodiments, the GHA is administered orally. In embodiments, the GHA is administered subcutaneously. In embodiments, the GHA is administered at a frequency ranging from once a day to twice a week. In embodiments, the GHA is administered at a dose of 1 mg to 1000 mg.

[0643] Diabetic Complications

[0644] Complications of diabetes such as retinopathy, nephropathy, and angiopathy are associated with the proliferation of microvascular endothelial cells stimulated by GH.

[0645] Diabetic retinopathy is one of the most common complications of diabetes and a leading cause of blindness. The proliferative form of retinopathy, characterized by retinal neovascularization, is more advanced and particularly prevalent in type 2 diabetes (T2D) patients with proliferative diabetic retinopathy compared to those with non-proliferative retinopathy or without any evidence of this complication.

[0646] GH expression has been detected in the human retina and vitreous fluid, underscoring a potential role for autocrine and paracrine GH actions. GH can directly stimulate the proliferation of human retinal microvascular endothelial cells in vitro. In vivo studies using transgenic mice expressing a GH antagonist have demonstrated that antagonizing GH can inhibit ischemia-induced retinal neovascularization. Patients with acromegaly show increased prevalence of proliferative retinopathy, and increased serum levels of GH and IGF-1 are observed in T2D patients with proliferative retinopathy. See Harvey et al. 2009, Neurosci. Lett. 455, 199-202; Harvey et al. 2016, Growth Horm. IGF Res. 29, 28-32; Rymaszewski et al. 1991, Proc. Natl Acad. Set. USA 88, 617-621; Smith 1997, Science 276, 1706-1709.

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[0648] In diabetic nephropathy, excess GH stimulates glomerular growth, affects the structure and function of the kidney, and is associated with glomerular podocyte dysfunction. Preclinical studies have shown promising results: GH antagonism preventing early renal changes in nonobese diabetic mice, and transgenic mice expressing a GH antagonist show reduced susceptibility to diabetic kidney disease. See Blutke et al. 2016. Physiol. Rep. 4, e 12709; Grunenwald et al. 2011. Ann. Endocrinol. (Paris) 72, 485-495; Kumar et al. 2010, Biol. Chem. 285, 31148-31156.

[0649] Given the preclinical evidence and effects of GH on kidney function, the use of growth hormone antagonists for treating diabetic nephropathy is a potential therapeutic approach. As GH and IGF-1 are implicated in the progression of diabetic nephropathy, GHAs disclosed herein may mitigate renal changes associated with diabetes by blocking GH action,.

[0650] Accordingly, in embodiments, the present disclosure provides methods of treating a complication of diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA disclosed herein. In embodiments, the complication of diabetes is diabetic retinopathy or diabetic nephropathy.

[0651] In embodiments, the GHA is used in combination with an anti-VEGF agent, such as ranibizumab or aflibercept, to reduce growth homione-mediated pathological angiogenesis in diabetic retinopathy.

[0652] In embodiments, the GHA is administered with a corticosteroid to mitigate inflammation and vascular leakage in diabetic retinopathy patients.

[0653] In embodiments, the GHA is administered with an angiotensin-converting enzyme (ACE) inhibitor, such as enalapril or lisinopril, to protect renal function by inhibiting growth homione-mediated pathways in diabetic nephropathy.

[0654] In embodiments, the GHA is administered with a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, such as dapagliflozin or empagliflozin, to enhance renal protection and manage growth hormone-related renal complications in diabetic nephropathy.

[0655] In embodiments, the GHA is administered in combination with an anti-VEGF therapy, such as bevacizumab, ranibizumab, aflibercept, or brolucizumab.

[0656] In embodiments, the GHA is administered as ocular drops. In embodiments, the GHA is administered by intravitreal injection. In embodiments, the GHA is administered at a frequency ranging from once daily to once monthly. In embodiments, the GHA is administered at a dose of 0.1 mg to 100 mg.

[0657] Reducing STAT phosphorylation

[0658] In embodiments, the present disclosure provides methods for reducing STAT phosphorylation, and more particularly, STAT3 phosphorylation, comprising administering to a subject in need thereof a -75- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0659] therapeutically effective amount of a GHA as disclosed herein, wherein the GHA disrupts GHR activation and thereby inhibits downstream signaling pathways involved in cell proliferation and survival and other functions.

[0660] In embodiments, the present disclosure provides methods for reducing STAT phosphorylation, and more particularly, STAT5 phosphorylation, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA as disclosed herein, wherein the GHA disrupts GHR activation and thereby inhibits downstream signaling pathways involved in cell proliferation and survival and other functions.

[0661] In embodiments, the subject has a cancer associated with aberrant STAT3 activation, selected from the group consisting of hepatocellular carcinoma, colorectal cancer, and prostate cancer. In embodiments, the GHA is administered in combination with an immune checkpoint inhibitor to enhance immune-mediated tumor suppression.

[0662] In embodiments, the subject has a cancer associated with aberrant STAT5 activation, selected from the group consisting of hepatocellular carcinoma, colorectal cancer, and prostate cancer. In embodiments, the GHA is administered in combination with an immune checkpoint inhibitor to enhance immune-mediated tumor suppression.

[0663] Inhibiting GH Dependent Vascular Complications

[0664] In embodiments, the present disclosure provides methods for treating a GH-dependent vascular complication, comprising administering to a subject in need thereof a therapeutically effective amount of any GHA disclosed herein, wherein the GHA is sufficient to inhibit GH-induced angiogenesis.

[0665] In embodiments, the vascular complication is diabetic retinopathy or macular edema, and preferably the GHA is administered intravitreally.

[0666] In embodiments, the vascular complication is cancer-associated angiogenesis, and the GHA is administered in combination with an anti-VEGF therapy.

[0667] Diagnostic Applications

[0668] Tire present disclosure further provides GHAs for use in diagnostic applications, including diagnosing disorders associated with GH dysregulation.

[0669] Current diagnostic approaches for GH-rclatcd disorders, such as acromegaly and GH insensitivity syndromes, rely heavily on biochemical tests like serum IGF-1 levels and GH suppression tests. However, these tests often yield ambiguous results, necessitating alternative methods to confirm diagnoses or differentiate between underlying causes. GHAs, by selectively inhibiting GH receptor activity without activating downstream signaling pathways, offer a novel diagnostic approach to address these limitations.

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[0671] In embodiments, a GHA disclosed herein can be used to confirm GH hypersecretion in an acromegaly disorder. For example, a significant reduction in IGF-1 levels following GHA administration indicates the presence of excessive GH activity, aiding in a definitive diagnosis of acromegaly.

[0672] In embodiments, a GHA disclosed herein can be used to evaluate the GH-dependency of a tumor. For example, a GHA disclosed herein can be used to distinguish between a non-GH secreting pituitary adenoma and an ectopic GH-secreting tumor. In such a case, a distinct reduction in IGF-1 levels following GHA administration would indicate the presence of a GH-dependent tumor.

[0673] In embodiments, a GHA disclosed herein can be used to assess GH sensitivity or insensitivity in a growth disorder. For example, in patients suspected with GH insensitivity, such as in Laron syndrome, administering a GHA can distinguish between reduced GH action due to receptor insensitivity or postreceptor signaling defects.

[0674] In embodiments, a GHA disclosed herein can be used to evaluate GH receptor activity and signaling defects, particularly in rare syndromes involving GH receptor mutations.

[0675] In embodiments, a GHA disclosed herein can be used to validate IGF-1 as a biomarker of GH activity. By inhibiting GH action, researchers can correlate IGF-1 reductions with GH receptor blockade, refining diagnostic criteria for GH-related disorders.

[0676] More generally, GHAs, by selectively blocking GH receptor activity, offer a targeted diagnostic tool with minimal off-target effects. Furthermore, they can enhance the diagnostic accuracy of current tests -especially in ambiguous or borderline cases. In addition, GHAs can help assess the dependency of a tumor on GH signaling, facilitating tailored therapeutic approaches.

[0677] Accordingly, in embodiments, the present disclosure provides a method of diagnosing acromegaly, comprising administering a growth hormone antagonist to a patient suspected of acromegaly and measuring a reduction in serum IGF-1 levels to confirm GH hypersecretion.

[0678] In embodiments, the present disclosure provides a method for differentiating GH-secreting Tumors, comprising administering a growth hormone antagonist and assessing the response in IGF-1 levels to differentiate between pituitary adenomas and ectopic GH-secreting tumors.

[0679] In embodiments, the present disclosure provides a method for testing GH sensitivity, comprising administering a growth hormone antagonist to assess GH receptor functionality in patients suspected of GH insensitivity or resistance.

[0680] In embodiments, the present disclosure provides a method for validating IGF-1 as a biomarker of GH activity, comprising administering a grow th hormone antagonist and correlating IGF-1 levels with GHR inhibition.

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[0682] Veterinary Applications

[0683] The present disclosure further encompasses the use of GHA compositions and formulations to treat a range of GH-related disorders in non -human animals, and, more particularly, veterinary subjects.

[0684] Excessive GH production in animals typically results from benign adenomas of the pituitary gland but may also arise from ectopic GH production by tumors or iatrogenic causes due to overadministration of GH or growth hormone-releasing hormone (GHRH).

[0685] Disorders associated with excess GH have been observed in various species, including dogs, cats, horses, and cattle, each presenting unique clinical manifestations. In dogs, acromegaly typically presents in middle-aged to older animals, who can exhibit enlarged cranial and paw dimensions, thickened skin, organomegaly, insulin-resistant diabetes, and cardiovascular complications. In cats, acromegaly often presents with enlarged paws and heads, organomegaly, and insulin resistance, resulting in challenging diabetes management. In horses, pituitary pars intermedia dysfunction (PPID), a rare cause of GH excess, can lead to abnormal fat distribution, muscle wasting, and excessive hair growth. In catle, rare cases of GH excess can manifest as increased body size and insulin resistance.

[0686] Diagnosing GH-related disorders in animals relies on a combination of clinical observations, blood tests for GH and insulin-like growth factor-1 (IGF-1) levels, glucose tolerance tests, and advanced imaging modalities such as CT or MRI to identify pituitary tumors. Existing treatment options, including surgical hypophysectomy, radiation therapy, and pharmacological agents like somatostatin analogs or dopamine agonists, are limited by their invasiveness, variable efficacy, and potential side effects.

[0687] Through administration of GHAs. including tailored fonnulations and delivery approaches, the current disclosure provides methods for directly inhibiting GH signaling by blocking GH interaction with the growth hormone receptor (GHR), thereby mitigating the downstream effects of GH overproduction.

[0688] In accordance with the disclosure, GHAs can be administered by various delivery routes depending on the indication and therapeutic strategy. In embodiments, such delivery routes can include systemic administration, such as by subcutaneous or intravenous routes, to broadly suppress systemic manifestation of GH activity associated with insulin resistance and organomegaly. In embodiments, delivery' routes can include localized administration, such as by direct injection to tumoral or ectopic sites of GH overexpression.

[0689] In accordance with the disclosure, GHA can be prepared in various formulations for delivery. For example, GHAs can be encapsulated in biodegradable polymers or hydrogels in sustained release formulations to provide extended therapeutic benefits. In other embodiments, GHA can be provided as protease resistant oral formulations for non-invasive administration in long-term disease management,

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[0691] especially in large animals. In other embodiments, GHAs can be provided as transdermal formulations, such as in microneedle patches, for convenient minimally invasive administration to companion animals.

[0692] Accordingly, in embodiments, the present disclosure provides a method for treating a disorder of excessive GH activity in a non-human animal or veterinary subject, comprising administering a GHA to a non-human animal in need thereof. The non-human animal or veterinary' subject can be a non-human mammal, such as a companion animal, e.g., a cat or dog, or a livestock animal.

[0693] In embodiments, the present disclosure provides a method of treating acromegaly in a companion animal, such as a dog or cat, comprising administering a therapeutically effective amount of a GHA.

[0694] In embodiments, tire GHA is an albumin (feline) GHA fusion protein, comprising an amino acid sequence of SEQ ID NO:20:

[0695] MKWVTFISLLLLFSSAYSRGVTRREAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKGCVAD QSAANCEKSLHELLGDKLCTVASLRDKYGEMADCCEKKEPERNECFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLG KYLYEIARRHPYFYAPELLYYAEEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKERLKCASLQKFGERAFKAWSV ARLSQKFPKAEFAEISKLVTDLAKIHKECCHGDLLECADDRADLAKYICENQDSISTKLKECCGKPVLEKSHCISEVERD ELPADLPPLAVDFVEDKEVCKNYQEAKDVFLGTFLYEYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPACYAHVFDE FKPLVEEPHNLVKTNCELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSRSLGKVGSKCCTHPEAERLSCAEDYLSW LNRLCVLHEKTPVSERVTKCCTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCTLPEAEKQIKKQSALVELLKHK PKATEEQLKTVMGDFGSFVDKCCAAEDKEACFAEEGPKLVAAAQAALAGGGSGGGSGGGSGGGSFPAMPLSELFAAAVLE

[0696] A|HLHQLAADTYKEFERAYIPEGQRYSl|NAQAAFCFSETIPAPiGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVF TNSLVEGTSDRVYEKLKDLERGIQALMRELEDGSPRGGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAEiYLR

[0697]

[0698] VMKCRRFVESSCiF (ALB-fGHA; SEQ ID NO:20).

[0699] SEQ ID NO:20 includes an N-terminal human serum albumin domain (MKWVTFISLLLLFSSAYSRGVTRREAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKGCVA DQSAANCEKSLHELLGDKLCTVASLRDKYGEMADCCEKKEPERNECFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFL GKYLYEIARRHPYFYAPELLYYAEEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKERLKCASLQKFGERAFKAWS VARLSQKFPKAEFAEISKLVTDLAKIHKECCHGDLLECADDRADLAKYICENQDSISTKLKECCGKPVLEKSHCI SEVER DELPADLPPLAVDFVEDKEVCKNYQEAKDVFLGTFLYEYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPACYAHVFD EFKPLVEEPHNLVKTNCELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSRSLGKVGSKCCTHPEAERLSCAEDYLSV VLNRLCVLHEKTPVSERVTKCCTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCTLPEAEKQIKKQSALVELLKH KPKATEEQLKTVMGDFGSFVDKCCAAEDKEACFAEEGPKLVAAAQAALA) followed by a peptide linker (GGGSGGGSGGGSGGGS), which is followed by a feline GHA domain ( FPAMPLSELFAA VLEA HLHQLAADTYKEFERAYIPEGQRYSI NAQAAFCFSETIPAP GKDEAQQRSDVELLRFSL

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[0701] LLIQSWLGPVQFLSRVFTNSLVLGTSDRVYEKLKDLERGIQALMRELEDGSPRGGQILKQTYDKFDTNLRSDDALLKNYG LLSCFKKDLHKAE|YLRVMKCRRFVESSC|F).

[0702] With respect to GHA1 to GHA4, the interacting residues at site 2. by homology to human GH (see alignment in Figure 17) are shown in underlined text and as mutated in SEQ ID NO:20, correspond to Ser8Glu, Asnl2Ala, Argl6Glu, PhelO2Leu, and Glul 17Arg in the feline GH sequence. The interacting residues at site 1, by homology to human GH (see alignment in Figure 13) are highlighted in gray and correspond to Gin 18, Gln45, Thr61, Thrl73, and Alal89 of the feline GH sequence.

[0703] In other embodiments, a mutant feline GHA has a site 2 modification to one or more residues selected from:

[0704] a substitution of serine (Ser; S) at position 8 with glutamate (Glu; E) or asparagine (Asn; N);

[0705] a substitution of asparagine (Asn; N) at position 12 with alanine (Ala; A), isoleucine (He; I), serine (Ser; S), or glutamine (Gin; Q);

[0706] a substitution of arginine (Arg; R) at position 16 with lysine (Lys; K), glutamate (Glu; E), leucine (Leu; L); alanine (Ala; A), or tryptophan (Trp; W);

[0707] a substitution of phenylalanine (Phe; F) at position 102 with Leucine (Leu; L) or Serine (Ser; S); and a substitution of glutamate (Glu: E) at position 117 with aspartate (Asp; D), arginine (Arg; R), valine (Vai; V), or alanine (Ala; A).

[0708] In other embodiments, a mutant feline GHA also includes a site 1 modification to one or more feline GH residues selected from:

[0709] a substitution of glutamine (Gin; Q) at position 18 with asparagine (Asn; N);

[0710] a substitution of glutamine (Gin; Q) at position 45 with asparagine (Asn; N);

[0711] a substitution of threonine (Thr; T) at position 61 with aspartate (Asp; D) or glutamate (Glu; E);

[0712] a substitution of threonine (Thr; T) at position 173 with aspartate (Asp; D) or glutamate (Glu; E); and a substitution of alanine (Ala; A) at position 189 with asparagine (Asn; N) or glutamine (Gin; Q).

[0713] With respect to GHA5 to GHA9, the interacting residues at site 2, by homology to human GH (see alignment in Figure 18) are shown in underlined text and correspond to Phe 10, Vall4, His21, and Glyl20 in the feline GH sequence. The interacting residues at site 1, by homology to human GH (see alignment in Figure 13) are highlighted in gray and correspond to Glnl8, Argl67. Lysl68, Hisl71, Lysl72. Aspl74, Tyrl76, and Vall79 in the feline GH sequence.

[0714] In embodiments, a mutant feline GHA (based on Set Two) has:

[0715] a site 2 modification to one or more feline GH residues selected from:

[0716] a substitution (mutation) of phenylalanine (Phe; F) at position 10 with alanine (Ala; A) or histidine (His; H), a substitution of Valine (Vai; V) at position 14 with tryptophan (Trp; W) or glycine (Gly; G),

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[0718] a substitution of histidine (His; H) at position 21 with asparagine (Asn; N), and

[0719] a substitution of glycine (Gly; G) at position 120 with a lysine (Lys; K) or arginine (Arg; R);

[0720] and a site 1 modification to one more feline residues selected from:

[0721] a substitution of glutamine (Gin; Q) at position 18 with aspartate (Asp: D,) or asparagine (Asn: N).

[0722] a substitution of arginine at position 167 with asparagine (Asn; N) or glutamate (Glu; E).

[0723] a substitution of lysine (Lys; K) at position 168 with alanine (Ala; A);

[0724] a substitution of histidine (His; H) at position 171 with a serine (Ser; S);

[0725] a substitution of lysine (Lys; K) at position 172 with an arginine (Arg; R);

[0726] a substitution of aspartate (Asp: D) at position 174 with a serine (Ser; S) or threonine (Thr; T); and a substitution of valine (Vai, V) at position 179 with a threonine (Thr; T).

[0727] In embodiments, a mutant feline GHA has one or more site 2 modifications and one or more site 1 modifications based on both Set One and Set Two GHAs. Thus, a mutant feline GHA may encompass one or more of the preceding substitutions at feline (site 2) residues 8. 10, 12, 14, 16, 21, 102. 107, 117. and 120, and one or more of the preceding substitutions at feline (site 1) residues 18, 45, 61, 167, 168, 171, 172, 173, 174, 179, and 189.

[0728] In embodiments, the veterinary subject is a cat or dog with acromegaly, and the GHA is administered as a long-acting fusion protein, as disclosed herein. In embodiments, the veterinary’ subject is a livestock animal, and the GHA is used to treat or control a growth-related metabolic disorder.

[0729] In embodiments, the present disclosure provides a method for treating a GH-related tumor in a non-human animal, comprising intralesional injection of a GHA into the tumor site.

[0730] In embodiments, tire present disclosure provides a method for managing insulin resistance and growth abnormalities in cattle by administering a sustained released GHA formulation. More particularly, the sustained release GHA formulation comprises a GHA encapsulated in a biodegradable polymer or hydrogel.

[0731] In embodiments, the present disclosure provides a combination therapy for treating pituitary pars intermedia dysfunction in a horse, comprising administering a GHA in combination yvith a dopamine agonist.

[0732] In embodiments, the GHA is administered at a frequency ranging from once daily to once every two months.

[0733] In embodiments, the GHA is administered at a dose of 0.01 mg / kg to 500 mg / kg.

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[0735] EXAMPLES

[0736] The following illustrative examples are representative of embodiments of the GHAs, compositions, formulations, and methods described herein and are not meant to be limiting in any way.

[0737] Example 1

[0738] Structural and Energetic Considerations for GHAs Targeting the GHR Complex Introduction

[0739] Growth hormone, also known as somatotropin, is a key regulator of body growth and metabolism in humans. The effects of growth hormone are mediated through its specific receptor, the growth hormone receptor (GHR), which is present on the cell surface in many tissues. Once growth hormone binds to its receptor, it triggers a series of biochemical events leading to the activation of specific genes that bring about growth and metabolic changes. The intricate nature of the GH-GHR interaction and its implications in health and disease underscores the importance of understanding this pathway at a molecular level.

[0740] GHR activation is a multi-step process that enables precise regulation of GH signaling. This activation begins with ligand binding, where a single GH molecule binds two GHR molecules sequentially, first via its "site 1" motif to one receptor and then through its "site 2" motif to a second receptor. Although GHRs may exist as preformed dimers, GH binding triggers conformational changes that ultimately lead to receptor activation. In particular, GH binding triggers a rotational change in the GHR transmembrane domains, leading to transphosphorylation and activation of two Janus kinase 2 (JAK2) kinases associated with the cytoplasm domains of the GHR. In turn, activated JAK2 phosphorylates tyrosines in the GHR cytoplasmic domains, facilitating recruitment of signaling molecules to the receptor.

[0741] The primary signaling cascades triggered by GH binding involve JAK-STAT, Ras-ERK, and PI13K-Akt pathways. These cascades ultimately result in transcriptional changes mediating GH growth -promoting activities, such as increasing IGF-1 levels. See, e.g., Waters 2016, IGF Research. 28, 6-10; Harding et al. 1996, J. Biol. Chem. 111, 6708-6712. Subsequently, elevated levels of GH and IGF-1 initiate feedback mechanisms, involving negative regulators such as SOCS proteins, which inhibit the release of growth hormone-releasing hormone (GHRH), promote the release of somatostatin, and consequently suppress further GH secretion from the pituitary7. Harvey 2010, Endocrine 38, 335-359.

[0742] This complex activation mechanism ensures the precise modulation of GH signaling, supporting various physiological functions, including growth, metabolic regulation, and tissue-specific activities. As excessive or dysregulated GH signaling is implicated in pathological conditions such as acromegaly and certain cancers, GH mutants that can antagonize the GH: GHR interactions can have substantial therapeutic value.

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[0744] Figure 1 depicts 3HHR, the previously solved 2.8 angstrom crystal structure of the complex between human GH and the extracellular domain of the human GHR. See de Vos et al. 1992, Science 255, 306-312. The crystal structure confirms that one GH molecule binds to 2 GHR molecules via two distinct and asymmetric binding sites. GH (Chain A) is a four-helix bundle shown in green, and tire extracellular GHR domains are shown in orange (Chain B) and purple (Chain C).

[0745] Structural and energetic contributions of interactions between chains in GHR complex With the 3HHR cry stal structure as a foundation, interactions at the interface between tire GH and GHR were analyzed using PDBePISA (Proteins, Interfaces, Structures and Assemblies), an interactive tool for exploring macromolecular surfaces. Molecular insights gleaned from the analysis were then applied to the targeted design of GH mutants targeting the GHR: GH: GHR complex.

[0746] Figure 2 shows the PISA interface list, presenting the interactions between chains of the human growth hormone-receptor complex in PDB 3HHR. The left and middle panels define the specific interacting chains, while the right panel shows the interface area, change in free energy, number of hydrogen bonds, and number of salt bridges. Also shown in the CSS (Complexation Significance Score) value, which provides an indication of the interface's significance for assembly fonnation. The score is defined as a maximal fraction of the total free energy of binding that belongs to the interface in stable assemblies.

[0747] As discussed in more detail below, the results reveal: (1) a significant interaction at tire AB interface (Site 1), indicating stable binding necessary for receptor activation; (2) a significant but slightly weaker interaction at the AC interface (Site 2), contributing to the overall stability and specificity of the complex; and (3) a strong interaction at the BC interface within the receptor complex.

[0748] The interface between chains A and B can be classified as a ligand-receptor interaction with high affinity: The interface between chain A (Growth Hormone) and chain B (Growth Hormone Receptor Site 1) in the 3HHR crystal structure represents the primary functional interaction driving receptor binding and activation. Referring to the PISA interface list in Figure 2, this interface has a surface area of 1254.3 A2, making it the largest interaction in the structure. The free energy change (AG) is -8.1 kcal / mol, with a AG P-value of 0.503, indicating a stable and significant interaction. The interface is strengthened by 17 hydrogen bonds and 9 salt bridges, which contribute to its high binding affinity and stability, with a CSS score of 1.000, confirming its biological importance. Table 1 summarizes the corresponding values from Figure 2.

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[0750] Table 1

[0751] Energetics of A-B Interaction

[0752] Buried Area Salt Row AG (kcal / mol) P-value H-Bonds CSS (Surface A2) Bridges

[0753] 1 -1254 -8.1 0.503 17 9 1.000

[0754]

[0755] The interface between chains A and C can be categorized as a ligand-receptor interaction with low affinity: In PDB 3HHR, the principal A-C interface occurs in row 2 of the PISA table, with a moderate interface area of —816 A2and a free energy change of 4.5 kcal / mol, indicating a meaningful but weaker contact than the main A-B interface. This interaction corresponds to “Site 2” in the hormone-receptor complex, where human growth hormone (Chain A) binds a second receptor molecule (Chain C). The other two listings (rows 3 and 6) potentially bearing on the A-C interaction are variants of the same subunits positioned by different crystal symmetry- operations: these have substantially smaller areas (554 A2and 308 A2) and lower free energy changes (-3.3 kcal / mol and -1.0 kcal / mol) than the row 2 listing, signifying weaker and likely non-biological cry stal contacts. Thus, among the A-C or C-A interfaces in 3HHR, the interface in Row 2 is the one with sufficient buried area and favorable energetics to be considered functionally relevant, while Row s 3 and 6 likely reflect cry stallographic artifacts. Table 2 summarizes the corresponding values from Figure 2.

[0756] Table 2

[0757] Comparative Energetics of Candidate A-C Interactions

[0758] Row Buried Area AG (kcal / mol) P-value H-Bonds Salt CSS (Surface A2) Bridges

[0759] 2 -816 -4.5 0.63 8 6 0.253

[0760] 3 -554 -3.3 0.55 8 0 0

[0761]

[0762] 6 -308 -1.0 0.64 2 0 0

[0763] In comparison to the A-C interface (~816 A2and -4.5 kcal / mol) in 3HHR, the A-B interface is both larger (—1254 A2of buried surface area) and more energetically favorable (about 8.1 kcal / mol). These differences align with the established notion that A-B is the high-affinity' “Site 1,” serving as the primary' binding interaction between growth hormone (chain A) and its receptor (chain B). By contrast, A-C is the second, weaker contact (“Site 2"). which although necessary for forming tire 1: 2 hormone-receptor complex does not provide as much stabilization. Moreover. PISA metrics such as the P-value and CSS indicate a

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[0765] stronger likelihood of A-B being an obligate, functionally important interface, whereas A-C, while still biologically relevant, scores lower and shows smaller crystallographic variants.

[0766] The interaction between chains B and C can be characterized as a Receptor-Receptor interaction: Tire interactions between chains B and C in the human growth hormone-receptor complex, as detailed in tire PISA interface list, reveal significant structural and energetic contributions. Interface 4 involves chains C and B with an interface area of 469.8 A2and a free energy change (AG) of -6.3 kcal / mol, stabilized by 7 hydrogen bonds and 1 salt bridge. Interface 7, also between chains C and B, has an interface area of 235.9 A2and a free energy change of -2.7 kcal / mol, supported by 2 hydrogen bonds. Additionally. Interface 8 involves chains C and B with an interface area of 185.6 A2and afiree energy change of -1.6 kcal / mol, stabilized by 2 hydrogen bonds. Summing these interactions, the total free energy change for all BC chain interactions is -10.6 kcal / mol, and the total interface surface area is 891.3 A2. These interactions underscore the role of chains B and C in ensuring the structural stability and functional integrity of the human growth hormone-receptor complex. Table 3 summarizes the corresponding values from Figure 2.

[0767] Table 3

[0768] Comparative Energetics of Candidate B-C Interactions

[0769] Buried Area Salt

[0770] Row AG (kcal / mol) P-value H-Bonds CSS (Surface A2) Bridges

[0771] 4 469.8 -6.3 0.227 7 1 0.268

[0772] 7 235.9 -2.7 0.264 2 0 0 8 185.6 -1.6 0.441 2 0 0

[0773]

[0774] Example 2

[0775] GHAs: Targeted Mutations Disrupting Interactions at the A-C Interface (Site 2) The rationale for designing GHAs, including the GHA1-GHA4 mutants, is based on insights from complex analysis, suggesting that that the interaction between the A and C chains is secondary to the primary high-affinity' interaction between chains A and B. This analysis suggested that the A-C interaction is comparatively weaker and likely follows the initial A-B binding, supporting approaches to selectively alter A-C interactions.

[0776] Reflecting these observations, a targeted strategy was conducted to develop GHAs with mutations in the A chain (GH) at specific interaction sites with the C chain (of the GHRthat can disrupt signaling through the A-C interface (Site 2) while preferably preserving or minimizing perturbations to the high-affinity A-B binding region.

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[0778] Detailed examination of the A-C interface, as summarized in Figure 3A, highlighted five key residues (positions 8, 12, 16, 103, and 119) as essential for A-C interaction: arginine (Arg), asparagine (Asn), arginine (Arg), tyrosine (Tyr), and glutamate (Glu). These residues contribute to hydrogen bonding and, in some cases, salt bridges with the C receptor chain. More particularly, Figure 3A describes the specific interactions between chain C of the Growth Honnone Receptor (designated as Structure 1) and the Growth Hormone Ligand (Chain A, designated as Structure 2), categorized into hydrogen bonds and salt bridges. The Hydrogen Bonds table details eight hydrogen bonding interactions, listing the residues and atoms involved from each structure, along with the distances between them in angstroms (A). The Salt Bridges table presents six salt bridge interactions between charged residues from chain C and chain A, specifying the residues and atoms involved and the distance betw een them in angstroms, reflecting ionic interactions that contribute to binding stability. No disulfide or covalent bonds were observed between the Growth Hormone Receptor and Growth Honnone Ligand in this interaction analysis.

[0779] These observations also underlie the design of mutant GHAs, comprising mutations at one or more of these residues. By altering hydrogen bonding, electrostatic, or other interactions involving such residues, such mutations may selectively modify' or weaken the A-C chain interactions without significantly compromising the A-B interaction.

[0780] From the listed hydrogen bonds and salt bridges, several interactions involve chain A residues that are currently polar or charged residues (e.g., Arg, Asn, Tyr, Glu) interacting with weak or neutral hydrogen bond partners on chain B (e.g., Ser, He) or charged residues on chain B (e.g., Arg, Asp, Glu). Introducing side chains that are less polar, oppositely charged, or bulky can potentially decrease the affinity for the targeted B residues by weakening hydrogen bonds or disrupting ionic (salt-bridge) interaction.

[0781] Targeted Mutations on Chain A to Disrupt the A-C Interface at Site 2:

[0782] 1. A: Arg8 interacting with C: Aspl26 and C: Ser219

[0783] Arg8 participates in multiple contacts and has a positively charged side chain. Substituting the Arg8 residue with an alanine residue will introduce a hydrophobic side chain that should weaken the interaction with the targeted C residues.

[0784] Other contemplated targeted substitutions / modifications / mutations include substituting Arg8 with a glutamate residue (as in GHA- 1) to introduce a negatively charged side chain; with a leucine residue (as in GHA-2) to introduce a hydrophobic side chain; with a glycine residue (as in GHA-3) to introduce a small residue lacking a side chain; or with a tryptophan residue (as in GHA-4) to introduce steric hindrance through a hy drophobic side chain with a bulky indole group.

[0785] 2. A: Asnl2 interacting with C: Arg43 and C: Aspl26

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[0787] Asnl2 participates in multiple contacts and has a polar side chain (CH2CONH2). Substituting the Asnl2 residue with a glutamine residue (as in GHA-4) introduces a longer side chain (CH2CH2CONH2) whose difference in length and relative charge may introduce steric hindrance and weaken the interactions with the targeted C chain residues.

[0788] Other contemplated targeted disruptions include substituting Asnl2 with an alanine residue (as in GHA-1) to introduce a non-polar side chain; with an isoleucine residue (as in GHA-2) to introduce a hydrophobic side chain; or with a serine residue (as in GHA-3) to introduce a small residue with a shorter polar chain.

[0789] 3. A: Arg 16 interacting with C: Glu44

[0790] Arg 16 is positively charged and forms a hydrogen body with Glu44. Substituting the Arg 16 residue with a lysine residue introduces a longer side chain with weaker hydrogen bonding potential that should weaken the interaction with the targeted C residue.

[0791] Other contemplated targeted disruptions include substituting Arg 16 with a glutamate residue (as in GHA-1) to introduce a negatively charged side chain; with a leucine residue (as in GHA-2) to introduce a hydrophobic side chain; with an alanine residue (as in GHA-3) to introduce a small residue with a short polar chain; or with a tryptophan residue (as in GHA-4) to introduce steric hindrance through a hydrophobic side chain with a bulky indole group.

[0792] 4. A: Tyrl03 interacting with C: Ilel65

[0793] Tyrl03 has a polar side chain comprising a phenyl moiety substituted with a hydroxyl group that participates in hydrogen bonding with He 165. Substituting the Tyrl03 residue with a phenylalanine (as in GHA-1 or GHA-4) introduces a side chain retaining the phenyl moiety but lacking the hydroxyl group. This change eliminates hydrogen-bonding potential while preserving hydrophobic stacking, which may maintain stability in nonpolar regions but alter binding dynamics where polar interactions are involved.

[0794] Other contemplated targeted disruptions include substituting Tyrl03 with a leucine residue (as in GHA-2) to introduce a hydrophobic side chain; or with a serine residue (as in GHA-3) to introduce a small residue with a shorter polar chain.

[0795] 5. A: Glul 19 interacting with C: Seri 02

[0796] Glul 19 has an acidic side chain (CH2CH2COOH) that interacts with Serl02. Substituting the Glul 19 residue with an aspartate residue introduces a shorter acid side chain (CH2COOH), whose difference in length and relative charge may weaken the interaction with S the targeted C residue.

[0797] Other contemplated targeted disruptions include substituting Glul 19 with an arginine residue (as in GHA-1 and in GHA-4) to introduce a basic side chain with a bulky guanidino group (-C(=NH)2HN2) that -87- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0798] can introduce steric hindrance basic side chain; with a valine residue (as in GHA-2) to introduce a hydrophobic side chain; or with an alanine residue (as in GHA-3) to introduce a small residue with a short polar chain.

[0799] Such variants, including GHA-1 through GHA-4, can be used in any of the compositions and methods of this disclosure. More generally, the present disclosure includes GHAs targeting one of more mutations at these and other positions. Mutations in these residues can alter hydrogen-bonding potential or electrostatic interactions in ways that can weaken the A-C binding, preferably without compromising the main A-B interaction. By modifying or weakening the A-C chain interaction, such antagonists can selectively interfere with downstream signaling.

[0800] The GHA 1 to GHA4 mutants thus serve as specific examples of targeted mutations at these five residues, designed to alter key bonding interactions at the A-C interface. Additional combinations of mutations at these or other sites can further provide a broader array of modified GHAs.

[0801] Example 3

[0802] GHAs: Additional Targeted Modifications Enhancing Interactions at the A-B Interface (Site 1) The AB interface (site 1) is the primary high-affmity binding site necessary for initial receptor engagement, whereas the AC interface (site 2) plays an important role in receptor dimerization and downstream signaling. As a strategy to further strengthen antagonist activity of GHAs herein, a targeted search was conducted for separate mutations in the B chain that could enhance or stabilize the A-B interface at Site 1. Combining such site 1 modifications with the previously described site 2 modifications can provide GHAs that can both outcompete GH for GHR binding and also prevent subsequent GHR signal transduction, offering highly effective therapeutic candidates. In other words, GHAs with targeted mutations at site 1 and site 2, in accordance with this disclosure, can enhance affinity at site 1 (to ensure the GHA effectively competes with endogenous GH for receptor binding) and weaken interactions at site 2 (to prevent proper receptor dimerization, as discussed in Example 2) thereby blocking JAK / STAT activation and GH-induced biological responses.

[0803] Detailed examination of the A-B interface highlights multiple residues involved in the interaction between the chains, contributing to hydrogen bonding and salt bridges between tire A and B chains. More particularly, Figure 3B lists the specific interactions between chain B of the Growth Hormone Receptor (designated as Structure 1) and Growth Hormone (chain A, designated as Structure 2), categorized into hydrogen bonds and salt bridges. Tire Hydrogen Bonds table details 17 hydrogen bonding interactions, listing the residues involved, along with tire distances between them in angstroms (A). The Salt Bridges table lists 9 salt bridge interactions between charged residues from the Growth Hormone Receptor and Growth Hormone, reflecting ionic interactions that contribute to binding stability. No disulfide or covalent bonds

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[0805] were observed between the Growth Hormone Receptor and Growth Hormone Ligand in this interaction analysis.

[0806] These observations also support the design of mutant GH variants, comprising mutations at one or more of these site 1 residues. By altering hydrogen bonding or electrostatic interactions involving such residues, such mutations may selectively enhance the A-B chain interaction at site 1. From the listed hydrogen bonds and salt bridges, several interactions involve Chain A residues that are currently weak or neutral hydrogen bond partners (e.g., Thr, Ser, Gly, His) interacting with strongly polar or charged residues on Chain B (e.g., Arg. Gin, Asn). Introducing side chains that are more polar or charged can potentially increase the affinity by creating more stable hydrogen bonds or converting a hydrogen bond into a stronger ionic (salt-bridge) interaction.

[0807] Targeted Mutations on Chain A to Enhance the A-B Interface at Site 1:

[0808] 1. A: Hisl8 interacting with B: Asn218

[0809] His 18 can form hydrogen bonds, but its protonation state can van’ depending on the pH. Asn218 is a good hydrogen-bonding partner, and introducing a residue with a more consistent hydrogen bond profile may strengthen this interaction. Substituting the His 18 residue with an asparagine or glutamine residue introduces a stable amide side chain to help ensure a strong, reliable hydrogen bond netw ork with Asn218 without the pH-dependent variability of histidine.

[0810] 2. A: Gln46 interacting with B: Trp76 and B: Thr77

[0811] Gln46 participates in multiple contacts with the B chain. Gin is already a good hydrogenbonding residue, but optional improvements are possible. For example, substituting the Gln46 residue with an asparagine residue could reduce side chain entropy and slightly strengthen the interaction by offering a more rigid hydrogen bonding geometry.

[0812] 3. A: Ser62 interacting with B: SerlO2 and B: Ilel03

[0813] Ser62 currently forms hydrogen bonds with Seri 02 and a backbone interaction with He 103. Substituting the Ser62 residue with an amide-containing residue, such as asparagine, provides additional hydrogen bonding possibilities that may lead to more favorable interactions at the Site 1 interface.

[0814] 4. A: Thrl75 interacting with B: Arg43

[0815] Currently, B: Arg43 forms hydrogen bonds with A: Thrl75 (through OG1). Substituting the Thrl75 residue with a negatively charged residue, such as aspartate or glutamate could form a stronger salt bridge (rather than the current simple hydrogen bond) with the guanidinium group on Arg43, increasing affinity at the Site 1 interface.

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[0817] 5. A: Glyl90 interacting with B: Glnl66

[0818] Glyl90 on Chain A provides only backbone hydrogen bonding capability with Chain B.

[0819] Substituting the Gly 190 residue with an asparagine or glutamine residue introduces a polar side chain that can form additional side-chain hydrogen bonds, potentially strengthening or stabilizing contacts with Gin 166.

[0820] In addition to hGH residues 18, 46, 62, 175, and 190 - which are preferably targeted herein for site 1 modifications - the PISA interface analysis also reveals other residues at the site 1 interface. These include the charged amino acids, Lys41, Arg64, Argl67, Lysl68, Aspl71, Glul74, and Argl78. While these charged residues are expected to confer existing strong electrostatic interactions with the B chain, optional improvements are possible (such as through the use of synthetic residues) and contemplated by this disclosure.

[0821] In sum, by introducing one or more of these site 1 mutations, the polarity and charge complementarity at the interface should increase, potentially resulting in higher binding affinity and a more stable complex between Chain A and Chain B.

[0822] More generally, combined targeted mutations at site 1 and site 2 of GHAs should prevent residual signaling that could result from partial receptor activation, ensuring tire GHA functions purely as a blocker. In other words, site 2 GHAs possessing such additional site 1 mutations should bind more tightly to site 1 than endogenous GH, further enhancing their capacity for competitive inhibition and therefore their potential as effective therapeutic candidates. As an example of enhancing binding affinity through targeted mutations in the growth hormone ligand that interact with the growth hormone receptor, residue 18 in the ligand was mutated from histidine (H) to asparagine (N). A structural model was generated and docked with the receptor, followed by interface analysis. While the number of hydrogen bonds remained unchanged at 17 in both models, the number of salt bridges increased from 9 to 13 in the mutant model.

[0823] Example 4

[0824] Bioinformatics Studies Showing Modulated GHA-GHR Interactions Introduction

[0825] This study evaluated the interaction dynamics between mutated forms of the GHA and the GHR. The aim was to determine if mutations in the GHA protein affect its binding to GHR in a way that disrupts normal ligand-receptor interactions, thereby potentially antagonizing GHR signaling. Understanding these altered interactions offers insights into the effectiveness of each GHA mutant as an antagonist, providing a foundation for therapeutic agents targeting GHR.

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[0827] It was hypothesized that GHA mutant proteins would interact with GHR in a manner that disrupts the normal ligand-receptor interactions, leading to altered chain interactions within the complex and subsequent antagonism of GHR signaling. Specifically, these mutations should impact the interaction energy between the chains, thereby weakening the stability of the complex required for effective GHR signaling.

[0828] Methods

[0829] To evaluate this hypothesis, the interaction energies and structural configurations of mutant GHA-GHR complexes were compared to that of the wild-type GHA: GHR complex (3HHR). The mutant GHAs evaluated were NT-GHA1, NT-GHA2, NT-GHA3, and NT-GHA4.

[0830] Each mutant-wild-type comparison was performed using a sequential set of bioinformatics tools: (1) generating three-dimensional models for each GHA mutant protein and saving them as Protein Data Bank (PDB) files; (2) docking the GHA mutant models to the receptor chains (B and C) of the reference 3HHR complex to form mutant-GHR complexes; (3) analyzing the protein-protein interfaces in the newly formed GHA mutant-GHR complexes to assess binding properties; and (4) comparing the interface properties and interaction energies of each mutant complex to those of the original 3HHR complex.

[0831] Results

[0832] As shown in Table 4 and Figure 4, the comparative analysis of all BC, AB, and AC interaction energies among the complexes of Growth hormone receptors (GHR) and GHA mutants reveals significant differences in their binding properties and potential as antagonists. Table 4 summarizes the calculated interaction energies of growth hormone antagonist (GHA) mutants in complexes with growth hormone receptor (GHR), providing insights into the binding dynamics of the BC, AB, and AC receptor subunit chains, along with the total interaction energy. The values serve as indicators of how specific mutations affect the stability and energetics of the receptor-ligand interface compared to the control structure (3HHR). Lower energy values suggest stronger interactions, while positive values indicate destabilization or loss of interaction. More particularly, for each mutant complex the BC, AB, and BC energy, as well as total energy, were increased, i.e., less negative, compared to the corresponding energies in the 3HHR complex.

[0833] Table 4

[0834] Comparative Chain Interaction Energies in Mutant and Wild-Type GHA: GHR Complexes Molecule Name BC Chains AB Chains AC Chains Total Energy Energy Energy Energy

[0835] 3HHR (Control) -10.6 -8.1 -8.8 -27.5

[0836]

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[0838] GHA1-GHR complex -6.3 -6.2 -6.3 -18.8

[0839] GHA2-GHR complex -6.3 3.9 0.2 -2.2

[0840] GHA3-GHR complex -6.3 -7.6 -8.4 -22.3

[0841] GHA4-GHR complex -6.3 -7.4 -1.9 -15.6

[0842]

[0843] Figure 4 graphically depicts these results, allowing a visual comparison of the paired and total interaction energy values in the mutant and wild-type complexes. The results highlight the impact of mutations on ligand-receptor interaction stability, with the mutant complexes generally displaying less favorable energy profiles compared to the wild-type.

[0844] More particularly, the NT-GHA1 through NT-GHA4 molecules exhibit distinct interaction energies across various chain pairs (BC, AB, and AC). As shown in the top graph (Figure 4A), wild-type ligand-receptor complex (3HHR) exhibits stronger negative interaction energies across all chain pairs, indicating robust binding, while the mutant forms (NT-GHA1, 2, 3, and 4) show variations in interaction strength, with some chains even exhibiting positive interaction energies, reflecting weakened binding. The bottom graph (Figure 4B) summarizes the total interaction energies for each molecule, showing that the wild-type structure (3HHR) has the most favorable total energy, while the mutant structures have comparatively lower total energies.

[0845] GHA1

[0846] The interaction energies of the GHA1-GHR complex are notably weaker than those of the control (3HHR) complex, indicating reduced binding affinity across all chain interactions (BC, AB. and AC).

[0847] In the 3HHR control complex, the interaction energies forthe BC, AB, and AC chains are -10.6 kcal / mol, -8.1 kcal / mol, and -8.8 kcal / mol, respectively, resulting in atotal energy of -27.5 kcal / mol. These strong negative interaction energies reflect a robust binding configuration necessary for effective GHR signaling.

[0848] In contrast, the GHA1-GHR complex exhibits reduced negative interaction energies, with BC, AB, and AC chain interactions measured at -6.3 kcal / mol. -6.2 kcal / mol, and -6.3 kcal / mol, respectively. This yields a total energy of -18.8 kcal / mol, significantly weaker than that of the control (-27.5 kcal / mol). The decrease in interaction strength across each chain interaction suggests that the GHA1 mutation disrupts the stability of the GH-GHR complex. This disruption likely impedes the ability of the complex to maintain the conformational integrity needed for signaling, making GHA1 a potential antagonist by competing with the natural ligand for GHR binding but failing to trigger the downstream signaling pathway.

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[0850] GHA2

[0851] GHA2 exhibits a unique interaction profile with a positive energy value for AB chains (3.9 kcal / mol), indicating a repulsive interaction, and a near-zero energy for AC chains (0.2 kcal / mol). The only significantly negative interaction is with the BC chains (-6.3 kcal / mol). The overall low total energy of -2.2 kcal / mol suggests that GHA2 is likely a weak binder with disruptive potential. The repulsive AB interaction could destabilize the target complex, making GHA2 an antagonist by preventing the formation of stable interactions and thus effectively inhibiting the function of the target protein.

[0852] GHA3

[0853] GHA3 has negative interaction energies across all chains, with the total energy of -22.3 kcal / mol. This suggests robust binding and significant interaction with the target protein complex. As an antagonist, GHA3 may bind tightly to the target, potentially outcompeting the natural ligand and stabilizing non-functional conformations. This strong binding could result in the effective inhibition of the target protein’s normal activity by maintaining a stable but inactive complex.

[0854] GHA4

[0855] GHA4 exhibits moderate interaction energies with BC and AB chains, but a relatively weak interaction with AC chains (-1.9 kcal / mol). The total energy of -15.6 kcal / mol indicates moderate binding affinity. As an antagonist, GHA4 might selectively interfere with certain interactions (specifically BC and AB) while allowing some degree of flexibility or disruption in others (AC). This selective binding could result in partial inhibition of the target protein’s activity, making GHA4 a potential partial antagonist that modulates rather than completely inhibits the target function.

[0856] These varied interaction profiles suggest that each GHA molecule has distinct mechanisms and efficacy as an antagonist, with potential applications depending on the specific therapeutic context.

[0857] Example 5

[0858] Bioinformatics Studies Supporting Enhanced Stability of GHA Fusions This study employed bioinformatics tools to evaluate whether fusing GHA1 to albumin or transferrin would enhance the half-life and stability of GHA 1 in mammalian, yeast, and Escherichia coli models, thereby assessing the potential suitability of fusion proteins for therapeutic applications where extended protein stability is desirable.

[0859] Introduction

[0860] Human GH (hGH) has a circulating half-life of 20-30 minutes, with rapid clearance by the liver and kidneys, and a biological half-life of 9-17 hours due to its indirect effects via IGF-1 stimulation. Factors influencing its half-life include the route of administration (e.g., subcutaneous lasting longer than intravenous), formulation differences, and individual considerations such as patient metabolism and hormone deficiency. Pharmacokinetically, subcutaneous administration peaks in 4-5 hours with a half-life of around 3

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[0862] hours, while intravenous administration has a half-life of approximately 21 minutes. Due to its short circulating half-life, hGH requires frequent dosing, often daily, with sustained effects maintained through its longer biological half-life. (See https: / / go.drugbank.com / drugs / DB00052)

[0863] Growth hormone antagonists (GHAs) hold significant therapeutic potential in treating conditions associated with excessive growth hormone (GH) activity, such as acromegaly, diabetes, and cancers.

[0864] However, a limitation in their therapeutic application is their typical short half-life, which necessitates frequent dosing to maintain effective plasma levels, potentially reducing patient compliance and therapeutic efficacy. Extending the half-life of GHAs can therefore be advantageous to enhance their clinical utility and reduce the frequency of administration.

[0865] Fusing GHAs to larger, long-circulating proteins such as albumin. Fc fragments, or transferrin is a promising approach to increase their half-life. This strategy leverages the pharmacokinetic properties of such carrier proteins, which resist rapid degradation and clearance, thereby prolonging the presence of the GHA in circulation. By their increased molecular size, protein fusions are also less prone to renal filtration, which is a common elimination pathway for smaller therapeutic proteins and peptides. Additionally, such fusions can provide steric hindrance against enzymatic degradation, further extending the antagonist's stability in the bloodstream.

[0866] The fusion of GHAs to carrier proteins therefore offer a potential means of achieving sustained therapeutic levels with reduced dosing frequency. Advantageously, this approach may enhance patient convenience and compliance and lead to more consistent therapeutic outcomes by maintaining antagonistic effects on GH signaling over extended periods. Ultimately, designing GHAs with enhanced half-life through protein fusions can address a significant limitation in GHA therapy, facilitating their use in long-term management of GH-related disorders.

[0867] Methods

[0868] The half-life of tested GHA fusions proteins was estimated using the ExPASy ProtParam (ProtParam) tool, which computes various physical and chemical parameters for a given protein query, including the molecular w eight, predicted pl, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (GRAVY).

[0869] ProtParam estimates the half-life of proteins using the N-end rule, which correlates the stability of a protein in vivo to its N-terminal amino acid. The predicted half-life was provided for three model organisms: human, yeast, and E. coli. This rule, established from experiments with engineered proteins containing different N-terminal residues, offers a general prediction based on the metabolic fate of proteins with specific amino acid residues at their amino terminus.

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[0871] The selected proteins were inputted as raw amino acid sequences to ProtParam, ensuring that space and numerical characters were ignored in alignment with ProtParam's sequence handling guidelines. For each protein, the complete sequence was analyzed without any modifications or assumptions about post-translational modifications, multimeric states, or N-terminal modifications. Tire half-life for each protein was calculated based on its N-terminal amino acid and documented in hours or minutes according to the ProtParam output for mammalian, yeast, and E. coli models.

[0872] Results

[0873] Table 5 shows the estimated half-life, as well other physical and chemical parameters, for the GHA1 protein by itself (SEQ ID NO: 1): Albumin-GHAl: a fusion of GHA1 at its N-terminus (via a GGGGSGGGSGGGGS linker) to a human albumin domain (SEQ ID NO:5); and Transferrin-GHAl: a fusion of GHA1 at its N-terminus (via a GGGGSGGGGSGGGGS linker to a human transferrin domain (SEQ ID NO:6).

[0874] Table 5

[0875] Comparative Stability and Half-Life Analysis of GHA1 and Fusions with Albumin and Transferrin Molecule

[0876] GHA1 Albumin-GHAl T ransferrin-GHAl Parameter

[0877] MW 22.86 kDa 100.87 kDa 110.75 kDa Theoretical pl 5.69 6.31 6.35

[0878] Instability Index137.58 36.90 36.23

[0879] Aliphatic Index281.68 72.65 74.05

[0880] GRAVY3-0.443 -0.379 -0.361

[0881] Estimated Half-Life

[0882] 1. Mammalian reticulocytes 1.1 h 30 h 30 h

[0883] (in vitro)

[0884] 2. Yeast 3 min > 20 h > 20 h

[0885] (in vivo)

[0886] 3. E. coli 2 min > 10 h > 10 h

[0887] (in vivo)

[0888]

[0889] 1All three molecules classified as stable.

[0890] - All 3 molecules predicted to have moderate thermostability.

[0891] 3All 3 molecules predicted to have a hydrophilic nature.

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[0893] All three proteins have instability indices below 40, moderate thermostability (aliphatic index), and a hydrophilic tendency indicated by negative GRAVY values. GHA1, with a shorter half-life, is less stable in mammalian, yeast, and bacterial models than the albumin and transferrin fusions, which show significantly extended half-lives.

[0894] The data highlight the enhancements achieved through fusions, including increased molecular weight, improved isoelectric point (pl), extended half-life across different biological models (mammalian reticulocytes, yeast, and E. coli), and maintained stability as indicated by the instability index. These results underscore the advantages of albumin- and transferrin-linked constructs in therapeutic applications, demonstrating superior pharmacokinetics and structural stability compared to the native GHA 1.

[0895] Example 6

[0896] Structural Models of GHA Fusions

[0897] Structural models of GHA fusions further support antagonist activity.

[0898] Figure 5 depicts a structural model of the human serum albumin-GHAl fusion protein, corresponding to SEQ ID NOTO. The ribbon diagram illustrates that the fusion protein is composed of three main parts: albumin, the linker, and GHA1. Albumin, the larger globular structure at the bottom, increases half-life and stability of the fusion protein. GHA1, positioned at the top, displays the expected helical structure and is connected to albumin via the linker. The linker provides flexibility and spatial separation between albumin and GHA1, helping to maintain the functionality of each component. This configuration allows GHA1 to retain its activity without interference from the albumin component, making this fusion protein design beneficial for enhanced stability and function in therapeutic applications.

[0899] Figure 6A depicts a structural model of GHA1 fused to an Immunoglobulin G1 Fc domain (Ig Fc), as encoded by SEQ ID NO: 19. The GHA1 domain is the functional region targeting grow th hormone receptors. The IgGl Fc region (IgG), outlined by the dashed lasso, enhances the stability and potential cellular uptake of the molecule. A flexible linker connects GHA1 and IgG Fc, allowing functional flexibility between the two domains. This fusion design aims to leverage GHAl's antagonistic properties with the delivery benefits provided by the IgG Fc fusion. Indeed, as described in experiment 3 of Example 7, GHA-1-IgG Fc, when combined with Sorafenib, significantly enhances inhibition of HepG2 cell proliferation.

[0900] Figure 6B provide a schematic structure of a GHA5 fused to an IgGl Fc domain (as described further in Example 12. Because the Fc domain can dimerize, the GHA-Fc domain is expected to form a dimer and therefore present two GHA5 molecules (as a bivalent molecule).

[0901] Example 7

[0902] Synergistic Anti-Tumor Activity of GHA1 Fusions with Sorafenib in a Human HCC Cellular Model -96- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0903] Introduction

[0904] Sorafenib has been a notable advancement in treating advanced hepatocellular carcinoma (HCC), but its use comes with several limitations. In terms of efficacy, sorafenib offers only modest survival benefits; the SHARP trial showed a median overall survival of 10.7 months for sorafenib compared to 7.9 months for placebo. See Rimassa et al. 2009, Expert Rev. Anticancer Ther. 9, 739-745. Only about 30-40% of patients respond to treatment, partly due to the genetic heterogeneity of HCC. Even for those who initially benefit, the effect is often short-lived, with resistance developing within six months. Resistance arises through various mechanisms, including epigenetic changes, altered transport processes, and modifications in the tumor microenvironment, which collectively diminish sorafenib’s long-term effectiveness. Safety and tolerability are also concerns, as sorafenib is associated with adverse events such as hand-foot skin reactions, fatigue, diarrhea, hypertension, and abdominal pain. In severe cases, these side effects lead to treatment discontinuation, reducing the overall benefit of the therapy. Moreover, sorafenib trials have strict eligibility criteria, limiting it applicability to HCC patients, and patients with advanced cirrhosis or poor performance status often experience minimal benefit. Pharmacokinetic variability further complicates its use, as differences in drug absorption and delivery' can impact effectiveness.

[0905] Lower doses of sorafenib may be clinically beneficial in addressing these limitations. Lower dosing could reduce the severity of adverse events, particularly skin and gastrointestinal toxicities, leading to better treatment adherence and fewer instances of discontinuation. Studies indicate that reduced doses do not significantly compromise survival outcomes, suggesting that lower doses may be just as effective as full doses, especially considering dose intensity does not strongly correlate with survival. Economically, fewer pills result in a lower overall cost, increasing accessibility. Prom a personalized treatment perspective, lower starting doses allow physicians to assess individual tolerability and adjust as needed. This approach may particularly benefit older patients and certain populations, such as East-Asian patients, who often start on lower doses. Lower dosing may also enable patients to remain on treatment longer, potentially leading to a higher cumulative dose and improved efficacy overtime. By reducing discontinuation rates and improving quality of life through fewer side effects, lower doses of sorafenib offer a balanced approach, enhancing the therapy's effectiveness, tolerability, and cost-efficiency in clinical practice.

[0906] Accordingly, the present study evaluated the synergistic effect of a GHA antagonist, in combination with sorafenib, in blocking GHR activity in a liver cancer proliferation assay. More particularly, this study evaluated whether GHA1 fusion proteins could enhance the therapeutic efficacy of Sorafenib in the inhibition of HepG2 liver cancer cell proliferation. GHA 1 -Albumin combines a growth hormone antagonist (GHA1) with albumin to improve stability, half-life, and delivery, enabling synergistic effects with suboptimal doses of sorafenib, a standard treatment for hepatocellular carcinoma (HCC). Similar benefits may be conferred by GHA1 fusions to transferrin and IgG Fc carrier proteins. Tire aim was to

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[0908] explore whether the fusion protein could augment sorafenib’s anti-proliferative activity through targeted disruption of growth hormone receptor (GHR) signaling pathways.

[0909] Methods

[0910] The study evaluated a sorafenib suboptimal dose alone, versus a sorafenib suboptimal dose in combination with a GHA, on HepG2 cell proliferation. In this study, three different GHA antagonist were evaluated - all fusions to GHA1: GHAl-Albumin (SEQ ID NO: 14), GHA1 -Transferrin (SEQ ID NO: 18). and GHAl-IgG Fc (SEQ ID NO: 19).

[0911] This study utilized a metabolic activity assay as a proxy for cell proliferation, allowing comparative analysis across treatment groups. The assay used in tire study measures a dye that correlates with the metabolic activity of the cells and therefore is an indicator of cell proliferation. The protocol is not designed to enumerate cells. The protocol allows determination of the relative effect of a treatment on cell proliferation as compared to another treatment. The study was conducted using an in vitro assay on HepG2 liver cancer cells. This assay measures cell metabolic activity using a colorimetric dye, which serves as an indicator of cell proliferation.

[0912] The rationale for employing a suboptimal Sorafenib dose (3 pM) was to create a baseline inhibition scenario w here potential enhancements by the particular GHA1 fusion could be effectively quantified. Sorafenib’s efficacy at optimal doses often obscures additive or synergistic effects of combination therapies. Therefore, suboptimal dosing provided a means to investigate NT-GHA1 -Albumin's role in enhancing therapeutic outcomes while potentially mitigating Sorafenib’s adverse effects, such as hand-foot syndrome and hypertension, associated with higher doses.

[0913] Initial studies were performed to determine media interference with the CellTiter 96® Assay Reagent. This study contains a vehicle control as a reference to cell growth with no treatments. This study comprised the following three experiments, collectively evaluating sorafenib in combination with GHAl-albumin (SEQ ID NO: 14). GHA 1 -transferrin (SEQ ID NO: 18), and GHA-IgG Fc (SEQ ID NO: 19):

[0914] Experiment 1 Experiment 2 Experiment 3

[0915] Control (Vehicle only) Control (Vehicle only) Control (Vehicle only) Sorafenib only (3 pM) Sorafenib only Sorafenib only

[0916] Sorafenib (3 pM) plus Sorafenib (3 pM) plus Sorafenib (3 pM) plus GHAl-Albumin (40, 80, 120, GHA 1 -Transferrin (40, 80, GHAl-IgG Fc (40, 80, and and 240 pg / mL) and 120 pg / mL) 240 pg / mL)

[0917]

[0918] Independent t-tests were performed to compare the effects of each combination therapy against Sorafenib alone. Statistical significance was determined at thresholds of p<0.05, p<0.01, and p<0.001. The dose-response relationship was assessed to validate the enhancement trend.

[0919] Results

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[0921] Experiment 1:

[0922] As shown in Figure 7, GHAl-Albumin enhanced sorafenib’s inhibitory effect on HepG2 cell proliferation. Compared to sorafenib alone, combination treatment showed statistically significant improvements at all GHAl-Albumin doses assessed: 40 pg / mL (p<0.05), 80 pg / mL (p<0.01), 120 pg / mL (pO. OOl). and 240 pg / mL (pO. OOl).

[0923] The data support a dose-response relationship, with greater concentrations of GHAl-Albumin yielding stronger inhibition of HepG2 proliferation. This effect underscores the therapeutic potential of GHAl-Albumin in augmenting Sorafenib's activity, particularly in scenarios requiring reduced dosing due to Sorafenib’s adverse effects.

[0924] A similar trend (on enhancing antiproliferative activity of Sorafenib) was observed with other GHA-albumin fusion proteins, specifically GHA2 -Albumin (SEQ ID NO: 15), GHA3-Albumin (SEQ ID NO: 16), and GHA4-Albumin (SEQ ID NO: 17).

[0925] In summary, GHA-albumin fusion proteins significantly enhance the anti-proliferative efficacy of sorafenib in HepG2 liver cancer cells. The findings support GHA-albumin fusion protein as a promising candidate for combination therapy, potentially offering improved clinical outcomes through reduced sorafenib dosing and enhanced tolerability. This study provides a foundation for further preclinical and clinical investigations into the synergistic mechanisms and therapeutic applications of GHA-based fusion proteins in oncology.

[0926] Experiment 2:

[0927] As shown in Figure 8, GHA1 -Transferrin enhanced sorafenib’s inhibitory effect on HepG2 cell proliferation. Compared to sorafenib alone, combination treatment showed statistically significant improvements at all GHA1 -Transferrin doses assessed: 40 pg / mL (p=0.000018); 80 pg / mL (p=0.000044); and 120 pg / mL (p<0.000042). These low p-values provide strong statistical evidence that the addition of GHA-1 -Transferrin at a range of concentrations enhances the anti-proliferative effect of sorafenib on the HepG2 cell line, suggesting that GHA-1 -Transferrin effectively contributes to the inhibition of cell growth in this context.

[0928] Overall, the findings suggest that GHA-1 -Transferrin, when combined with sorafenib, offers a significant advantage in reducing cell proliferation over Sorafenib alone, with consistent efficacy across the tested concentrations. Even at the lowest concentration evaluated (40 pg / mL). GHA-1 -Transferrin achieves a comparable level of cell proliferation reduction when paired with sorafenib, providing a stable and potentially optimal dosage range for this combination treatment in inhibiting HepG2 proliferation. This suggests that GHA-1 -Transferrin can potentiate the effects of sorafenib, offering an improved therapeutic strategy for liver cancer treatment.

[0929] Experiment 3:

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[0931] As shown in Figure 9, GHAl-IgG Fc enhanced sorafenib’s inhibitory effect on HepG2 cell proliferation. Compared to sorafenib alone, combination treatments showed significantly reduced cell proliferation (p<0.05) at all GHAl-IgG Fc doses tested, with the greatest statistical significance at the 80 pg / mL concentration. The results demonstrates that GHA-l-IgG Fc, when combined with sorafenib, significantly enhances inhibition of HepG2 cell proliferation. This combination could potentially offer a more effective therapeutic approach for inhibiting liver cancer cell growth by targeting the grow th hormone receptor pathway.

[0932] Discussion

[0933] The studies explored the enhancement of sorafenib’s anti-proliferative effects on HepG2 liver cancer cells by combining it with various fusion proteins designed to inhibit growth hormone receptor (GHR) pathways. Each study focused on a different fusion protein: GHAl-Albumin, GHA1 -Transferrin, or GHAl-IgG Fc, each employing distinct enhancements for stability and half-life to improve GHAl’s potential as a GHR inhibitor. The combination of sorafenib with GHAl-Albumin showed a dose-dependent inhibitory effect, with statistical significance strengthening as GHAl-Albumin concentrations increased, indicating a clear dose-response relationship. Meanwhile, GHA1 -Transferrin demonstrated potent antiproliferative effects even at the lowest concentration, suggesting that efficacy plateaued across doses and potentially identifying an optimal therapeutic range. Similarly, GHAl-IgG Fc also significantly enhanced Sorafenib’s effects, with the strongest inhibition observed at 80 pg / mL.

[0934] Collectively, these studies underscore the potential of using GHA1 -based fusion proteins (and other GHA fusions disclosed herein) to augment sorafenib’s efficacy in treating liver cancer, with each protein showing a promising ability to inhibit cell proliferation through targeted GHR pathw ay interference. These findings lay a foundation for the clinical potential of such combination therapies in improving treatment outcomes for liver cancer.

[0935] Example 8

[0936] Inhibition of GH- Induced STAT3 Phosphorylation in a Human HCC Cellular Model by a GHA1

[0937] Fusion Protein

[0938] Introduction

[0939] The rationale for this study arises from the need to develop novel therapeutic agents that antagonize growth signaling pathways, specifically those involving STAT3, a key transcription factor in promoting cell proliferation, survival, and tumorigenesis. Aberrant activation of STAT3 has been implicated in various cancers and growth-related disorders, where it supports uncontrolled cell growth and apoptosis resistance. Growth hormone is one factor known to activate STAT3, and targeting this pathway may offer a novel approach to inhibit pathological cell growth.

[0940] -100- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0941] In this context, GHA1 is hypothesized to act as a growth antagonist by inhibiting STAT3 phosphorylation and subsequent activation. Reducing STAT3 activity could counteract the growthpromoting effects of GH, providing therapeutic benefits in diseases driven by excessive growth signaling. HepG2 cells, a human liver cancer cell line, were chosen for this study due to their functional GH receptor and relevance as a model for studying growth pathways like STAT3, which are often dysregulated in cancers, including hepatocellular carcinoma. By using HepG2 cells. GHAl’s effects on STAT3 modulation were evaluated in a cancer-relevant context, adding translational value to the findings.

[0942] The objective of the study was to determine whether GHA1 could inhibit GH-induced STAT3 phosphorylation in HepG2 cells in a dose-dependent manner. Positive results would provide a foundation for further development of GHA1 as a STAT3 -targeted growth antagonist, with potential applications in treating cancers and other growth-related pathologies where STAT3 plays a pivotal role.

[0943] Methods

[0944] To assess the effect of GHAl-Albumin (SEQ ID NO: 14) on STAT3 phosphorylation in HepG2 cells, HepG2 cells were cultured in DMEM with 10% fetal bovine serum at 37°C and 5% CO₂ and seeded in 6-well plates until they reached 70-80% confluency. Cells were then treated with 1 pg / mL growth hormone (GH) to stimulate STAT3 phosphorylation, with varying concentrations of GHAl-Albumin (40, 80, and 160 pg / mL) added to observe a potential dose-dependent effect. Control groups included cells treated with GH alone and untreated cells. After 24 hours, whole-cell lysates were collected using a lysis buffer with protease and phosphatase inhibitors and protein concentrations were determined using the Quick Protein Assay Kit. Proteins were separated by 12% SDS-PAGE, transferred onto PVDF membranes, and blocked with 5% nonfat dry milk to prevent non-specific binding. Membranes were incubated overnight at 4°C with anti-160 pg / mL; lane 1) showing the greatest decrease in phospho-STAT3 levels. The negative control, as expected, showed minimal phospho-stat3 signal. These findings suggest that GHAl-Albumin effectively reduces GH-induced STAT3 phosphorylation in a dose-dependent manner, implying a potential inhibitory role of GHA1 on STAT3 activation in GH signaling pathways in HepG2 cells.

[0945] This data highlights the specificity of GHA1 (and fusion proteins thereof) as a growth hormone antagonist by demonstrating its targeted action on GH-induced STAT3 phosphorylation in HepG2 cells. By -101- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0946] selectively inhibiting GH-stimulated STAT3 phosphorylation, GHA 1 shows potential as a targeted therapy aimed at curtailing GH-mediated pathways without broad, non-specific inhibition of STAT3, which could impact essential cellular functions beyond tumor growth suppression.

[0947] This specificity is further underscored by the dose-dependent response observed in the western blot analysis, where increasing concentrations of GHA1 consistently reduced phospho-STAT3 levels, peaking at the highest dose tested. This dose-dependent inhibition not only supports GHAl's specificity in modulating STAT3 activity in the GH pathway but also highlights its potential for controlled therapeutic applications, where therapeutically effective dosages can be adjusted to optimize its inhibitory effects while minimizing off-target actions. Thus, these findings support the development of GHA1, including GHAl-Albumin, as a STAT3 -targeted therapeutic with potentially significant clinical implications for cancers and other disorders driven by excessive STAT3 activation.

[0948] Example 9

[0949] Anti-Tumor Activity of a GHA1 Fusion Protein in a Mouse Liver Cancer Model Introduction

[0950] Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal forms of liver cancer, characterized by aggressive tumor growth and poor prognosis. Current therapeutic options, including targeted kinase inhibitors like sorafenib, offer limited efficacy with modest survival benefits and are often accompanied by significant side effects. This underscores the need for novel therapeutic approaches that can more effectively inhibit tumor progression while minimizing adverse effects.

[0951] Growth hormone (GH) and its receptor (GHR) have been implicated in the progression and resistance mechanisms of several cancers, including HCC. GH signaling promotes tumor cell proliferation, survival, and angiogenesis, primarily through pathways such as JAK-STAT, MAPK, and PI3K-Akt.

[0952] Antagonizing GH activity represents a promising strategy to disrupt these oncogenic pathways.

[0953] Growth hormone antagonist 1 (GHA1) has been designed to block GH receptor signaling selectively. Preliminary in vitro studies have demonstrated that GHA1 effectively inhibits cell proliferation in HepG2 liver cancer cells. However, the efficacy of GHA1 in vivo remains to be validated.

[0954] This study evaluated the therapeutic potential of GHAl-Albumin (SEQ ID NO: 14) in an in vivo HepG2 xenograft model. Using nude mice, a preclinical model for human liver cancer, the study investigated whether systemic administration of GHA1 can significantly inhibit tumor growth. The experimental design included the administration of GHA1 at a therapeutically relevant dose (200 mg / kg) to assess its effect on tumor volume reduction compared to vehicle-treated controls.

[0955] By exploring the anti-tumor efficacy of GHA1 in this model, the study tested the use of GHAs as a viable therapeutic approach for HCC.

[0956] Methods

[0957] -102- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0958] Swiss nu-nu / Ncr nude mice (5-6 weeks old) were obtained from The Jackson Laboratory (Bar Harbor, ME). HepG2 cells (5.0×106) from ATCC (Manassas, VA) were suspended in a 1:1 mixture of PBS and Matrigel (354234; Coming Life Sciences, Tewksbury, MA). The cell suspension was subcutaneously injected into the flanks of each mouse. Tumor growth became visible approximately 7 days post-inoculation.

[0959] Tumor dimensions (length and width) were measured using digital calipers, and tumor volumes were calculated using the formula:

[0960] Volume = π / 6 x length x width2.

[0961] Once tumor volumes reached 70-90 mm3, mice were randomly assigned to two groups: (1) Control group - vehicle; and Treatment group - GHAl-Albumin (200 mg / kg, administered twice weekly). The experiment was terminated after 32 days or earlier if signs of excessive tumor burden appeared. Tumor growth curves were generated using average tumor volumes at each time point. Statistical analyses were conducted using one-way ANOVA.

[0962] Results

[0963] As shown in Figure 11, treatment with GHAl-Albumin significantly reduced tumor volume, compared to control, revealing about a one-third decrease by day 32.

[0964] Figure 12 depicts this relative inhibition effect over time: Tumor growth inhibition increases steadily during the early phase of treatment, reaching about 28% by Day 8, indicating early responsiveness.

[0965] Inhibition peaks between day 12 and 16, reaching a maximum of about 42%, representing a period of high therapeutic efficacy. From Day 16 onward, the inhibition stabilizes at about 35%, indicating sustained efficacy over time. By Day 32, the tumor inhibition is approximately 32%, demonstrating a continued reduction in tumor progression. This pattern underscores the value of dosing or combination strategies to maintain or enhance the peak inhibition. Overall, the GHAl-Albumin treatment data demonstrates consistent efficacy in slowing tumor growth, including during the early and intermediate phases, making it a promising therapeutic candidate for conditions involving tumor proliferation. The stabilization of the inhibition curve further underscores its potential for combination therapy to optimize long-term outcomes.

[0966] Overall, the data suggest that GHAl-Albumin (100 mg / kg) effectively inhibits tumor growth in this model, generally supporting the use of GHAs disclosed herein in therapeutic applications.

[0967] Example 10

[0968] Inhibition of GH- Induced STAT5 Phosphorylation by GHA5-GHA9 Fusion Proteins Introduction

[0969] Growth hormone (GH) signaling is highly relevant in liver biology because hepatocytes are a principal physiologic target of GH and respond through the canonical GH receptor (GHR) — > JAK2 STAT5 pathway. Accordingly, STAT5 phosphorylation (pSTAT5) provides a direct and sensitive functional readout of GHR activation and downstream signal transduction.

[0970] -103- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0971] Testing candidate GH-pathway inhibitors in Hepal-6 cells offers several advantages. First, Hepal-6 is a murine liver-derived cell line that provides a hepatocyte-relevant context in which GH-dependent signaling can be evaluated under controlled conditions. Second, because Hepal-6 is of mouse-origin, it enables assessment of inhibitor activity in a species-matched system that is aligned with subsequent evaluation in murine in vivo efficacy models (e.g., syngeneic tumor studies), thereby reducing uncertainty associated with interspecies differences in ligand-receptor interactions and pathway pharmacology. Third, Hepal-6 cells typically exhibit robust GH responsiveness, allowing clear discrimination between compounds with strong versus partial antagonistic effects based on suppression of GH-induced pSTAT5.

[0972] More particularly, this study assessed whether each of GHA-5, GHA-6, GHA-7, GHA-8, and GHA-9 could inhibit GH-induced STAT5 phosphorylation in Hepal-6 cells in a dose-dependent manner. These results provide further support for efficacy of STAT5-targeted GHAs, with potential applications in treating cancers and other growth-related pathologies where STAT5 plays a key role.

[0973] Methods

[0974] Hepal-6 (ATCC CRL-1830) cells were seeded in 6-well plates and subjected to serum starvation for 24 hours. The GHA5-GHA9 test inhibitors correspond to SEQ ID NOS:26-30. Each test inhibitor was then added to the cells at a final concentration of 160 pg / mL. After two (2) hours of inhibitor exposure, recombinant GH (Xiamen Amoytop Biotech Co.) was added to a final concentration of 2 pg / mL to stimulate GH signaling. Cells were harvested 20 minutes after GH stimulation.

[0975] Cell lysates were analyzed by Western blot to quantify phosphorylated STAT5 (pSTAT5), using an anti-pSTAT5 antibody (Cell Signaling Technology, Cat# 9314). GAPDH was used as the loading control using an anti-GAPDH antibody (Abclonal, Cat# AC002). Following enhanced chemiluminescence (ECL) development, band intensities were quantified using ImageJ software. Optical density values were normalized to GAPDH to control for loading differences. The non-treated (NT) group was assigned a reference value of 1, and relative pSTAT5 levels were calculated to determine inhibitory effects on GH-induced STAT5 phosphorylation. Data were tabulated and visualized using Microsoft Excel.

[0976] Results

[0977] Figure 13 shows representative pSTAT5 and GAPDH immunoblots (A) and a corresponding bar graph showing pSTAT5 levels normalized to GAPDH (B). GH stimulation produced a strong induction of STAT5 phosphorylation in Hepal-6 cells, increasing normalized pSTAT5 from 1.00 (NT: No Treatment) to 6.80-fold. Co-treatment with GHA variants reduced GH-induced pSTAT5 to varying degrees, demonstrating functional antagonism of GH signaling in a hepatocyte-relevant cell context. Table 6 provides summarizes the results from these experiments.

[0978] Table 6

[0979] Fold-Induction of pSTAT5 and Inhibition of GH Response

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[0981] Condition pSTAT5 / GAPDH (fold vs NT) Inhibition vs GH Activation1GH 6.80 0

[0982] GH + GHA5 2.12 80.7

[0983] GH + GHA6 3.11 63.6

[0984] GH + GHA7 2.78 69.2

[0985] GH + GHA8 3.00 65.5

[0986]

[0987] GH + GHA9 1.84 85.5

[0988] 1Inhibition calculated relative to the GH-induced signal above baseline (i.e., comparing (GH - NT) to (Treatment - NT)).

[0989] Collectively, these data demonstrate that the tested GHA constructs can attenuate GH-driven STAT5 phosphorylation in Hepal-6 cells, supporting their use as growth hormone pathway antagonists.

[0990] Example 11

[0991] Inhibition of GH- Induced Proliferation by GHA5-GHA9 Fusion Proteins Introduction

[0992] Growth hormone (GH) signaling through the GH receptor (GHR) is a key regulator of hepatic biology and can promote cellular growth programs via downstream pathways that include JAK2 / STAT5 / STAT3 and broader pro-proliferative signaling networks. Accordingly, compounds that inhibit GH pathway signaling are expected to reduce GH-driven functional outcomes, including cell proliferation, in GH-responsive liver-derived cells.

[0993] The Hepal-6 cell line provides a practical and biologically relevant system for assessing GH pathway antagonism because it is murine liver-derived, demonstrates measurable responses to GH stimulation, and supports translational alignment with mouse in vivo models (e.g., syngeneic tumor studies) used to evaluate efficacy and pharmacology. While phosphorylation assays (e g., pSTAT5) provide an early mechanistic readout of pathway inhibition, a cell proliferation assay provides a complementary functional endpoint that integrates signaling effects over time and can better reflect downstream biological consequences of GH pathway modulation.

[0994] Accordingly, the GH-induced proliferation assay described herein was designed to (i) establish a GH-responsive growth phenotype in Hepal-6 cells, (ii) quantify the capacity of candidate inhibitors to suppress this phenotype under defined stimulation conditions, and (iii) enable rank-ordering of inhibitors and variants based on their ability to reduce GH-driven proliferation relative to a GH-only control.

[0995] Methods

[0996] Hepal-6 (ATCC CRL-1830) cells were seeded in 384-well plates in starvation medium and incubated for 24 hours. Test inhibitors, as in Example 10, were then added to the wells at a final

[0997] -105- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO

[0998] concentration of 160 pg / mL. After two (2) hours of inhibitor exposure, recombinant GH was added to a final concentration of 2 pg / mL. Wells receiving GH alone (without inhibitor) served as the stimulated control.

[0999] Cell proliferation was quantified at 48 hours following GH addition using a Cell Growth (ATP) Assay Kit (Luminescence) according to tire manufacturer's instructions. Each condition was tested in quadruplicate, and the mean luminescence signal for each condition was used for subsequent statistical analysis.

[1000] Results

[1001] As shown in Figure 14, GH stimulation produced a high ATP -dependent luminescence signal in cells treated with just GH (control). Co-treatment with GHA constructs reduced the GH-driven proliferation signal, consistent with functional antagonism of GH pathway activity. In a representative dataset, GHA5 reduced the proliferation readout to approximately 62.9% of the GH-only control (approximately 37.1% inhibition).

[1002] Additional GHA variants also reduced the GH-induced signal, including GHA6 (approximately 70.0% of control; approximately 30.0% inhibition), GHA7 (approximately 76.8% of control; approximately 23.2% inhibition), GHA9 (approximately 75.4% of control; approximately 24.6% inhibition), and GHA9 (approximately 78.0% of control; approximately 22.0% inhibition).

[1003] Nonparametric analysis demonstrated statistically significant differences across groups and confirmed reductions relative to the GH-only control. Overall group differences were significant (Kruskal-Wallis p = 0.000165). Pairwise vs control (Holm-adjusted): GHA5 (0.000941), GHA 6, 7, and 8 (0.00527), GHA9 (0.04536).

[1004] Collectively, the Hepal-6 studies in Examples 10 and 11 demonstrate that the disclosed GHA molecules demonstrate antagonist activity in both canonical GH signaling by reducing pSTAT5, and in GH-driven cell growth by reducing cell proliferation.

[1005] Example 12

[1006] Inhibition of GH-Induced STAT5 Phosphorylation by GHA5 and GHA5-Fc. Introduction

[1007] As noted above, GH signaling is highly relevant in liver biology because hepatocytes are a principal physiologic target of GH and respond through the canonical GH receptor (GHR) → JAK2 → STAT5 pathway. Accordingly, STAT5 phosphorylation (pSTAT5) provides a direct and sensitive functional readout of GHR activation and downstream signal transduction. As further described above, e.g., in Examples 10 and...

Claims

Attorney Docket No. 1362.0003-1WOWhat is claimed is:

1. A growth hormone mutant antagonist (GHA), comprising a modification:to two or more residues at positions 10, 14, 21, and 120 of a human growth hormone (GH), selected from:a substitution (mutation) of phenylalanine (Phe; F) at position 10 with alanine (Ala; A) or histidine (His; H),a substitution of methionine (Met; M) at position 14 with tryptophan (Trp; W) or glycine (Gly; G),a substitution of histidine (His; H) at position 21 with asparagine (Asn; N), and a substitution of glycine (Gly; G) at position 120 with a lysine (Lys; K) or arginine (Arg; R); and to two or more residues at positions 18, 167, 168, 171, 172, 174, 176, and 179 of the human GH. selected from:a substitution of histidine at position 18 with aspartate (Asp: D,) or asparagine (Asn; N), a substitution of arginine at position 167 with asparagine (Asn; N) or glutamate (Glu: E), a substitution of lysine (Lys; K) at position 168 with alanine (Ala; A);a substitution of aspartate (Asp; D) at position 171 with a serine (Ser; S);a substitution of lysine (Lys; K) at position 172 with an arginine (Arg; R);a substitution of glutamate (Glu; E) at position 174 with a serine (Ser; S) or threonine (Thr; T);a substitution of phenylalanine (Phe; F) at position 176 with a tyrosine (Tyr; Y); and a substitution of isoleucine (He, I) at position 179 with a threonine (Thr; T).

2. Hie GHA of claim 1. wherein the modification comprises:two or more mutations, or three or more mutations selected from the group consisting of PhelOAla (or PhelOHis), Metl4Trp (or Metl4Gly), His21Asn, and Glyl20Lys (or Gly 120Arg); and three or more mutations, four or more mutations, five or more mutations, six or more mutations, or seven or more mutations selected from the group consisting ofHisl8Asp (or His 18 Asn), Argl67Asn (or Argl67Glu), Lysl68Ala, Asp 171 Ser. Lysl72Arg, Glul74Ser (or Glul74Thr), Phel76Tyr. and Ilel79Thr.Attorney Docket No. 1362.0003-1WO3. The GHA of claim 1 or claim 2, wherein the modification comprises a substitution at each of residues 18, 21, 120, 167, 171, and 174, selected from Hisl8Asp (orHisl8Asn), His21Asn, Glyl20Lys (or Glyl20Arg), Argl67Asn (or Argl67Glu), Aspl71Ser, and Glul74Ser (or Glul74Thr).

4. The GHA of claim 3, further comprising one or more mutations at residues 10, 14, 168, 172, 176, and 179, selected from PhelOAla (or PhelOHis), Metl4Trp (or Metl4Gly), Lysl68Ala, Lysl72Arg, Phel76Tyr, and Ilel79Thr.

5. The GHA of any of claims 1 to 4, wherein the GHA comprises the amino acid sequence of GHA5 (SEQ ID NO:21), GHA6 (SEQ ID NO:22), GHA7 (SEQ ID NO:23). GH8 (SEQ ID NO:24). or GHA9 (SEQ ID NO:25).

6. The GHA of any one of claims 1 to 5,wherein the modifications at positions 10, 14, 21, and 120 are within an A chain of the GH, targeting residues interacting at an interface (site 2) between the A chain and a C chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations disrupt GH binding at the A- C interface; andwherein the modifications at positions 18, 167, 168, 171. 172, 174. 176, and 179 are within an A chain of the GH, targeting residues interacting at an interface (site 1) between the A chain and a B chain of a growth hormone receptor (GHR) in a GH: GHR complex, wherein the targeted mutations enhance or stabilize GH binding at the A-B interface.

7. The GHA of any one of claims 1 to 6, wherein the GHA is a fusion protein that further comprises a carrier protein.

8. The GHA of claim 7, wherein the carrier protein is joined to an N-terminus or a C -terminus of the GHA.

9. Hie GHA of claim 7 or 8, wherein the carrier protein is selected from a human serum albumin, a human transferrin, a human IgG Fc domain, and a human growth hormone binding protein.

10. The GHA of claim 9. wherein the human serum albumin comprises an amino acid sequence of SEQ ID NO:5.

11. The GHA of claim 9, wherein the human transferrin comprises an amino acid sequence of SEQ ID NO:6, or residues 20 to 698 of SEQ IDNO:6.

12. The GHA of claim 9, wherein the human IgG Fc domain is a human IgGl Fc domain comprising an amino acid sequence of SEQ ID NO:7.-144- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO13. The GHA of claim 9 or 12, wherein the human IgG Fc domain comprises one or more effector reducing mutations selected from the group consisting of L14A, L15A, N77A, and P109G (referring to SEQ ID NO: 7).

14. The GHA of claim 9, 12, or 13, wherein the human IgG Fc domain comprises one or more effector reducing mutations that enhance binding affinity to neonatal Fc receptor (FcRn) relative to wild-type Fc, selected from the group consisting of M32Y / S34T / T36E (YTE), M208L / 224S (LS), and combinations thereof (referring to SEQ ID NOY).

15. The GHA of claim 9, wherein the human IgG Fc domain comprises an S228P stabilizing mutation (based on EU numbering).

16. The GHA of claim 9, wherein the IgG Fc domain is an engineered human IgG Fc domain, and more particularly, comprises an amino acid sequence of SEQ ID NO:34.

17. The GHA of any one of claims 9 or 12 to 16, wherein the IgG Fc fusion protein is a homodimer comprising two identical polypeptide chains, each chain comprising the GH antagonist polypeptide linked to the Fc domain.

18. Hie GHA of claim 9. wherein the human growth honnone binding protein comprises an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9.

19. The GHA of any one of claims 7 to 18. wherein the carrier protein is joined to the GHA via a peptide linker.

20. The GHA of claim 19, wherein the peptide linker comprises glycine residues.

21. The GHA of claim 19, wherein the peptide linker comprises serine residues.

22. The GHA of claim 19, wherein the peptide linker comprises glycine and serine residues.

23. The GHA of any one of claims 20 to 22, wherein the peptide linker has a length of less than 20 amino acids.

24. The GHA of any one of claims 20 to 22, wherein the peptide linker has a length of 5 to 30 amino acids.

25. The GHA of any one of claims 20 to 22, wherein the peptide linker has a length of greater than 20 amino acids.

26. The GHA of any one of claims 20 to 22, wherein the peptide linker has the formula (GGGGS)n, (GGSGG)n, or (GGSGGS)n, or (GGSGGS)n, wherein n is independently 1 to 10, 1 to 8, 1 to 6, 1 to-145- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO4, 1 to 3, or 1 to 2, and more particularly, wherein the peptide linker comprises GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGG.

27. The GHA of claim 26, wherein the GHA comprises an amino acid sequence of any one of SEQ ID NOS: 26-33.

28. The GEIA of any one of claims 1 to 27, wherein the GEIA comprises a targeting (homing) domain.

29. The GHA of claim 28, wherein the targeting domain is transferrin-receptor binding peptide T12 (TBP), having the sequence THRPPMWSPVWP.

30. Tire GHA of claim 29, wherein the TBP binds to a site on a human transferrin receptor distinct from that of native transferrin.

31. The GHA of claim 29 or 30 for use in liver-targeted delivery of the GHA.

32. The GHA of any one of claims 1 to 31. wherein the GH comprises a cleavable peptide sequence.

33. The GHA of claim 32, wherein the cleavable peptide sequence is cleaved by a cathepsin, a matrix metalloproteinase (MMP), furin, legumain, or a caspase.

34. The GHA of claim 33, wherein the cleavable peptide sequence is selected from PLGLAG, GPLGVRG, GFLG, RVRR, and AAN, and more particularly, is PLGLAG35. The GHA of claim 34, wherein the cleavable peptide sequence is cleaved by one or more MMPs.

36. The GHA of claim 35, wherein the cleavable peptide sequence is PLGLAG.

37. The GHA of claim 36, wherein PLGLAG is cleaved by MMP-2 and MMP-9.

38. A GHA comprising PLGLAG for use in cancer targeted drug deli ven. and more particularly wherein the GHA comprises an amino acid sequence of SEQ ID NO:33.

39. The GHA for use in claim 38, wherein one or more MMPs, such as MMP-1 and MMP-2, are overexpressed in a tumor microenvironment of the targeted cancer.

40. A pharmaceutical composition comprising: a GHA of any one of claims 1 to 39; and a pharmaceutically acceptable excipient.

41. The pharmacal composition of claim 40, prepared as a fonnulation for delivery of the GHA as a protein.-146- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO42. The pharmaceutical composition of claim 41, wherein the formulation comprises a liquid formulation, a lyophilized formulation, a microparticle formulation, a nanoparticle formulation, or a depot formulation.

43. The pharmaceutical composition of claim 41 or 42, wherein the GHA is optionally conjugated to a high molecular molecule, such as polyethylene glycol (PEG).

44. The pharmacal composition of claim 40, prepared as a fonnulation for delivery of the GHA as a nucleic acid encoding the GHA.

45. The pharmaceutical composition of claim 44, wherein the nucleic acid in the formulation is an RNA molecule encoding the GHA.

46. The pharmaceutical composition of claim 45, wherein the formulation comprises lipid nanoparticles (LNPs) encapsulating the RNA.

47. The pharmaceutical formulation of claim 45, wherein the formulation comprises polymeric nanoparticles encapsulating the RNA.

48. Hie pharmacal fonnulation of claim 46 or 47, wherein the nanoparticles are coated with a mucoadhesive polymer.

49. The pharmacal composition of claim 46, wherein the LNPs are modified with polyethylene glycol (PEG).

50. The pharmaceutical composition of claim 45, wherein the formulation comprises a hydrogel matrix encapsulating the RNA.

51. The pharmaceutical composition of claim 45, wherein the formulation is a dry powder formulation suitable for a non-invasive administration route or for reconstitution.

52. The pharmaceutical formulation of any one of claims 45 to 51, wherein the RNA is chemically modified to include pseudouridine or 5 -methylcytidine.

53. The pharmaceutical composition of claim 44, wherein the nucleic acid in the formulation is a DNA molecule encoding the GHA.

54. The pharmaceutical composition of claim 53, wherein the DNA molecule is delivered by a viral vector.-147- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO55. The pharmaceutical composition of claim 54, wherein the viral vector is an adeno-associated virus, lentivirus, adenovirus, or retrovirus vector.

56. The pharmaceutical formulation of claim 54 or 55, wherein the viral vector is coated with PEG or a mucoadhesive polymer.

57. The pharmaceutical formulation of claim 54 or 55, wherein the viral vector is encapsulated in lipid nanoparticles (LNPs).

58. Hie pharmacal formulation of claim 54 or 55, wherein the formulation is encapsulated in a hydrogel matrix.

59. The pharmacal formulation of claim 54 or 55, wherein the fonnulation is encapsulated in biodegradable microspheres.

60. A method of treating a condition associated with excessive (or aberrant) GH activity or GHR activation, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA, or composition thereof, of any one of claims 1 to 59.

61. The method of claim 60, wherein the condition is acromegaly or gigantism.

62. The method of claim 61, wherein the GHA, or composition thereof, is administered in combination with a somatostatin analog, such as octreotide or lanreotide, to achieve synergistic suppression of growth honnone and insulin-like growth factor 1 (IGF-1) levels in the subject, and more particularly, reduces cancer risk in the subject.

63. The method of claim 61, wherein the GHA, or composition thereof, is co-administered with a dopamine agonist, such as cabergoline or bromocriptine, to provide an enhanced treatment regimen for acromegaly, particularly in patients with partial resistance to somatostatin analogs.

64. The method of claim 61, wherein the GHA, or composition thereof, is administered in conjunction with a radiotherapy or a surgical intervention to improve long-term management of acromegaly by¬ mitigating residual growth hormone receptor activity post-treatment.

65. Hie method of claim 60, wherein the condition is a cancer, more particularly a cancer characterized by overexpression of GH or GHR.

66. The method of claim 65, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), prostate cancer, colorectal cancer, breast cancer, endometrial cancer, lung cancer, glioblastoma, and melanoma.-148- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO67. The method of claim 65 or 66, wherein the method reduces circulating IGF-1 levels in the subject to below an age- and sex-adjusted normal range, thereby suppressing IGF-1 -mediated tumor proliferation and survival signaling.

68. Hie method of any one of claims 65 to 67, wherein the cancer exhibits autocrine or paracrine growth hormone expression, and the GHA antagonizes endocrine IGF-1 signaling and / or local autocrine / paracrine GH signaling within the tumor.

69. A method of claim 65 or 66, wherein the method comprises sensitizing a tumor to cytotoxic chemotherapy, comprising co-administering to a subject in need thereof (a) a therapeutically effective amount of the GHA, or composition thereof; and (b) one or more cytotoxic chemotherapy agents; wherein administration of the GHA enhances the anti-tumor efficacy of the cytotoxic agent relative to the cytotoxic agent administered alone.

70. The method of claim 69, wherein the one or more cytotoxic chemotherapy agents are selected from the group consisting of doxorubicin, cisplatin, carboplatin, paclitaxel, docetaxel, 5 -fluorouracil, gemcitabine, and combinations thereof.

71. The method of claim 65 or 66, wherein the GHA, or composition thereof, reduces expression or activity of one or more multidrug resistance transporters in tumor cells.

72. The method of claim 65 or 66, wherein the GHA, or composition thereof, is administered with a kinase inhibitor, such as sorafenib or lenvatinib, for treating a GHR-positive hepatocellular carcinoma.

73. The method of claim 65 or 66, wherein the GHA, or composition thereof, is co-administered with an immune checkpoint inhibitor, such as a PD-1 inhibitor, preferably pembrolizumab or nivolumab, a PD-L1 inhibitor, preferably avelumab or durvalumab, or a CTLA-4-1 inhibitor, preferably ipilimumab or trcmclimumab, to enhance immune -mediated suppression of tumor growth in a cancer expressing a GHR.

74. The method of claim 65 or 66, wherein the GHA, or composition thereof, is administered in combination with an anti-angiogenic agent, preferably bevacizumab, to suppress growth hormone- mediated vascular proliferation in a solid tumor.

75. The method of claim 65 or 66, wherein the GHA, or composition thereof, is administered in combination with a radiation therapy to enhance radiosensitivity in a cancer characterized by an overactive GH signaling pathway.-149- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO76. The method of claim 65 or 66, wherein the GHA, or composition thereof, is administered in combination with a targeted anti-cancer agent selected from the group consisting of anti-HER2 agents (trastuzumab, pertuzumab), anti-EGFR agents (cetuximab, erlotinib), mTOR inhibitors (everolimus, temsirolimus), CDK4 / 6 inhibitors (palbociclib, ribociclib), and PI3K inhibitors (alpelisib); wherein the combination overcomes or prevents resistance to the targeted anti-cancer agent mediated by compensatory GH / IGF-1 pathway activation.

77. The method of any one of claims 65 to 76, wherein the GHA comprises the amino acid sequence of any one of SEQ ID NOS:26-33, and more, particularly, SEQ ID NO:32 or SEQ ID NO:33.

78. The method of claim 66, wherein the cancer is HCC.

79. The method of claim 78, wherein the subject has an advanced or an unresectable HCC.

80. The method of claim 78, wherein the GHA, or composition thereof, is administered with a first line therapy selected from sorafenib, lenvatinib, a combination of atezolizumab and bevacizumab, and a combination of durvalumab and tremelimumab.

81. The method of claim 78, wherein the GHA, or composition thereof, is administered with a second line therapy selected from regorafenib, cabozantinib, ramucirumab, nivolumab, pembrolizumab, and a combination of ipilimumab and nivolumab.

82. The method of claim 78, wherein the GHA, or composition thereof, is administered as a third line therapy.

83. The method of claim 78, wherein the subject has an intermediate stage HCC.

84. The method of claim 83, wherein the GHA, or composition thereof, is administered in combination with a locoregional therapy, such as TACE, radioembolization, or an ablative procedure, to improve tumor control.

85. The method of claim 78, wherein the subject has an early stage HCC.

86. The method of claim 85, wherein the GHA, or composition thereof, is administered prior to a surgery, as a neoadjuvant therapy to reduce tumor size before resection or is administered after surgery as an adjuvant therapy to target residual microscopic disease, thereby reducing recurrence rates.

87. The method of any one of claims 65 to 86, wherein upregulated GH signaling resulting from the GH activity or GHR activation comprises increased phosphorylation of STAT5, STAT3. or Akt in tumor cells.-150- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO88. The method of claim 60, wherein the condition is insulin resistance or type 2 diabetes mellitus.

89. The method of claim 88, wherein the GHA, or composition thereof, is administered with insulin or an insulin analog to improve glycemic control in a subject with GH-mediated insulin resistance.

90. The method of claim 88, wherein the GHA, or composition thereof, is administered with a GLP-1 receptor agonist, such as semaglutide or dulaglutide, to address both hyperglycemia and grow th honnone-related metabolic complications in diabetes.

91. Hie method of claim 88, wherein the GHA, or composition thereof, is administered with an insulin sensitizer selected from metfonnin and pioglitazone.

92. The method of claim 88, wherein the GHA, or composition thereof, is administered with metformin to enhance insulin sensitivity and mitigate the effects of GH-induced hepatic glucose production in patients with type 2 diabetes.

93. The method of claim 60, wherein the condition is diabetic retinopathy or diabetic nephropathy.

94. The method of claim 93, wherein the GHA, or composition thereof, is used in combination with anti- VEGF agent, such as ranibizumab or aflibercept, to reduce grow th hormone-mediated pathological angiogenesis in diabetic retinopathy.

95. The method of claim 93, wherein the GHA, or composition thereof, is administered with a corticosteroid to mitigate inflammation and vascular leakage in diabetic retinopathy.

96. The method of claim 93, wherein the GHA, or composition thereof, is co-administered with an angiotensin-converting enzyme (ACE) inhibitor, such as enalapril or lisinopril, to protect renal function by inhibiting a growth hormone-mediated pathway in diabetic nephropathy.

97. The method of claim 93, wherein the GHA, or composition thereof, is combined with an angiotensin receptor blocker (ARB), such as losartan or valsartan, to reduce proteinuria and preserve kidney function in diabetic nephropathy.

98. The method of claim 93, wherein the GHA, or composition thereof, is administered with a sodiumglucose cotransporter-2 (SGLT-2) inhibitor, such as dapagliflozin or empagliflozin, to enhance renal protection and manage growth hormone-related renal complications in diabetic nephropathy.

99. The method of claim 93, wherein the GHA, or composition thereof, is administered in combination with an anti-VEGF therapy, such as bevacizumab, ranibizumab, aflibercept, or brolucizumab.-151- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO 100. A method of treating a GH-dependent vascular complication, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA, or composition thereof, of any one of claims 1 to 59, wherein tire GHA is sufficient to inhibit GH-induced angiogenesis.

101. Tire method of claim 100, wherein the vascular complication is diabetic retinopathy or macular edema.

102. The method of claim 100, wherein the vascular complication is cancer-associated angiogenesis, and the GHA is administered in combination with an anti-VEGF therapy.

103. A method for reducing STAT3 phosphorylation, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA, or composition thereof, of any one of claims 1 to 59, wherein the GHA disrupts GHR activation and thereby inhibits a downstream signaling pathway involving STAT3.

104. Tire method of claim 103, wherein the subject has a cancer associated with aberrant STAT3 activation, selected from the group consisting of hepatocellular carcinoma, colorectal cancer, and prostate cancer.

105. The method of claim 103 or 104, wherein the GHA is administered with an immune checkpoint inhibitor to enhance immune-mediated tumor suppression.

106. A method for reducing STAT3 phosphorylation, comprising administering to a subject in need thereof a therapeutically effective amount of a GHA, or composition thereof, of any one of claims 1 to 59, wherein the GHA disrupts GHR activation and thereby inhibits a downstream signaling pathway involving STAT5.

107. Tire method of claim 106, wherein the subject has a cancer associated with aberrant STAT5 activation, selected from the group consisting of hepatocellular carcinoma, colorectal cancer, and prostate cancer.

108. The method of claim 106 or 107, wherein the GHA is administered with an immune checkpoint inhibitor to enhance immune-mediated tumor suppression.

109. The method of any one of claims 60 to 108, wherein the GHA is delivered via a route of administration selected from enteral, topical, parenteral, intravenous, oral, ocular, transdermal, intrathecal / epidural, rectal / vaginal, subcutaneous, intramuscular, intranasal, pulmonary, intravitreal, intradermal, and intratumoral administration.-152- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO110. The method of claim 109, wherein the GHA is delivered in a lipid nanoparticle (LNP) formulation optimized for a selected route of administration, providing controlled release and prolonged therapeutic effect.

111. Tire method of claim 109, wherein the GHA is administered intravenously in a nanoparticle- encapsulated formulation designed for rapid systemic absorption and protection against enzymatic degradation.

112. Tire method of claim 109, wherein the GHA, more particularly, a PEGylated or carrier-fused growth GHA, is administered subcutaneously as a long-acting injectable formulation.

113. The method of claim 109, wherein the GHA is administered subcutaneously in a hydrogel-based matrix designed for localized sustained release at the administration site.

114. The method of claim 109, wherein the GHA is administered intramuscularly as a depot formulation comprising biodegradable microspheres for sustained release over a period of at least one month.

115. The method of claim 109, wherein the GHA is administered intramuscularly in a hydrogel-based matrix designed for localized sustained release at tire administration site.

116. Tire method of claim 109, wherein the GHA is administered orally in an enteric-coated formulation with protease inhibitors, conferring protection against gastrointestinal degradation and enhancing bioavailability.

117. The method of claim 109, wherein the GHA is administered intranasally in a mucoadhesive formulation for rapid absorption through the nasal mucosa, providing a non-invasive alternative for acute management of GH-related disorders.

118. The method of claim 109, w herein the GHA is administered transdermally using a microneedle patch for sustained release, enhancing patient compliance by reducing the need for frequent dosing.

119. Tire method of claim 109, w herein the GHA is administered via pulmonary inhalation using a dry powder inhaler or nebulized solution, enabling systemic absorption through the respiratory epithelium.

120. The method of claim 109, wherein the GHA is administered intravitreally for the treatment of a growth honnone-induced vascular complication, such as diabetic retinopathy or macular edema.

121. The method of claim 109. wherein the GHA is administered intratumorally for targeted inhibition of GHR activity in a GH-dependent cancer.-153- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO 122. The method of claim 109, wherein the GHA is administered using a dual-delivery system comprising both oral and transdermal routes to maintain steady-state therapeutic levels over an extended duration.

123. Tire method of claim 109, wherein the GHA is delivered using a patient-controlled delivery device, such as a wearable infusion pump, enabling precise dosing adjustments based on real-time monitoring of GH or IGF-1 levels.

124. A method of delivering a GHA to a subject in need thereof, comprising administering a synthetic mRNA encoding a GHA of any one of claims 1 to 59, wherein the mRNA is encapsulated in lipid nanoparticles (LNPs) or polymeric nanoparticles, and wherein the mRNA is translated in situ to produce the active GHA and inhibit GHR activity.

125. The method of claim 124, wherein the mRNA is delivered via a hydrogel-based system for localized, controlled release over time.

126. Tire method of claim 124, wherein the mRNA is delivered intranasally using mucoadhesive polymer coated lipid nanoparticles (LNPs) or polymeric nanoparticles,127. A method of delivering a GHA to a subject in need thereof, comprising administering a viral DNA vector encoding the GHA of any one of claims 1 to 59. selected from adeno-associated virus, lentivirus, or adenovirus, wherein the vector targets specific tissues, leading to sustained expression of the GHA to inhibit growth hormone receptor activity.

128. The method of claim 127, w herein the viral DNA vector encoding the GHA is delivered intratumorally to achieve localized inhibition of grow th hormone receptor activity in GH-dependent tumors.

129. The method of claim 127, wherein the viral DNA vector is modified with tissue-specific promoters to drive expression of the GHA in liver or tumor cells.

130. The method of claim 127, wherein the viral DNA vector is encapsulated in lipid nanoparticles (LNPs) for enhanced stability and targeted delivery.

131. The method of claim 127, w'herein the viral DNA vector is co-administered with an immune checkpoint inhibitor to enhance anti-tumor efficacy in a growth hormone-dependent cancer.

132. The method of claim 127, w herein the viral DNA vector is administered using a hydrogel matrix for localized and sustained release at an administration site.-154- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO 133. A pharmaceutical formulation comprising a GHA of any one of claims 1 to 59, wherein the GHA is formulated as a solution, lyophilized powder, or suspension, and includes one or more excipients selected from a stabilizer, a cryoprotectant, a buffering agent, and a preservative to enhance stability, solubility, or shelf-life.

134. Tire fonnulation of claim 133, wherein the stabilizer is trehalose or sucrose and the buffering agent maintains a pH range of about 6.5 to about 7.5.

135. The formulation of claim 133. wherein the GHA is formulated as a lyophilized powder, which is reconstituted with sterile water for injection or saline prior to administration.

136. The formulation of any one of claims 133 to 135, wherein the GHA is PEGylated to enhance halflife and reduce immunogenicity of the GHA.

137. A sustained-release formulation of a GHA of any one of claims 1 to 59, wherein the GHA is encapsulated in biodegradable microspheres, wherein the microspheres are composed of a polymer selected from poly(lactic-co-glycolic acid) (PLGA), chitosan, or hyaluronic acid, providing controlled release of the GHA over a period of at least one week.

138. The formulation of claim 137. wherein the polymer is PLGA with about a 50:50 lactic -to-glycolic ratio to achieve a release duration of about 1 to 3 months.

139. The formulation of claim 137. wherein the microspheres include a targeting ligand for delivery to a liver or tumor tissue expressing a growth hormone receptor.

140. The formulation of any one of claims 137 to 139, wherein the kinetics of controlled release are adjusted by varying the molecular weight of the polymer or incorporating a hydrophilic excipient.

141. A nanoparticle-based formulation of a GHA of any one of claims 1 to 59, wherein the GHA is encapsulated in lipid nanoparticles (LNPs) or polymeric nanoparticles, and the fonnulation includes a surfactant, a stabilizer, and a targeting ligand to enhance delivery, bioavailability, and stability.

142. The formulation of claim 141, wherein the lipid nanoparticles (LNPs) are modified with polyethylene glycol (PEG) to reduce immune recognition and enhance circulation time.

143. The formulation of claim 141. wherein the polymeric nanoparticles include targeting ligands such as antibodies or peptides for enhanced delivery to growth hormone receptor-expressing cells.

144. The formulation of any one of claims 141 to 143, wherein the encapsulation efficiency of the GHA is at least 85%.-155- 4920-7107-4970, v. 4Attorney Docket No. 1362.0003-1WO145. An oral formulation of a GHA of any one of claims 1 to 59, comprising an enteric coating, a protease inhibitor, and an absorption enhancer, designed to protect the GHA from gastrointestinal degradation and facilitate systemic absorption.

146. Tire formulation of claim 145, wherein the enteric coating is composed of a polymer selected from Eudragit® (basic methacrylate copolymer or poly((dimethylaminoethyl methac late)-co-(methyl methacrylate)-co-(butyl methacrylate)) or cellulose acetate phthalate to resist degradation in the stomach.

147. The formulation of claim 145, wherein the absorption enhancer is sodium caprate or a bile salt to improve intestinal uptake.

148. The formulation of claim 145, wherein the protease inhibitor is aprotinin or soybean trypsin inhibitor to protect the GHA from enzymatic degradation.

149. A transdermal patch formulation of a GHA of any one of claims 1 to 59, comprising a hydrogel matrix or microneedles for sustained and controlled release of the GHA through the skin, allowing for non-invasive administration.

150. The formulation of claim 149. wherein the microneedles are composed of a biocompatible material selected from silicon, stainless steel, and polylactic acid.

151. The formulation of claim 149. wherein the hydrogel matrix includes carbopol or polyvinyl alcohol to enhance stability and sustained release.

152. The formulation of claim 149, wherein the patch includes a permeation enhancer such as dimethyl sulfoxide (DMSO) or ethanol to improve skin penetration of the GHA.

153. A GHA, or composition thereof, wherein the GHA shares at least 80% homology with a GHA of any one of claims 1 to 59.

154. A GHA, or composition thereof, wherein the GHA shares at least 85% homology with a GHA of any one of claim 1 to 59.

155. A GHA, or composition thereof, wherein the GHA shares at least 90% homology with a GHA of any one of claims 1 to 59.

156. A GHA, or composition thereof, wherein the GHA shares at least 95% homology with a GHA of any one of claims 1 to 59.

157. A GHA, or a composition thereof, wherein the GHA shares at least 97% homology with the GHA of any one of claims 1 to 59.-156- 4920-7107-4970, v. 4Attomey Docket No. 1362.0003-1WO158. A GHA, or a composition thereof, wherein the GHA shares at least 99% homology with the GHA of any one of claims 1 to 59.

159. A therapeutically effective amount of the GHA, or a composition thereof, of any one of claims 153 to 158, for use in the method of any one of claims 60 to 132.

160. A therapeutically effective amount of the GHA, of composition thereof, of any one of claims 153 to 158, for use in the formulation of any one of claims 133 to 152.

161. A polynucleotide, comprising a DNA molecule, RNA molecule, modified DNA molecule, or modified RNA molecule, encoding a GHA of any one of claims 1 to 59 or 133 to 152.

162. A cell or cell-line encoding a GHA of any one of claims 153 to 157.-157- 4920-7107-4970, v. 4

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