Pharmaceutical compositions and methods of using same

Abstract

Provided herein are pharmaceutical compositions containing active agents that are soluble VEGFR-3 trapping molecules. Also provided herein are therapeutic methods and uses that include the pharmaceutical compositions, particularly in ocular diseases and disorders, and port devices that include the pharmaceutical compositions.

Description

[Technical Field]

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 374,366, filed September 1, 2022, the entire contents of which are incorporated herein by reference. The present disclosure relates to pharmaceutical compositions containing active agents that are soluble VEGFR-3 trapping molecules, and to therapeutic methods and uses including the pharmaceutical compositions, particularly in ocular diseases and disorders, as well as port devices including the pharmaceutical compositions. [Background technology]

[0002] Loss or reduction of vision is an extremely debilitating condition and can have a serious impact on an individual's quality of life. Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in the elderly. The "wet" form of this disorder occurs when abnormal blood vessels grow within the macula, causing scarring of the macula by leaking blood or fluid, resulting in blindness. A more severe eye disorder associated with vascular leakage is diabetic macular edema (DME), in which leaking fluid accumulates in the macula, resulting from vascular damage in individuals with diabetic retinopathy.

[0003] Current therapies for AMD and DME include treatment with laser therapy (e.g., laser photocoagulation) and administration of medications including ranibizumab (Lucentis®), aflibercept (Eylea®, Zaltrap®), brolucizumab (Beovu®), and corticosteroids such as triamcinolone.

[0004] Vascular endothelial growth factor (VEGF) proteins and their receptors play important roles in vasculogenesis, the development of the embryonic vasculature from early differentiated endothelial cells; angiogenesis, the process by which new blood vessels form from pre-existing blood vessels; and lymphangiogenesis, the process by which new lymphatic vessels form. Furthermore, dysfunction of the endothelial cell regulatory system is a key feature of cancer and many other diseases associated with abnormalities in vasculogenesis, angiogenesis, and lymphangiogenesis.

[0005] Therapies aimed at blocking VEGF / PDGF signal transduction via receptors have been approved for the treatment of ocular conditions, including AMD and DME, and cancer. For example, the above-mentioned aflibercept is a VEGF inhibitor consisting of a portion of the extracellular domain of human VEGF receptor 1 and human VEGF receptor 2 fused to the Fc portion of human IgG1. It acts by binding to circulating VEGF-A and VEGF-B and placental growth factor (PlGF), which normally bind to VEGFR-1 and VEGFR-2, and is therefore referred to as a VEGFR-1 / VEGFR-2 capture molecule.

[0006] Another therapy under development for the treatment of ocular symptoms is OPT-302, a VEGFR-3 trapping molecule that comprises a portion of human VEGF receptor 3 extracellular domain, which is soluble in body fluids (e.g., blood and plasma) and binds to the circulating VEGF proteins that normally bind to VEGFR-3, namely, VEGF-C and VEGF-D.OPT-302 has completed a phase 2b clinical trial in wet age-related macular degeneration (wet AMD) and a phase 2a clinical trial for DME, and is currently undergoing a phase 3 clinical trial for wet AMD.Soluble VEGFR-3 trapping molecules, such as OPT-302, are described, for example, in International Publication No. WO2014 / 124487 and International Publication No. WO2015 / 123715, the entire contents of which are incorporated herein by reference.

[0007] However, drug discovery and development is a long and complex process, and after identifying a therapeutic agent, there can often be significant barriers to bringing the drug to market and obtaining approval to treat patients. For example, there can be significant challenges in developing a pharmaceutical formulation of the active agent that provides required properties such as safety, acceptable stability of the active agent, acceptable stability of the formulation, retention of sufficient activity over time, ease of administration, and avoidance of administration site reactions. For some therapeutic agents, despite efforts to identify a suitable formulation, special storage conditions, such as low or ultra-low temperature conditions, may be required to achieve an acceptable shelf life of the formulation.

[0008] There remains a need for additional pharmaceutical formulations to treat conditions such as wet AMD and DME. For example, there also remains a need for formulations of soluble VEGFR-3 trapping molecules, such as OPT-302, that offer good storage stability properties.

[0009] Where any prior art publication is referred to herein, it should be understood that such reference does not constitute an acknowledgement that the publication forms part of the common general knowledge in the art in Australia or any other country. Summary of the Invention

[0010] OPT-302, a soluble VEGFR-3 capture molecule, tends to form dimers when present in some aqueous formulations, which is associated with reduced purity and binding activity.For example, the aqueous composition of OPT-302 developed has been found to rapidly form high levels of OPT-302 dimers, and therefore must be stored at very low temperatures, such as -20°C, to achieve acceptable shelf life.

[0011] The present study identified a pharmaceutical formulation of OPT-302 that unexpectedly provides good stability characteristics and does not require storage at -20°C to achieve acceptable formulation shelf life. Thus, in a first aspect, an active agent that is a soluble VEGFR-3 capture molecule, present at a concentration ranging from 5 mg / mL to 250 mg / mL; trehalose, buffering agents, and water An aqueous pharmaceutical composition comprising: The pH is in the range of 6.5 to 8.0, Aqueous pharmaceutical compositions are provided that contain trehalose at a concentration of at least 7.0% w / v and / or that are free of added sodium chloride.

[0012] In some embodiments, the pharmaceutical composition does not contain added sodium chloride. In some embodiments, the pharmaceutical composition comprises trehalose at a concentration of at least 7.0% w / v.

[0013] In some embodiments, trehalose is present at a concentration of up to 20% w / v, 8.5% w / v to 15% w / v, or about 10.9% w / v trehalose.

[0014] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand-binding polypeptide comprising immunoglobulin-like domains 1-3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain.

[0015] In some embodiments, the ligand-binding polypeptide comprises an amino acid sequence defined by positions 25-329 of SEQ ID NO:1, except that positions in the polypeptide corresponding to positions 104-106 of SEQ ID NO:1 are not identical to NXS or NXT, and the ligand-binding polypeptide retains four N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO:1, positions 166-168 of SEQ ID NO:1, positions 251-253 of SEQ ID NO:1, and positions 299-301 of SEQ ID NO:1, and is glycosylated at all four of the N-glycosylation sequon sites.

[0016] In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defined by amino acids 99-330 of SEQ ID NO:2. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 6, or an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 3, or an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 4, or an amino acid sequence defined by positions 1 to 546 of SEQ ID NO: 5, or an amino acid sequence defined by positions 1 to 546 of SEQ ID NO: 6.

[0017] In some embodiments, the ligand-binding polypeptide comprises an amino acid sequence defined by positions 25-329 of SEQ ID NO:1, and the ligand-binding polypeptide possesses five N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO:1, positions 104-106 of SEQ ID NO:1, positions 166-168 of SEQ ID NO:1, positions 251-253 of SEQ ID NO:1, and positions 299-301 of SEQ ID NO:1, and is glycosylated at the five N-glycosylation sequon sites.

[0018] In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defined by amino acids 99-330 of SEQ ID NO:2. In some embodiments, the soluble VEGFR-3 capture molecule has the amino acid sequence set forth in SEQ ID NO:7 or has the amino acid sequence defined by positions 1-547 of SEQ ID NO:7.

[0019] In some embodiments, the active agent is present in a concentration of up to 120 mg / mL. In some embodiments, the active agent is present in a concentration of about 40 mg / mL or about 80 mg / mL or about 120 mg / mL.

[0020] In some embodiments, the pH of the composition ranges from 7.2 to 7.8, hi some embodiments, the pH of the composition is about 7.5. In some embodiments, the buffering agent is sodium phosphate. In some embodiments, the buffering agent is present at a concentration ranging from 5 mM to 100 mM. In some embodiments, the buffering agent is present at a concentration ranging up to 50 mM. In some embodiments, the buffering agent is present at a concentration of about 10 mM.

[0021] In some embodiments, the composition includes a surfactant. In some embodiments, the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate. In some embodiments, the surfactant is present at a concentration ranging from 0.005% to 0.2% w / v. In some embodiments, the surfactant is present at a concentration of about 0.01% w / v.

[0022] In some embodiments, the composition has an osmolality in the range of 300 mOsm / kg to 1000 mOsm / kg. In some embodiments, the composition has an osmolality in the range of 350 mOsm / kg or greater. In some embodiments, the composition has an osmolality in the range of 400 mOsm / kg or greater. In some embodiments, the composition has an osmolality in the range of 400 mOsm / kg to 600 mOsm / kg.

[0023] In some embodiments, the composition is substantially free of sodium chloride. In some embodiments, the composition does not include an additional sugar. In some embodiments, the composition does not include an additional osmolality adjusting agent.

[0024] In some embodiments, the composition comprises: an active agent at a concentration of about 40 mg / ml, which is a soluble VEGFR-3 capture molecule comprising a ligand-binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand-binding polypeptide comprising immunoglobulin-like domains 1-3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain; trehalose at a concentration of about 10.9% w / v; sodium phosphate at a concentration of about 10 mM, Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v, and water and the pH of this aqueous pharmaceutical composition is about 7.5.

[0025] In a further aspect, an active agent which is a soluble VEGFR-3 capture molecule; trehalose, and buffer The freeze-dried pharmaceutical composition for reconstitution comprises the above compound, wherein the weight ratio of trehalose to active substance is in the range of 1:3 to 40:1.

[0026] In some embodiments, the weight ratio of trehalose to active agent ranges from 1:1 to 7.5:1, 1:1 to 5:1, or 2.1:1 to 4.5:1, In some embodiments, the weight ratio of trehalose to active agent is about 2.7:1.

[0027] In some embodiments, the buffering agent is sodium phosphate. In some embodiments, the buffering agent is sodium phosphate, and the weight ratio of sodium phosphate to active agent is in the range of 1:3 to 1:1000, or 1:3 to 1:200, or 1:5 to 1:100. In some embodiments, the buffering agent is sodium phosphate, and the weight ratio of sodium phosphate to active agent is about 0.03:1.

[0028] In some embodiments, the composition comprises a surfactant, hi some embodiments, the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

[0029] In a further aspect, there is also provided a reconstituted pharmaceutical composition obtained by mixing a lyophilized pharmaceutical composition as defined herein with an aqueous diluent. In some embodiments of the above-defined aspects, the pharmaceutical composition is formulated for intravitreal injection.

[0030] In a further aspect, there is provided a method of inhibiting angiogenesis in a subject comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein. In a further aspect, there is provided a method for treating and / or preventing a disease or disorder associated with abnormalities in angiogenesis, vasculogenesis, and / or lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein.

[0031] In a further aspect, there is provided the use of a VEGF-C trapping molecule and / or a VEGF-D trapping molecule or a salt thereof for the manufacture of a pharmaceutical composition as defined herein for the treatment and / or prevention of a disease or disorder associated with abnormalities in angiogenesis, vasculogenesis, and / or lymphangiogenesis.

[0032] In a further aspect, there is provided a pharmaceutical composition as defined herein for use in the treatment and / or prevention of a disease or disorder associated with abnormalities in angiogenesis, vasculogenesis and / or lymphangiogenesis.

[0033] In some embodiments of the methods, uses, and pharmaceutical compositions for use, the disease or disorder is an ocular disease or disorder. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the ocular disease or disorder is selected from the group consisting of macular degeneration, diabetic retinopathy, macular edema, retinal vein occlusion, and macular telangiectasia. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the ocular disease or disorder is wet age-related macular degeneration. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the ocular disease or disorder is diabetic macular edema. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the pharmaceutical composition is administered in combination with an additional active substance. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the additional active substance is an anti-VEGF-A substance or an anti-VEGF-B substance. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the additional active agent is selected from the group consisting of ranibizumab, aflibercept, bevacizumab, and brolucizumab.

[0034] In some embodiments of the methods, uses, and pharmaceutical compositions for use, the pharmaceutical composition is administered intravitreally. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the pharmaceutical composition is administered using a port device that is implanted in the eye, the port device containing a reservoir for the pharmaceutical composition and allowing for controlled release of the active agent into the vitreous of the eye.

[0035] In a further aspect, there is also provided a port device for intraocular implantation comprising a reservoir containing a pharmaceutical composition as defined herein, the port device allowing for controlled release of an active agent into the vitreous of the eye.

[0036] In some embodiments, the port device includes a semipermeable membrane that allows passive diffusion of the active agent into the vitreous of the eye. In some embodiments, the port device includes a septum that allows for the replenishment of the reservoir with additional pharmaceutical composition using a needle. [Brief explanation of the drawings]

[0037] [Figure 1] 1 shows a chart depicting the results of stability testing for OPT-302 compositions according to the present disclosure and comparative compositions. The percent formation levels of high molecular weight species of the active agent over time were determined for the compositions at 37° C. [Figure 2] 2 shows a chart illustrating the results of stability testing for OPT-302 compositions according to the present disclosure. The percent formation level of active monomer over time was determined for the compositions at 25° C. [Figure 3] 3 shows a chart illustrating the results of stability studies for OPT-302 compositions according to the present disclosure. The level of active agent monomer formation over time was determined for the compositions at 5° C. DETAILED DESCRIPTION OF THE INVENTION

[0038] definition Throughout this specification, the word "comprise" or variations thereof, such as "comprises" or "comprising," unless the context otherwise requires, will be understood to imply the inclusion of the specified element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0039] As used herein, the term "and / or," e.g., "X and / or Y," shall be understood to mean either or both "X and Y" and "X or Y," and shall be understood to provide explicit support for both meanings or for either meaning.

[0040] As used herein, the term about refers to + / - 10%, more preferably + / - 5% of the indicated value, unless stated to the contrary. As used herein, the terms "a," "an," and "the" include both singular and plural aspects unless the context clearly dictates otherwise.

[0041] As used herein, the phrase "at least one of," when used in conjunction with a list of items, means that various combinations of one or more of the listed items may be used, or only one item in the list may be required.

[0042] As used herein, the term "subject" refers to any living organism susceptible to a disease or condition. For example, the subject may be an animal, a mammal, a primate, a livestock animal (e.g., sheep, cow, horse, pig), a companion animal (e.g., dog, cat) or an experimental animal (e.g., mouse, rabbit, rat, guinea pig, hamster). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal.

[0043] As used herein, the terms "treating" and "treatment" include one or more of the following: curing a disease or disorder, reducing the severity of a disease or disorder, preventing or slowing the progression of a disease or disorder, and alleviating symptoms associated with a disease or disorder.

[0044] As used herein, the terms "preventing" and "prevention" include one or more of the following: preventing a subject from developing a disease or disorder, delaying the onset of a disease or disorder, and preventing the onset of a disorder or condition.

[0045] The term "therapeutically effective amount," as used herein, refers to administering a pharmaceutical composition comprising a soluble VEGFR-3 capture molecule in an amount sufficient to treat or prevent the disorder or condition being treated.

[0046] The term "identity," as known in the art, refers to the relatedness between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness of nucleic acid molecules or polypeptide sequences, as the case may be, as determined by the match between a stretch of two or more nucleotides or two or more amino acid sequences. "Identity" measures the percent of perfect matches between two or more short sequences, with gap alignment (if necessary), as specified by a particular mathematical model (i.e., "algorithm") in a computer program. Algorithms suitable for determining percent identity in the present disclosure include BLASTP and BLASTN, using the most commonly and accepted default parameters.

[0047] The term "component domain," as used herein, refers to a domain within a ligand-binding molecule that is derived from or based on a protein domain within the extracellular portion of a receptor protein. For example, each of the Ig domains (D1-D7) of VEGFR-3 constitutes a component domain. Reference to a component domain herein includes both the complete native wild-type domain as well as variants thereof with insertions, deletions, and / or substitutions that substantially retain the functional properties of the intact domain. It will be readily apparent to those skilled in the art that numerous variants of the above domains (e.g., Ig domains) can be obtained that retain substantially the same functional properties as the wild-type domain.

[0048] active substance The pharmaceutical compositions of the present disclosure include an active agent that is a soluble VEGFR-3 trapping molecule. Vascular endothelial growth factor receptor 3 (VEGFR-3; formerly known as Flt4) is a receptor for VEGF-C and VEGF-D ligands and is found primarily on endothelial cells of blood and lymphatic vessels. It is primarily involved in angiogenesis and lymphangiogenesis. Soluble VEGFR-3 capture molecules are useful in the treatment of diseases and disorders associated with abnormalities in angiogenesis and / or vascular permeability, angiogenesis, and / or lymphangiogenesis, such as many ocular disorders, including wet age-related macular degeneration and diabetic macular edema. They also have applications in the treatment of other disease manifestations associated with abnormalities in angiogenesis and / or lymphangiogenesis (e.g., cancer).

[0049] The term "soluble," as used herein with respect to an active agent, means that the active agent has sufficiently high solubility in biological fluids, such as blood, plasma, and / or vitreous humor, so that it is available for binding to circulating VEGF-C. In some embodiments, the soluble VEGFR-3 capture molecule has a plasma solubility of at least 1 mg / mL, or at least 2 mg / mL, or at least 5 mg / mL, or at least 10 mg / mL, or at least 20 mg / mL, or at least 30 mg / mL, or at least 40 mg / mL.

[0050] The active substance is a VEGFR-3 capture molecule. As referred to herein, a VEGFR-3 capture molecule is a molecule that can bind to circulating VEGF-C and / or VEGF-D. In some embodiments, the soluble VEGFR-3 capture molecule binds to human VEGF-C with a KD of about 1 nM or less (e.g., 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM or less). In some embodiments, the soluble VEGFR-3 capture molecule binds to human VEGF-D with a KD of about 5 nM or less (e.g., 2 nM, 1 nM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM or less).

[0051] The binding affinity of VEGF-C and VEGF-D can be determined by any suitable assay.For example, binding affinity can be determined by ELISA or surface plasmon resonance.This type of technology is described, for example, in International Publication No. 2014 / 124487(A1), the entire content of which is incorporated herein by reference.

[0052] In some embodiments, the soluble VEGFR-3 capture molecule is or comprises a polypeptide. In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide. For example, the ligand-binding polypeptide can be or include a fragment of a growth receptor tyrosine kinase extracellular domain (ECD). In some embodiments, the fragment can differ from the wild-type sequence so as not to eliminate growth factor binding, and the fragment is preferably modified as described herein to improve its therapeutic properties for administration to a subject / patient in need thereof.

[0053] VEGF-C and VEGF-D bind with high affinity to at least one VEGF receptor (or receptor heterodimer) selected from VEGFR-2 and VEGFR-3 and stimulate its phosphorylation. Preferred ligand-binding polypeptides do not simply bind to target growth factors. Preferred ligand-binding polypeptides also inhibit the growth factor(s) to which they bind from stimulating phosphorylation of at least one (and preferably all) of the receptor tyrosine kinases to which they bind. Stimulation of tyrosine phosphorylation is easily measured using in vitro cell-based assays and antibodies.

[0054] A ligand-binding polypeptide "specific" for a particular growth factor is a ligand-binding molecule that specifically recognizes the activated form of the growth factor (e.g., the form found circulating in the body). Preferably, the ligand-binding polypeptide also specifically binds other forms of the growth factor. By way of example, VEGF-C (and VEGF-D) are translated as prepromolecules with extensive amino- and carboxy-terminal propeptides that are cleaved to yield the "fully processed" form of VEGF-C (or VEGF-D) that binds to and stimulates VEGFR-2 and VEGFR-3. A ligand-binding polypeptide specific for VEGF-C (or VEGF-D) binds at least the fully processed form of VEGF-C (or VEGF-D), and preferably also binds the partially processed and unprocessed forms.

[0055] SEQ ID NO: 1 comprises the amino acid sequence of human VEGFR-3, with positions 1 to 24 of SEQ ID NO: 1 corresponding to the putative signal peptide, and positions 25 and beyond of SEQ ID NO: 1 corresponding to the putative mature form of the receptor without the putative signal peptide.

[0056] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide comprising a portion of the extracellular domain (ECD) of human VEGFR-3. The ECD of human VEGFR-3 contains seven immunoglobulin-like domains. Domains 1-3 are involved in ligand binding, and domains 4-7 are involved in the structural rearrangements required for receptor dimerization.

[0057] The complete ECD of VEGFR is not required for ligand (growth factor) binding. The ECD of VEGFR-3 has six intact Ig-like domains and one truncated Ig-like domain, and D5 of VEGFR-3 is post-translationally cleaved to form VEGFR-3 with disulfide-linked subunits (Veikkola, T et al., Cancer Res. 60:203-212 (2000)). In some embodiments, receptor fragments containing at least the first three Ig-like domains of this family are sufficient to bind to ligands. Soluble receptors capable of binding to VEGF-C and VEGF-D, thereby inhibiting the activity of VEGF-C or VEGF-D, or signaling through VEGFR-3, are also disclosed in International Publication Nos. WO 2000 / 023565, WO 2000 / 021560, WO 2002 / 060950, and WO 2005 / 087808, the disclosures of which are incorporated herein in their entireties. Soluble receptors, optionally with modifications as described herein, are contemplated as soluble VEGFR-3 capture molecules of the present disclosure.

[0058] The table below defines the approximate boundaries of the Ig-like domain of human VEGFR-3. Such boundaries are important because the selected boundaries can be used to form ligand-binding molecules and can affect the binding properties of the resulting construct.

[0059] [Table 1]

[0060] In some embodiments, the ligand-binding polypeptide comprises a portion of the amino acid sequence of at least one of immunoglobulin-like domain 1, immunoglobulin-like domain 2, and immunoglobulin-like domain 3 of the ECD of human VEGFR-3.

[0061] In some embodiments, the ligand-binding polypeptide comprises substantially all or all of the amino acid sequence of at least one of immunoglobulin-like domain 1, immunoglobulin-like domain 2, and immunoglobulin-like domain 3 of the ECD of human VEGFR-3.

[0062] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide, which is a purified or isolated ligand-binding polypeptide comprising a first amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to the sequence of amino acids defined by positions 47-115 of SEQ ID NO: 1 or positions 25-115 of SEQ ID NO: 1. The aforementioned segment of SEQ ID NO: 1 approximately corresponds to or comprises the first immunoglobulin-like domain of the extracellular domain (ECD) of human VEGFR-3 ("VEGFR-3 D1").

[0063] In some embodiments, the soluble VEGFR-3 capture molecule comprises a purified or isolated ligand-binding polypeptide that comprises an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to the sequence of amino acids defined by positions 47-115 of SEQ ID NO:1 or positions 25-115 of SEQ ID NO:1, except that the polypeptide positions corresponding to positions 104-106 of SEQ ID NO:1 are not identical to NXS or NXT (where X represents any amino acid).

[0064] Ig-like domains 1-3 of VEGFR-3 contain five putative N-glycosylation sites (referred to herein as the N1, N2, N3, N4, and N5 sequons of VEGFR-3, respectively). N1 corresponds to amino acids 33-35 of SEQ ID NO: 1, N2 corresponds to amino acids 104-106 of SEQ ID NO: 1, N3 corresponds to amino acids 166-168 of SEQ ID NO: 1, N4 corresponds to amino acids 251-253 of SEQ ID NO: 1, and N5 corresponds to amino acids 299-301 of SEQ ID NO: 1. In some embodiments, the ligand binding molecules described herein comprise a modification in the N2 sequon of the molecule.

[0065] In some embodiments, this putative glycosylation sequon at positions 104-106 is removed from the amino acid sequence of the ligand-binding polypeptide. The term "removal," as used in this context, refers to a change (by substitution, deletion, or insertion) in the primary amino acid sequence at at least one position that disrupts the NXT sequon motif. In one variation, the amino acid corresponding to position 104 of SEQ ID NO: 1 may be deleted and replaced with another amino acid (e.g., glutamine, aspartic acid, glutamic acid, arginine, and lysine).

[0066] For example, in some embodiments, the amino acid of the ligand-binding molecule corresponding to position 104 of SEQ ID NO: 1 is deleted and replaced with another amino acid. Conservative substitutions are preferred. In some embodiments, the amino acid corresponding to position 104 of SEQ ID NO: 1 is deleted and replaced with an amino acid selected from the group consisting of glutamine, aspartic acid, glutamic acid, arginine, and lysine. In embodiments in which the N2 sequon of SEQ ID NO: 1 is modified as described above, the N1 sequon, N3 sequon, N4 sequon, and N5 sequon of SEQ ID NO: 1 preferably have unchanged amino acid sequences.

[0067] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide, which is a purified or isolated ligand-binding polypeptide sequence comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to the sequence of amino acids defined by positions 154-210 of SEQ ID NO: 1. The sequence of amino acids defined by the polypeptide positions corresponding to positions 154-210 approximately corresponds to or comprises the second immunoglobulin-like domain of the ECD of human VEGFR-3 ("VEGFR-3 D2").

[0068] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide, which is a purified or isolated ligand-binding polypeptide sequence comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to the sequence of amino acids defined by positions 248-314 of SEQ ID NO: 1. The sequence of amino acids defined by the polypeptide positions corresponding to positions 248-314 approximately corresponds to or comprises the third immunoglobulin-like domain of the ECD of human VEGFR-3 ("VEGFR-3 D3").

[0069] In some embodiments, the ligand-binding polypeptide comprises substantially all or all of the amino acid sequence of immunoglobulin-like domain 1 and immunoglobulin-like domain 2 of the ECD of human VEGFR-3. In some embodiments, the ligand-binding polypeptide comprises substantially all or all of the amino acid sequence of immunoglobulin-like domain 2 and immunoglobulin-like domain 3 of the ECD of human VEGFR-3. In some embodiments, the ligand-binding polypeptide comprises substantially all or all of the amino acid sequence of immunoglobulin-like domain 1, immunoglobulin-like domain 2, and immunoglobulin-like domain 3 of the ECD of human VEGFR-3.

[0070] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide comprising immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain.

[0071] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide, which is a purified or isolated ligand-binding polypeptide sequence comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to or identical to the sequence of amino acids 25 to 329 of SEQ ID NO:1.

[0072] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide comprising an amino acid sequence defined by positions 25 to 329 of SEQ ID NO:1, wherein the ligand-binding polypeptide possesses five N-glycosylation sequon sites corresponding to positions 33 to 35 of SEQ ID NO:1, positions 104 to 106 of SEQ ID NO:1, positions 166 to 168 of SEQ ID NO:1, positions 251 to 253 of SEQ ID NO:1, and positions 299 to 301 of SEQ ID NO:1, and is glycosylated at the five N-glycosylation sequon sites.

[0073] In some embodiments, the soluble VEGFR-3 capture molecule comprises a purified or isolated ligand-binding polypeptide comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to or identical to the sequence of amino acids defined by positions 25 to 329 of SEQ ID NO:1, except that the polypeptide positions corresponding to positions 104 to 106 of SEQ ID NO:1 are not identical to NXS or NXT (where X represents any amino acid).

[0074] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide comprising an amino acid sequence defined by positions 25 to 329 of SEQ ID NO:1, except that the polypeptide positions corresponding to positions 104 to 106 of SEQ ID NO:1 are not identical to NXS or NXT, and the ligand-binding polypeptide retains four N-glycosylation sequon sites corresponding to positions 33 to 35 of SEQ ID NO:1, positions 166 to 168 of SEQ ID NO:1, positions 251 to 253 of SEQ ID NO:1, and positions 299 to 301 of SEQ ID NO:1, and is glycosylated at all four of the N-glycosylation sequon sites.

[0075] In some embodiments, this putative glycosylation sequon at positions 104-106 is removed from the amino acid sequence of the ligand-binding polypeptide. The term "removal," as used in this context, refers to a change (by substitution, deletion, or insertion) in the primary amino acid sequence at at least one position that disrupts the NXT sequon motif. In one variation, the amino acid corresponding to position 104 of SEQ ID NO: 1 may be deleted and replaced with another amino acid (e.g., glutamine, aspartic acid, glutamic acid, arginine, and lysine).

[0076] Constructs that include additional Ig-like domains of VEGFR-3 that are linked to ligand-binding polypeptides are contemplated. For example, soluble VEGFR-3 capture molecules can include some or substantially all or all of the seven Ig-like domains of VEGFR-3.

[0077] In embodiments in which the ligand-binding polypeptide comprises an amino acid sequence substantially corresponding to two or more component domains of VEGFR-3, the component domains may be linked directly to one another or may be linked via one or more spacers. Preferably, the component domains are linked by one or more spacers.

[0078] For example, the ligand-binding polypeptide may optionally include sequences before the most N-terminally located Ig-like domain, between the Ig-like domains, and / or after the most C-terminally located Ig-like domain.

[0079] In one embodiment, the spacer comprises one or more peptide sequences between the component domains that are 1 to 100 amino acids in length, preferably 1 to 50 amino acids in length. In one embodiment, the spacer between two component domains consists essentially of peptide sequences that naturally bind to the component domains of native VEGFR-3.

[0080] In embodiments in which the ligand-binding polypeptide comprises an amino acid sequence that approximately corresponds to or includes consecutive component domains of VEGFR-3 (e.g., D1-D2 or D1-D2-D3), the component domains may be linked via one or more spacers that comprise one or more peptide sequences between the component domains that are 1 to 100 amino acids in length, preferably 1 to 50 amino acids in length.

[0081] In some embodiments, the spacer between two component domains consists essentially of a peptide sequence corresponding to the peptide sequence connecting each of the consecutive component domains of a native VEGFR-3. In some embodiments, the spacer between two consecutive component domains comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to, or identical to, the sequence of amino acids connecting the consecutive domains of a native VEGFR-3.

[0082] In embodiments in which the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide comprising multiple component domains of VEGFR-3, e.g., component domains D1, D2, and D3, the component domains may be directly linked to each other or may be linked via one or more spacers. Generally, the term "spacer" refers to one or more molecules, e.g., nucleic acids or amino acids, or non-peptide moieties, e.g., polyethylene glycol or disulfide bridges, that can be inserted between one or more component domains to form a covalent bond. Spacer sequences can be used to create desired target sites between components for ease of manipulation. Spacers can also be created to enhance expression of the ligand-binding polypeptide by host cells, reduce steric hindrance so that a component or group of components can adopt an optimal tertiary structure and / or properly interact with a target molecule, and for other purposes. For information about spacers and methods for identifying desirable spacers, see, e.g., George et al. (2003) Protein Engineering 15:871-879, which is incorporated herein by reference. The spacer sequence may comprise one or more amino acids naturally associated with a receptor component, or it may be an additional sequence used to enhance expression of the ligand-binding polypeptide, generate a specific desired site of interest, allow the component domains to form an optimal tertiary structure, and / or enhance the interaction of the component or group of components with a target molecule. In one embodiment, the spacer comprises one or more peptide sequences between one or more components, 1 to 100 amino acids in length, preferably 1 to 50 amino acids in length. In a preferred embodiment, the spacer between two component domains consists essentially of amino acids naturally associated with a receptor component in a wild-type receptor. For example, in the case of a ligand-binding polypeptide comprising multiple component domains from the same receptor, such as D1, D2, and D3 of VEGFR-3, which are adjacent to each other in the native receptor, in one embodiment, the domains are linked to each other using a spacer corresponding to the naturally occurring amino acid binding sequence (e.g., D1 to D2 and D2 to D3).

[0083] In some variations, each ligand-binding polypeptide is expressed as a fusion with a fusion partner protein, for example, an immunoglobulin constant region, and the heterologous fusion partners combine to form the ligand-binding molecule.

[0084] In some embodiments, the ligand binding molecule is a polypeptide comprising a portion of a human VEGFR-3 ECD, which portion binds to one or both of human VEGF-C and human VEGF-D and includes at least the first, second, and third Ig-like domains of the VEGFR-3 ECD, wherein the amino acid sequence of the VEGFR-3 ECD fragment is modified from wild-type VEGFR-3 to remove the second putative N-linked glycosylation sequon in wild-type VEGFR-3, and the polypeptide does not include VEGFR-3 Ig-like domains 4-7, and preferably does not include any transmembrane domains, and preferably does not include any intracellular domains.

[0085] In addition to monomeric constructs, the present disclosure also includes multimeric ligand-binding constructs comprising two or more ligand-binding molecules described herein that are covalently or non-covalently linked to one another to form dimeric or multimeric structures. In some variations, the binding occurs between VEGFR-3-like sequences of the ligand-binding polypeptides, while in other variations, the binding occurs between heterologous polypeptides that bind to one or both of the VEGFR-3-like sequences.

[0086] Reference herein to a ligand-binding polypeptide described herein includes reference to variants thereof. In some embodiments, the ligand-binding polypeptide is a variant. In other embodiments, the ligand-binding polypeptide is not a variant.

[0087] The VEGFR-3 from which the ligand-binding polypeptide can be derived includes splice variants and naturally occurring allelic variants. Allelic variants are well known in the art and represent alternative forms or nucleic acid sequences, including substitution, deletion, or addition of one or more nucleotides, but without causing any substantial change in the functionality of the encoded polypeptide. Exemplary allelic variants of VEGFR-3 have been reported in the literature, for example, http: / / www.uniprot.org / uniprot / P35916, and include 149, 378, 494, 527, and 641 in ECD. Standard methods for producing such polypeptides can be easily used, including site-directed mutagenesis of polynucleotides or specific enzymatic cleavage and ligation. Similarly, the use of peptidomimetic compounds, or compounds in which one or more amino acid residues are replaced with non-naturally occurring amino acids or amino acid analogs that retain binding activity, is also contemplated.

[0088] Preferably, when amino acid substitution is used, the substitution is conservative. That is, an amino acid is replaced with an amino acid of similar size and similar charge characteristics. As used herein, the term "conservative substitution" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative substitutions include the substitution of a hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine, for another hydrophobic residue, or the substitution of a polar residue for another polar residue, such as arginine for lysine, glutaric acid for aspartic acid, or glutamine for asparagine. Neutral hydrophilic amino acids that can be substituted for each other include asparagine, glutamine, serine, and threonine. The term "conservative substitution" also includes the use of a substituted amino acid instead of an unsubstituted amino acid.

[0089] Alternatively, conservative amino acids may be grouped as described by Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY, pp. 71-77 (1975)), as shown below. Non-polar (hydrophobic) A. Aliphatic: A, L, I, V, P, B. Aromatic: F, W, C. Sulfur content: M, D.Borderline:G. Uncharged - Polar A. Hydroxyl: S, T, Y, B. Amides: N, Q, C. Sulfhydryl: C, D.Borderline:G. Positively charged (basic): K, R, H. Negative charge (acidic): D, E.

[0090] Soluble VEGFR-3 capture molecules can include, for example, a fusion partner (e.g., a heterologous peptide, etc.) that confers desirable properties (e.g., prolongs serum half-life, increases solubility in aqueous media, and / or enables targeting of specific cell populations, e.g., tumor cells or retinal cells, etc.).

[0091] In some embodiments, a fusion partner is any heterologous moiety that enhances the functionality of a ligand-binding polypeptide. Thus, for example, a fusion partner may increase the solubility, regulate clearance, facilitate targeting to a specific cell or tissue type, enhance biological activity, aid in production and / or recovery, enhance pharmacological properties, or enhance the pharmacokinetic (PK) profile of the ligand-binding polypeptide. With regard to an enhanced PK profile, this may be achieved by, for example, enhancing the serum half-life, tissue penetration, lack of immunogenicity, or stability of the ligand-binding molecule. In some embodiments, the fusion partner is selected from the group consisting of a multimerization component, a serum protein, or a molecule capable of binding a serum protein.

[0092] In some embodiments, the fusion moiety comprises an immunoglobulin-derived domain, eg, from human IgG, Ig, or IgA. In some variations, the soluble VEGFR-3 capture molecule comprises an immunoglobulin constant domain or a fragment thereof. In some embodiments, the soluble VEGFR-3 capture molecule comprises a human immunoglobulin G domain or a fragment thereof. The amino acid sequence of a human immunoglobulin 1 heavy chain constant domain is set forth in SEQ ID NO: 2. In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defined by amino acids 99 to 330 of SEQ ID NO: 2.

[0093] In some embodiments, the soluble VEGFR-3 capture molecule comprises an immunoglobulin constant domain fragment comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to or identical to the sequence of amino acids 99 to 330 of SEQ ID NO:2.

[0094] In some embodiments, the immunoglobulin constant domain fragment may exclude the C-terminal amino acid residues, for example, in some embodiments, the immunoglobulin constant domain fragment has an amino acid sequence defined by amino acids 99 to 329 of SEQ ID NO:2.

[0095] The immunoglobulin-derived amino acid sequence can be linked to the C-terminus or N-terminus of the ligand-binding polypeptide, preferably the C-terminus. Cells transfected with DNA encoding the immunoglobulin light chain fusion protein and the immunoglobulin heavy chain fusion protein each express a heavy / light chain heterodimer containing the ligand-binding polypeptide. Both ligand-binding polypeptides advantageously contain native or heterologous signal peptides to facilitate secretion from the cell when initially synthesized, although the signal sequence may be cleaved, for example, during secretion. Variations of any of the foregoing embodiments that include a signal peptide are contemplated. The native signal peptide of human VEGFR-3 comprises residues 1-24 of SEQ ID NO: 1. Numerous other signal peptide proteins are taught in the literature.

[0096] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide fused (as a single polypeptide chain) to the Fc portion of human immunoglobulin G (IgG).

[0097] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide (as a single-chain polypeptide) fused to the Fc portion of human immunoglobulin G (IgG), with a single amino acid substitution in the second Ig-like domain to remove the N-glycosylation site.

[0098] In some embodiments, the ligand-binding polypeptides described herein optionally include a linker, e.g., the Factor Xa linker sequence DPIEGRGGGGG (SEQ ID NO: 8), that connects a fusion partner, such as a heterologous peptide, to the ligand-binding polypeptide. In other embodiments, the ligand-binding molecule comprises a polypeptide in which the C-terminal amino acid of the ligand-binding polypeptide is directly linked to the N-terminal amino acid of the heterologous peptide fusion partner by a peptide bond. In some embodiments, the ligand-binding polypeptide and the heterologous peptide are linked by an amide bond (directly or via a linker polypeptide) to form a single-chain polypeptide.

[0099] In some embodiments, the soluble VEGFR-3 capture molecule is OPT-302. OPT-302 has the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the soluble VEGFR-3 capturing molecule comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence defined by amino acids 1 to 536 of SEQ ID NO: 3.

[0100] In some embodiments, the soluble VEGFR-3 capturing molecule comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence defined by amino acids 1 to 536 of SEQ ID NO: 4.

[0101] In some embodiments, the soluble VEGFR-3 capturing molecule comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence defined by amino acids 1 to 546 of SEQ ID NO: 5.

[0102] In some embodiments, the soluble VEGFR-3 capturing molecule comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence defined by amino acids 1 to 546 of SEQ ID NO: 6.

[0103] In some embodiments, the soluble VEGFR-3 capturing molecule is VGX-300. VGX-300 has the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the soluble VEGFR-3 capturing molecule comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the soluble VEGFR-3 capturing molecule has an amino acid sequence defined by amino acids 1 to 546 of SEQ ID NO: 7.

[0104] If necessary, the soluble VEGFR-3 capture molecule described herein can comprise functional region that facilitates purification or production.The specific example of such additional amino acid sequence includes GST sequence or His tag sequence.In some variants, the region that facilitates purification is removed for the formulation of pharmaceutical use composition.

[0105] The soluble VEGFR-3 capture molecule can be chemically modified to impart desired properties (e.g., glycosylation, PEGylation, etc.), and such modifications preferably do not substantially reduce the growth factor binding affinity or specificity of the ligand binding molecule.

[0106] The polypeptides may be modified by, for example, glycosylation, amidation, carboxylation or phosphorylation, or by the formation of acid addition salts, amides, esters, particularly C-terminal esters, and N-acyl derivatives.

[0107] In some embodiments, the soluble VEGFR-3 capture molecules described herein optionally include at least one PEG (polyethylene glycol) moiety attached to the molecule. For example, in some embodiments, a PEG of approximately 20-40 kDa is attached to the amino terminus of the ligand-binding molecule. As used herein, polyethylene glycol is intended to encompass any form of PEG, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol, that can be used to derivatize other proteins. PEG is a linear or branched neutral polyether available in a wide range of molecular weights and is soluble in water and most organic solvents. PEG is effective in excluding other polymers or peptides when present in water, primarily due to its highly dynamic and hydrophilic chain mobility, which creates a water shell or hydration layer when bound to other proteins or polymer surfaces. PEG is nontoxic, non-immunogenic, and approved by the Food and Drug Administration for internal use.

[0108] Polypeptides can be conjugated to reporter groups, including but not limited to radiolabels, fluorescent labels, enzymes (e.g., catalyzing calorimetric or fluorometric reactions), substrates, solid matrices or carriers (e.g., biotin or avidin).Examples of analogs are described in International Publication No. 98 / 28621, and Olofsson et al., Proc.Nat'l.Acad.Sci.USA, 95:1 709-1 714 (1998), U.S. Patent Nos. 5,512,545 and 5,474,982, U.S. Patent Application Publication Nos. 20020164687 and 20020164710, the entire contents of each of which are incorporated herein by reference.

[0109] In some variations, the ligand binding molecule comprises a signal peptide that directs secretion of the molecule from cells expressing it. Soluble VEGFR-3 capture molecule can be prepared by any suitable process.For example, can be transfected with the vector comprising the polynucleotide sequence encoding the amino acid sequence of soluble VEGFR-3 capture molecule into cell line (for example, eukaryotic cell line, Chinese hamster ovary cell line), and then cultured to express capture molecule.The method for producing and purifying soluble VEGFR-3 capture molecule is described in International Publication No. 2014 / 124487 (A1), International Publication No. 2015 / 123715 (A1) and International Publication No. 2002 / 060950 (A1), the entire contents of each of which are incorporated herein by reference.

[0110] The soluble VEGFR-3 capture molecule is present in the aqueous pharmaceutical composition at a concentration ranging from 5 mg / mL to 250 mg / mL. In some embodiments, the soluble VEGFR-3 capture molecule is present at a concentration of 10 mg / mL or more, 20 mg / mL or more, 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, 110 mg / mL or more, 120 mg / mL or more, 130 mg / mL or more, 140 mg / mL or more, 150 mg / mL or more, 160 mg / mL or more, 170 mg / mL or more, 180 mg / mL or more, 190 mg / mL or more, 200 mg / mL or more, 210 mg / mL or more, 220 mg / mL or more, 230 mg / mL or more, or 240 mg / mL or more. In some embodiments, the soluble VEGFR-3 capturing molecule is present at a concentration of up to 240 mg / mL, up to 230 mg / mL, up to 220 mg / mL, up to 210 mg / mL, up to 200 mg / mL, up to 190 mg / mL, up to 180 mg / mL, up to 170 mg / mL, up to 160 mg / mL, up to 150 mg / mL, up to 140 mg / mL, up to 130 mg / mL, up to 120 mg / mL, up to 110 mg / mL, up to 100 mg / mL, up to 90 mg / mL, up to 80 mg / mL, up to 70 mg / mL, up to 60 mg / mL, up to 50 mg / mL, up to 40 mg / mL, up to 30 mg / mL, up to 20 mg / mL or up to 10 mg / mL. Preferably, the soluble VEGFR-3 capture molecule is present at a concentration of up to 120 mg / mL, more preferably up to 100 mg / mL, even more preferably up to 80 mg / mL, even more preferably up to 60 mg / mL, and even more preferably up to 40 mg / mL.

[0111] In some embodiments, the soluble VEGFR3 capture molecule is 5 mg / mL to 120 mg / mL, 5 mg / mL to 100 mg / mL, 5 mg / mL to 80 mg / mL, 5 mg / mL to 60 mg / mL, 5 mg / mL to 50 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 120 mg / mL, 10 mg / mL to 100 mg / mL, 10 mg / mL to 80 mg / mL, 10 mg / mL to 60 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 40 mg / mL g / mL, 20mg / mL~120mg / mL, 20mg / mL~100mg / mL, 20mg / mL~80mg / mL, 20mg / mL~60mg / mL, 20mg / mL~50mg / mL, 20mg / mL~40mg / mL, 30 Present in concentrations of mg / mL to 120 mg / mL, 30 mg / mL to 100 mg / mL, 30 mg / mL to 80 mg / mL, 30 mg / mL to 60 mg / mL, 30 mg / mL to 50 mg / mL, or 30 mg / mL to 40 mg / mL.

[0112] In some embodiments, the soluble VEGFR-3 capturing molecule is present at a concentration of about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL or about 120 mg / mL.

[0113] excipients The aqueous pharmaceutical composition of the present disclosure contains trehalose. Trehalose is a disaccharide consisting of two glucose units linked by a 1,1-glycosidic bond. Typically, the glucose unit present in trehalose is an α-glucose unit. Trehalose is also known as α,α-trehalose, α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside, α-D-glucopyranoside-α-D-glucopyranoside, and D-(+)-trehalose. Trehalose has the CAS number 6138-23-4. Trehalose has the following chemical structure:

[0114] [ka]

[0115] Forms of trehalose include anhydrous and dihydrate forms. The molar mass of anhydrous trehalose is 342.3 g / mol, and the molar mass of dihydrate trehalose is 378.3 g / mol. Trehalose can be obtained from various suppliers, such as Pfanstiehl (www.pfanstiehl.com). Trehalose dihydrate is also available from Sigma Aldrich, Merck, Fisher Scientific, Acros, and Alfa Aesar.

[0116] In some embodiments, the aqueous pharmaceutical composition of the present disclosure comprises trehalose at a concentration of at least 7.0% w / v. Regardless of the form of trehalose used to produce the formulation (e.g., anhydrous trehalose or trehalose dihydrate), the % w / v of trehalose refers to the weight percent of trehalose, e.g., not including any associated solvates (e.g., dihydrate). For example, a 10% w / v solution of trehalose can be prepared by using 10 g of anhydrous trehalose and dissolving it in water to make 100 ml. Alternatively, a 10% w / v solution of trehalose can be prepared by using 11.1 g of trehalose dihydrate and dissolving it in water to make 100 ml.

[0117] In some embodiments, trehalose is present at a concentration of up to 20% w / v, or up to 15% w / v, or up to 14% w / v, or up to 13% w / v, or up to 12% w / v, or up to 11% w / v, hi some embodiments, trehalose is present at a concentration of at least 7.5% w / v, at least 8.0% w / v, at least 8.5% w / v, at least 9% w / v, at least 9.5% w / v, at least 10% w / v, or at least 10.5% w / v. In some embodiments, the trehalose is between 7.0% w / v and 20% w / v, or between 7.0% w / v and 15% w / v, or between 7.0% w / v and 14% w / v, or between 7.0% w / v and 13% w / v, or between 7.0% w / v and 12% w / v, or between 7.5% w / v and 20% w / v, or between 7.5% w / v and 15% w / v, or between 7.5% w / v and 14% w / v, or between 7.5% w / v and 13% w / v, or 7. 5%w / v~12%w / v or 8.0%w / v~20%w / v or 8.0%w / v~15%w / v or 8.0%w / v~14%w / v or 8.0%w / v~13%w / v or 8.0%w / v~12%w / v or 8.5%w / v~20%w / v or 8.5%w / v~15%w / v or 8.5%w / v~14%w / v or 8.5%w / v~13%w / v or 8.5%w / v~12% w / v or 9% w / v~20% w / v or 9% w / v~15% w / v or 9% w / v~14% w / v or 9% w / v~13% w / v or 9% w / v~12% w / v or 9.5% w / v~20% w / v or 9.5% w / v~15% w / v or 9.5% w / v~14% w / v or 9.5% w / v~13% w / v or 9.5% w / v~12% w / v or 10 It is present at a concentration of %w / v~20%w / v or 10%w / v~15%w / v or 10%w / v~14%w / v or 10%w / v~13%w / v or 10%w / v~12%w / v or 10.5%w / v~20%w / v or 10.5%w / v~15%w / v or 10.5%w / v~14%w / v or 10.5%w / v~13%w / v or 10.5%w / v~12%w / v.In some embodiments, trehalose is about 7.0% w / v, about 7.5% w / v, about 8.0% w / v, 8.5% w / v, about 9% w / v, about 9.5% w / v, about 10% w / v, about 10.1% w / v, about 10.2% w / v, about 10.3% w / v, about 10.4% w / v, about 10.5% w / v, about 10.6% w / v, about 10.7% w / v, about 10.8% w / v, about 10.9% w / v, about 11% w / v, about 11.1% w / v, about 11.2% w / v, about 11.3% w / v, about 11.4% w / v, about 11.5% w / v, about 11.6% w / v, about 11.7% w / v, about 11.8% w / v, about 11.9 ... 0.2% w / v, about 11.3% w / v, about 11.4% w / v, about 11.5% w / v, about 11.6% w / v, about 11.7% w / v, about 11.8% w / v, about 11.9% w / v, about 12% w / v, about 12.5% ​​w / v, about 13% w / v, about 13.5% w / v, about 14% w / v, about 14.5% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v or about 20% w / v.

[0118] Aqueous pharmaceutical compositions contain water. Typically, sterilized, highly purified water, such as water for injection, is used. The aqueous pharmaceutical composition contains a buffer, for example, to maintain a pH within a desired range. Any suitable buffer may be utilized. Examples of buffers include phosphate buffers (e.g., sodium dihydrogen phosphate, disodium phosphate), amino acids such as histidine buffers (e.g., histidine hydrochloride), citrate buffers (e.g., sodium citrate), Tris(2-amino-2-(hydroxymethyl)propane-1,3-diol), and acetate buffers (e.g., sodium acetate). In some embodiments, the buffer is a Tris or phosphate buffer. In some embodiments, the buffer is a phosphate buffer, which may be, for example, a mixture of acidic and basic forms of phosphate. In some embodiments, the buffer is sodium phosphate, for example, a mixture of sodium dihydrogen phosphate and disodium phosphate.

[0119] The buffering agent may be present at a suitable concentration, for example, at a concentration of up to 100 mM, up to 90 mM, up to 80 mM, up to 70 mM, up to 60 mM, up to 50 mM, up to 40 mM, up to 30 mM, up to 20 mM, or up to 10 mM. In some embodiments, the buffering agent is present at a concentration of at least 5 mM or at least 10 mM. In some embodiments, the buffering agent is present at a concentration ranging from 5 mM to 100 mM, 5 mM to 80 mM, 5 mM to 70 mM, 5 mM to 60 mM, 5 mM to 50 mM, 5 mM to 40 mM, 5 mM to 30 mM, or 5 mM to 20 mM. In some embodiments, the buffering agent is present at a concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0120] It has been found that aqueous pharmaceutical compositions containing soluble VEGFR-3 capturing molecules having a high pH have improved stability with respect to dimer formation. The aqueous pharmaceutical composition has a pH in the range of 6.5 to 8.0. In some embodiments, the pH of the composition is in the range of 6.5 to 7.0, 7.0 to 7.5, 7.5 to 8.0, 7.2 to 7.8, 7.3 to 7.7, or 7.4 to 7.6. In some embodiments, the pH of the composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0121] In some embodiments, the aqueous pharmaceutical composition comprises a surfactant. When a surfactant is included, it can be, for example, an ionic surfactant (e.g., cationic, anionic, or zwitterionic) or a nonionic (e.g., neutral) surfactant. Examples of surfactants include polyoxyethylene (20) sorbitan monolaurate (e.g., sold under the brand names Polysorbate 20® and Tween 20®), polyoxyethylene (20) sorbitan monooleate (e.g., sold under the brand names Polysorbate 80® and Tween 80®), polyethylene glycol, and poloxamer (e.g., a copolymer of poly(propylene oxide) and poly(ethylene oxide)) (e.g., sold under the brand name Pluronic F68®). In some embodiments, the aqueous pharmaceutical composition comprises a surfactant that is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

[0122] If a surfactant is included, it may be present at a concentration ranging from, for example, 0.005% to 0.2% w / v. In some embodiments, the surfactant is present at a concentration of 0.005% to 0.1% w / v, or 0.005% to 0.05% w / v, or 0.005% to 0.02% w / v. In some embodiments, the surfactant is present at a concentration of about 0.005% w / v, or about 0.006% w / v, or about 0.007% w / v, or about 0.008% w / v, or about 0.009% w / v, or about 0.01% w / v, or about 0.011% w / v, or about 0.012% w / v, or about 0.013% w / v, or about 0.014% w / v, or about 0.015% w / v, or about 0.016% w / v, or about 0.017% w / v, or about 0.018% w / v, or about 0.019% w / v, or about 0.02% w / v.

[0123] In some other embodiments, the aqueous pharmaceutical composition does not include a surfactant. Aqueous pharmaceutical compositions containing soluble VEGFR-3 trapping molecules and having relatively high osmolality have been found to provide good stability characteristics. Osmolality relates to the concentration of osmotically active particles in a solution and is typically defined in units of mOsm / kg.

[0124] Thus, in some embodiments, the aqueous pharmaceutical composition has an osmolality of at least 300 mOsm / kg, or at least 350 mOsm / kg, or at least 400 mOsm / kg, hi some embodiments, the aqueous pharmaceutical composition has an osmolality of at most 1000 mOsm / kg, or at most 900 mOsm / kg, or at most 800 mOsm / kg, or at most 700 mOsm / kg, or at most 600 mOsm / kg, or at most 500 mOsm / kg. In some embodiments, the aqueous pharmaceutical composition has an osmolality in the range of 300 mOsm / kg to 1000 mOsm / kg, or 350 mOsm / kg to 1000 mOsm / kg, or 400 mOsm / kg to 1000 mOsm / kg, or 300 mOsm / kg to 800 mOsm / kg, or 350 mOsm / kg to 800 mOsm / kg, or 400 mOsm / kg to 800 mOsm / kg, or 300 mOsm / kg to 600 mOsm / kg, or 350 mOsm / kg to 600 mOsm / kg, or 400 mOsm / kg to 600 mOsm / kg.

[0125] Aqueous pharmaceutical compositions can contain relatively few components and still provide good stability characteristics. As defined herein, a tonicity agent is a substance that affects the osmotic pressure of a pharmaceutical composition. An isotonicity agent is typically included to adjust the osmotic pressure of the composition to a desired value.

[0126] In some embodiments, the aqueous pharmaceutical composition contains an additional tonicity agent, while in other embodiments, the aqueous pharmaceutical composition does not contain an additional tonicity agent. The term additional tonicity agent refers to a substance that substantially affects the osmotic pressure of the pharmaceutical composition and is other than the active substance, trehalose, buffer, water, and surfactant (if present). Examples of isotonicity agents include sugars (e.g., sucrose, dextrose), certain salts (e.g., sodium chloride, potassium chloride), and polyols (e.g., mannitol, sorbitol, glycerin).

[0127] In some embodiments, the pharmaceutical composition comprises less than 50 mM sodium chloride, or less than 25 mM sodium chloride, or less than 10 mM sodium chloride, or less than 5 mM sodium chloride.

[0128] In some embodiments, the pharmaceutical composition does not contain added sodium chloride.As used herein, the term "does not contain added sodium chloride" means that sodium chloride is not added during the preparation of the pharmaceutical composition.Nevertheless, it is understood that the pharmaceutical composition may contain a small amount of sodium chloride, and for example, when adjusting the pH of the formulation by adding hydrochloric acid and sodium hydroxide, a small amount of sodium chloride may be formed.

[0129] In some embodiments, the aqueous pharmaceutical composition is substantially free of sodium chloride. In some embodiments, the composition contains less than 1 mM sodium chloride, or less than 0.5 mM sodium chloride, or less than 0.2 mM sodium chloride, or less than 0.1 mM sodium chloride, or less than 0.05 mM sodium chloride. In some embodiments, the aqueous pharmaceutical composition contains no detectable sodium chloride.

[0130] In some embodiments, the aqueous pharmaceutical composition is substantially free of additional sugars (i.e., substantially free of sugars other than trehalose (e.g., monosaccharides or disaccharides) and any sugars that form part of the active ingredient). In some embodiments, the aqueous pharmaceutical composition comprises less than 1 mM additional sugars, 0.5 mM additional sugars, less than 0.2 mM additional sugars, 0.1 mM additional sugars, or less than 0.05 mM additional sugars. In some embodiments, the aqueous pharmaceutical composition comprises no detectable additional sugars.

[0131] In some embodiments, the aqueous pharmaceutical composition does not contain any components beyond water, a soluble VEGFR-3 capture molecule, a phosphate buffer, and a surfactant (except for impurities that may be present in such components).

[0132] In some embodiments, the aqueous pharmaceutical composition comprises: an active agent that is a soluble VEGFR-3 capture molecule, present at a concentration ranging from 5 mg / mL to 120 mg / mL; trehalose at a concentration ranging from 8.5% w / v to 20% w / v; a buffering agent at a concentration ranging from 5 mM to 20 mM; optionally a surfactant, and water The aqueous pharmaceutical composition has a pH in the range of 6.5 to 8.0.

[0133] In some embodiments, the aqueous pharmaceutical composition comprises: an active agent that is a soluble VEGFR-3 capture molecule, present at a concentration ranging from 20 mg / mL to 80 mg / mL; trehalose at a concentration ranging from 9% w / v to 13% w / v; a buffering agent at a concentration ranging from 5 mM to 20 mM, which is sodium phosphate; a surfactant that is polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v, and water The aqueous pharmaceutical composition has a pH in the range of 7.0 to 8.0.

[0134] In some embodiments, the aqueous pharmaceutical composition consists of or consists essentially of the active agent, trehalose, a buffering agent, and a surfactant. In some embodiments, the aqueous pharmaceutical composition consists of or consists essentially of the active agent, trehalose, sodium phosphate buffer, and polyoxyethylene (20) sorbitan monolaurate.

[0135] In some embodiments, the aqueous pharmaceutical composition comprises: an active agent at a concentration of about 40 mg / ml, which is a soluble VEGFR-3 capture molecule comprising a ligand-binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand-binding polypeptide comprising immunoglobulin-like domains 1-3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain; trehalose at a concentration of about 10.9% w / v; a buffering agent at a concentration of about 10 mM which is sodium phosphate; Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v, and water and the pH of the aqueous pharmaceutical composition is about 7.5.

[0136] In some embodiments, the aqueous pharmaceutical composition comprises: an active substance at a concentration of about 40 mg / ml, which is OPT-302 or VGX-300; trehalose at a concentration of about 10.9% w / v; a buffering agent at a concentration of about 10 mM which is sodium phosphate; Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v, and water and the pH of the aqueous pharmaceutical composition is about 7.5.

[0137] In some embodiments, the aqueous pharmaceutical composition comprises: an active substance comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, at a concentration of about 40 mg / ml; trehalose at a concentration of about 10.9% w / v; a buffering agent at a concentration of about 10 mM which is sodium phosphate; Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v, and water and the pH of the aqueous pharmaceutical composition is about 7.5.

[0138] In some embodiments, the aqueous pharmaceutical composition comprises: an active substance comprising or consisting of the amino acid sequence of SEQ ID NO: 7, at a concentration of about 40 mg / ml; trehalose at a concentration of about 10.9% w / v; a buffering agent at a concentration of about 10 mM which is sodium phosphate; Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v, and water and the pH of the aqueous pharmaceutical composition is about 7.5.

[0139] The aqueous pharmaceutical composition may be prepared in advance and provided to a hospital, surgery, etc. as a pre-prepared aqueous pharmaceutical formulation, or may be provided as a solid composition, e.g., a lyophilized pharmaceutical composition for reconstitution with water before use.

[0140] therefore, an active agent which is a soluble VEGFR-3 capture molecule; trehalose, and buffer The present invention also provides a freeze-dried pharmaceutical composition for reconstitution, comprising the above compound, wherein the weight ratio of trehalose to active substance is in the range of 1:3 to 40:1.

[0141] Also provided is a reconstituted pharmaceutical composition obtained by mixing a lyophilized pharmaceutical composition as defined herein with an aqueous diluent. In use, an appropriate amount of aqueous diluent is added to the lyophilized pharmaceutical composition to obtain the desired concentrations of the constituents (eg, desired concentrations of active agent, trehalose, and / or buffer).

[0142] A lyophilized pharmaceutical composition containing trehalose and active agent in a weight ratio ranging from 1:3 to 40:1 corresponds, after reconstitution with an appropriate amount of aqueous diluent, to a reconstituted pharmaceutical composition containing 5 to 250 mg / mL of active agent and 8.5% w / v to 20% w / v of trehalose.

[0143] In some embodiments, the weight ratio of trehalose to active agent in the lyophilized pharmaceutical composition ranges from 1:1 to 7.5:1, 1:1 to 5:1, or 2.1:1 to 4.5:1. In some embodiments, the weight ratio of trehalose to active agent in the lyophilized pharmaceutical composition ranges from 0.7:1 to 3.3:1 or from 0.8:1 to 3:1.

[0144] In some embodiments, the weight ratio of trehalose to active agent in the lyophilized pharmaceutical composition is about 2.7:1, or about 1.4:1, or about 0.9:1. As mentioned above, trehalose is a disaccharide consisting of two glucose units linked by a 1,1-glycosidic bond. Typically, the glucose unit present in trehalose is an α-glucose unit. Trehalose is also known as α,α-trehalose, α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside, α-D-glucopyranoside-α-D-glucopyranoside, and D-(+)-trehalose. Trehalose has the CAS number 6138-23-4. Trehalose has the following chemical structure:

[0145] [ka]

[0146] Forms of trehalose include anhydrous and dihydrate forms. The molar mass of the anhydrous form of trehalose is 342.3 g / mol, and the molar mass of the dihydrate form of trehalose is 378.3 g / mol.

[0147] Trehalose can be obtained from a variety of suppliers, such as Pfanstiehl (www.pfanstiehl.com), and trehalose dihydrate is available from Sigma Aldrich, Merck, Fisher Scientific, Acros, and Alfa Aesar.

[0148] The lyophilized pharmaceutical composition includes a buffer. Any suitable buffer may be utilized. Examples of buffers include phosphate buffers (e.g., sodium dihydrogen phosphate, disodium phosphate), amino acids such as histidine buffers (e.g., histidine hydrochloride), citrate buffers (e.g., sodium citrate), Tris(2-amino-2-(hydroxymethyl)propane-1,3-diol), and acetate buffers (e.g., sodium acetate). In some embodiments, the buffer is a Tris or phosphate buffer. In some embodiments, the buffer is a phosphate buffer, which may be, for example, a mixture of acidic and basic forms of phosphate. In some embodiments, the buffer is sodium phosphate, for example, a mixture of sodium dihydrogen phosphate and disodium phosphate.

[0149] In some embodiments, the weight range of buffer to active agent ranges from 1:420 to 3:1, or 1:200 to 1:1.8, or 1:26 to 1:40. In some embodiments, the buffering agent is sodium phosphate, and the weight ratio of sodium phosphate to active agent ranges from 1:3 to 1:1000, or 1:3 to 1:200, or 1:5 to 1:100.

[0150] In some embodiments, the buffering agent is sodium phosphate, and the weight ratio of sodium phosphate to active agent is about 0.03:1. In some embodiments, the lyophilized pharmaceutical composition comprises a surfactant. When a surfactant is included, it can be, for example, an ionic surfactant (e.g., cationic, anionic, or zwitterionic) or a nonionic (e.g., neutral) surfactant. Examples of surfactants include polyoxyethylene (20) sorbitan monolaurate (e.g., sold under the brand names Polysorbate 20® and Tween 20®), polyoxyethylene (20) sorbitan monooleate (e.g., sold under the brand names Polysorbate 80® and Tween 80®), polyethylene glycol, and poloxamer (e.g., a copolymer of poly(propylene oxide) and poly(ethylene oxide)) (e.g., sold under the brand name Pluronic F68®). In some embodiments, the lyophilized pharmaceutical composition comprises a surfactant that is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

[0151] The process of lyophilization typically involves multiple steps, eg, a freezing step and one or more drying steps (eg, a primary drying step involving sublimation and a secondary drying step involving desorption).

[0152] The lyophilized pharmaceutical composition can include, for example, one or more lyophilization excipients.Examples of lyophilization excipients include cryoprotectants and lyoprotectants.Examples of lyophilization excipients include sugars such as sucrose, mannitol, and dextrose, polymer excipients such as polyvinylpyrrolidone, and amino acids such as glycine.

[0153] In some embodiments, the lyophilized pharmaceutical composition does not contain any additional lyophilization excipients (ie, excluding the active agent, trehalose, buffer, and surfactant, if present).

[0154] As discussed above, in use, an appropriate amount of aqueous diluent is added to the lyophilized pharmaceutical composition to obtain the desired concentrations of the components (e.g., the desired concentrations of the active substance, trehalose, and / or buffer). The aqueous diluent can be, for example, water. Typically, sterilized, highly purified water, such as water for injection, is used.

[0155] The amount of aqueous diluent may be added so that the concentration of the active agent in the reconstituted pharmaceutical composition is typically in the range of 5 mg / mL to 250 mg / mL, or, for example, 20 mg / mL to 120 mg / mL or about 40 mg / mL or about 80 mg / mL or about 120 mg / mL.

[0156] By way of further example, the amount of aqueous diluent may be added such that the concentration of trehalose in the reconstituted pharmaceutical composition is typically at least 8.5% w / v, for example, in the range of 8.5% w / v to 20% w / v, or 8.5% w / v to 15% w / v, or 9% w / v to 13% w / v, or 10% w / v to 12% w / v, or about 10.9% w / v.

[0157] By way of still further example, the amount of aqueous diluent may be added such that the concentration of buffer is typically up to 100 mM, e.g., up to 50 mM or up to 20 mM, or at least 5 mM, or in the range of 5 mM to 50 mM or in the range of 5 mM to 20 mM, or about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM or about 50 mM.

[0158] Typically, after reconstitution, the pH of the reconstituted lyophilized pharmaceutical composition is in the range of 6.5 to 8.0, e.g., 6.5 to 7.0, 7.0 to 7.5, 7.5 to 8.0, 7.2 to 7.8, 7.3 to 7.7, or 7.4 to 7.6. In some embodiments, the pH of the reconstituted lyophilized pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0159] The above discussion regarding aqueous pharmaceutical compositions and the required nature and amounts, and optional characteristics, e.g., active agent, trehalose, buffer, surfactant, pH, osmolality, and tonicity agent, also applies to lyophilized pharmaceutical compositions and reconstituted lyophilized pharmaceutical compositions, as appropriate.

[0160] Preparation of the Composition The pharmaceutical compositions of the present disclosure can be prepared by any suitable method, for example, an aqueous solution of purified soluble VEGFR-3 capture molecules can be optionally subjected to a buffer exchange process step, filtration, and / or mixing with other excipients.

[0161] In some embodiments, an aqueous pharmaceutical composition according to the present disclosure may be prepared by mixing an aqueous solution of a soluble VEGFR-3 capturing molecule with an aqueous solution of trehalose, then subjecting the resulting mixture to UF-DF using a buffer comprising trehalose and sodium phosphate, followed by mixing with a surfactant (e.g., polyoxyethylene (20) monolaurate).

[0162] In the case of a lyophilized formulation, the aqueous pharmaceutical composition described above may be prepared and then subjected to lyophilization. For example, the aqueous composition may be subjected to low temperature conditions so that the mixture freezes, and low pressure conditions so that water is removed by sublimation.

[0163] Composition characteristics Soluble VEGFR-3 capture molecules have poor stability characteristics and tend to form dimers or other high molecular weight aggregates upon storage in aqueous pharmaceutical compositions, resulting in reduced purity, activity, and shelf life, or the need for low temperature storage conditions.

[0164] However, as demonstrated by the following examples, the aqueous pharmaceutical compositions of the present disclosure have demonstrated unexpectedly improved stability characteristics, reduced dimer formation, and maintained good levels of binding activity to VEGF-C and VEGF-D over time.

[0165] In some embodiments, the pharmaceutical composition forms less than 6%, or less than 5%, or less than 4% dimerized active agent after storage at 25° C. for a period of 2 months. In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5% dimerized active agent after storage at 25° C. for a period of 3 months.

[0166] In some embodiments, the pharmaceutical composition forms less than 5%, or less than 4%, or less than 3%, or less than 2% dimerized active agent after storage at 5° C. for a period of 2 months. In some embodiments, the pharmaceutical composition forms less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% dimerized active agent after storage at 5° C. for a period of 3 months.

[0167] In some embodiments, the pharmaceutical composition forms less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% dimerized active agent after storage at 5° C. for a period of 6 months. In some embodiments, the pharmaceutical composition forms less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at 5° C. for a period of 12 months.

[0168] In some embodiments, the pharmaceutical composition forms less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at 5° C. for a period of 18 months.

[0169] In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at 5° C. for a period of 24 months.

[0170] In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4% dimerized active agent after storage at 5° C. for a period of 30 months.

[0171] In some embodiments, the pharmaceutical composition forms less than 5%, or less than 4%, or less than 3%, or less than 2% dimerized active agent after storage at -20°C for a period of 2 months. In some embodiments, the pharmaceutical composition forms less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at -20°C for a period of 3 months.

[0172] In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at -20°C for a period of 6 months.

[0173] In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at -20°C for a period of 12 months.

[0174] In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at -20°C for a period of 18 months.

[0175] In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at -20°C for a period of 24 months.

[0176] In some embodiments, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of dimerized active agent after storage at -20°C for a period of 30 months.

[0177] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage at 5°C for a period of 2 months compared to the binding activity of the composition at 0 months.

[0178] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage at 5°C for a period of 3 months compared to the binding activity of the composition at 0 months.

[0179] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage at 5°C for a period of 6 months compared to the binding activity of the composition at 0 months.

[0180] In some embodiments, the pharmaceutical composition retains at least 80% of its binding activity to VEGF-C and / or VEGF-D after storage at 5°C for a period of 12 months, compared to the binding activity of the composition at 0 months.

[0181] In some embodiments, the pharmaceutical composition retains at least 70% of the binding activity to VEGF-C and / or VEGF-D after storage at 5°C for a period of 18 months compared to the binding activity of the composition at 0 months.

[0182] In some embodiments, the pharmaceutical composition retains at least 60% of the binding activity to VEGF-C and / or VEGF-D after storage at 5°C for a period of 24 months compared to the binding activity of the composition at 0 months.

[0183] In some embodiments, the pharmaceutical composition retains at least 60% of the binding activity to VEGF-C and / or VEGF-D after storage at 5°C for a period of 30 months compared to the binding activity of the composition at 0 months.

[0184] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage at -20°C for a period of 2 months compared to the binding activity of the composition at 0 months.

[0185] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage at -20°C for a period of 3 months compared to the binding activity of the composition at 0 months.

[0186] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage at -20°C for a period of 6 months compared to the binding activity of the composition at 0 months.

[0187] In some embodiments, the pharmaceutical composition retains at least 80% of its binding activity to VEGF-C and / or VEGF-D after storage at -20°C for a period of 12 months, compared to the binding activity of the composition at 0 months.

[0188] In some embodiments, the pharmaceutical composition retains at least 80% of its binding activity to VEGF-C and / or VEGF-D after storage at -20°C for a period of 18 months, compared to the binding activity of the composition at 0 months.

[0189] In some embodiments, the pharmaceutical composition retains at least 80% of its binding activity to VEGF-C and / or VEGF-D after storage at -20°C for a period of 24 months, compared to the binding activity of the composition at 0 months.

[0190] In some embodiments, the pharmaceutical composition retains at least 80% of its binding activity to VEGF-C and / or VEGF-D after storage at -20°C for a period of 30 months, compared to the binding activity of the composition at 0 months.

[0191] As defined herein, the shelf life of a pharmaceutical composition is a period of several months during which the extent of dimerization of the active agent upon storage is less than 10% and the binding activity to VEGF-C and / or VEGF-D remains at least 70% of the binding activity achieved at 0 months.

[0192] In some embodiments, the pharmaceutical composition has a shelf life of at least 2 months or at least 3 months when stored at 25°C. In some embodiments, the pharmaceutical composition has a shelf life of at least 3 months, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months when stored at 5°C.

[0193] In some embodiments, the pharmaceutical composition has a shelf life of at least 3 months, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months when stored at -20°C.

[0194] In some embodiments, the pharmaceutical composition remains physically stable, i.e., there is no significant phase separation or precipitation of solid material, for at least 3 months upon storage at 25°C. In some embodiments, the pharmaceutical composition remains physically stable for at least 3 months, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months upon storage at 5° C., i.e., there is no significant phase separation or precipitation of solid material.

[0195] In some embodiments, the pharmaceutical composition remains physically stable for at least 3 months, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months upon storage at −20° C., i.e., there is no significant phase separation or precipitation of solid material.

[0196] Therapeutic Uses and Methods The pharmaceutical compositions of the present disclosure have application in the therapy of diseases and / or disorders in which inhibition of the interaction of VEGF-C and / or VEGF-D with the VEGFR-3 receptor results in a therapeutic response. For example, the pharmaceutical compositions of the present disclosure are useful in inhibiting angiogenesis and have application in the therapy of diseases and / or disorders associated with abnormalities in angiogenesis, vasculogenesis, and / or lymphangiogenesis.

[0197] Angiogenesis is the formation of new blood vessels. Angiogenesis is the formation of new blood vessels from existing blood vessels, and plays a role in various conditions, including cancer and eye disorders such as age-related macular degeneration. Lymphangiogenesis is the formation of lymphatic vessels from existing lymphatic vessels, and excessive lymphatic vessel formation is associated with various conditions, including edema, tumor metastasis, and lymphangiomatosis.

[0198] Accordingly, the present disclosure also provides a method of inhibiting angiogenesis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein. The present disclosure also provides a method for treating and / or preventing a disease or disorder associated with abnormal angiogenesis, vasculogenesis, and / or lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein. Also provided is the use of a soluble VEGFR-3 capture molecule for the manufacture of a pharmaceutical composition as defined herein for the treatment and / or prevention of a disease or disorder associated with abnormal angiogenesis, vasculogenesis, and / or lymphangiogenesis. Also provided herein is a pharmaceutical composition as defined herein for use in the treatment and / or prevention of a disease or disorder associated with abnormal angiogenesis, vasculogenesis, and / or lymphangiogenesis.

[0199] In some embodiments, the disease or disorder is an ocular disease or disorder. In some embodiments, the ocular disease or disorder is selected from the group consisting of macular degeneration, diabetic retinopathy, macular edema, retinal vein occlusion, and macular telangiectasia. In some embodiments, the ocular disease or disorder is wet age-related macular degeneration. In some embodiments, the ocular disease or disorder is diabetic macular edema.

[0200] In addition, pharmaceutical compositions containing soluble VEGFR-3 capture molecules can be applied to the therapy of other diseases and / or disorders associated with abnormalities in angiogenesis, vasculogenesis, and / or lymphangiogenesis. In some embodiments, the disease or disorder is cancer, such as colorectal cancer, lung cancer, breast cancer, glioblastoma, ovarian cancer, cervical cancer, and kidney cancer.

[0201] Administration Pharmaceutical compositions may be administered by any suitable route, eg, compatible with the disease or disorder to be treated.

[0202] In some embodiments, for example, when the disease or disorder is an ocular condition, the pharmaceutical composition may be administered intravitreally. In some embodiments, the pharmaceutical composition is administered by intravitreal injection. Intravitreal injection includes administration into the vitreous humor of the eye.

[0203] In some embodiments, the pharmaceutical composition is administered using an implantation device that is implanted intraocularly and allows for controlled release of the active agent into the vitreous of the eye. In some embodiments, the pharmaceutical composition is administered using a port device that is implanted in the eye, the port device containing a reservoir for the pharmaceutical composition and allowing for controlled release of the active agent into the vitreous of the eye.

[0204] In some embodiments, the pharmaceutical composition may be administered intravenously. In some embodiments, the pharmaceutical composition may be administered subcutaneously. In some embodiments, the pharmaceutical composition may be administered intramuscularly. In some embodiments, the pharmaceutical composition may be administered intrathecally.

[0205] In some embodiments, the pharmaceutical composition is administered by injection. In some embodiments, the pharmaceutical composition is administered by infusion. Thus, the pharmaceutical compositions may be formulated for injection (eg, intravitreal, subcutaneous, intravenous, intramuscular, or intrathecal injection) or for administration using an intraocular implant device.

[0206] In use, the pharmaceutical composition comprising soluble VEGFR-3 trapping molecule utilizes suitable dosage and administration schedule.The amount and frequency of administration can depend on factors including the type of disease or disorder, whether the active substance is administered for prophylactic or therapeutic purposes, the age, weight, sex, and health of the person to be treated, the route of administration, and whether the soluble VEGFR-3 trapping molecule is administered in combination with other active substances.In addition, the pharmacogenomic information of a particular patient (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic agent) can affect the dosage that the patient uses.

[0207] Dosage form The pharmaceutical composition can be administered in a variety of dosage forms. Accordingly, the present disclosure also includes a container containing the pharmaceutical composition. The present disclosure also includes a kit containing a container containing the pharmaceutical composition, and optionally including instructions for administering the pharmaceutical composition to a subject.

[0208] For example, in the case of an aqueous pharmaceutical formulation, the pharmaceutical composition may be provided in a vial or bottle and administered using a syringe. In a further example, the aqueous pharmaceutical composition may be provided in a pre-filled syringe. Thus, in some embodiments, the kit includes a device for administering the pharmaceutical composition.

[0209] When multiple doses are delivered (for example, to the same subject over time or to different subjects), for convenience, for example, a container (for example, a divided bottle or container with multiple wells) can be used, which contains multiple separate portions, each containing a unit dose of the pharmaceutical composition. Alternatively, a kit can be used, which contains multiple containers, each containing a unit dose of the pharmaceutical composition.

[0210] As noted above, in some embodiments, the pharmaceutical composition may be administered utilizing a port device that may be implanted in the eye. Accordingly, there is also provided a port device for intraocular implantation that includes a reservoir containing a pharmaceutical composition as defined herein, the port device allowing for controlled release of an active agent into the vitreous of the eye.

[0211] In use, the port device can be implanted, for example, through the surface of the eye, such as the sclera, so that it can release the active agent into the vitreous, but at least a portion of the device is accessible for reservoir replenishment.

[0212] In some embodiments, the port device can include a reservoir chamber connected to a membrane, an opening, a diffusion barrier, a diffusion mechanism, and / or a porous structure for controlled release of the active agent. For example, this can include a semipermeable membrane, such as a titanium-containing semipermeable membrane, that allows passive diffusion of the active agent into the vitreous of the eye.

[0213] Port devices typically include one or more retention elements to hold the device in place, for example, through the sclera. The port device may, for example, extend through the sclera but be covered by the conjunctiva.

[0214] The port device may be refillable, eg, may include a septum (eg, a silicone septum) that allows for refilling of the reservoir with additional pharmaceutical composition using a refill element, eg, a needle.

[0215] In some embodiments, the port device is configured to receive a quantity of pharmaceutical composition sufficient to deliver a therapeutic dose of the active agent for up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, or up to 6 months.

[0216] Examples of port devices are disclosed in WO 2012 / 019176, WO 2012 / 065006, and WO 2014 / 152959, the contents of each of which are incorporated herein by reference in their entirety.

[0217] Also provided herein are kits comprising: (i) a port device for intraocular implantation, the port device comprising a reservoir for containing a pharmaceutical composition as defined herein, the port device allowing for controlled release of an active agent into the vitreous of the eye; and (ii) a container for filling the port device, the container comprising an amount of a pharmaceutical composition as defined herein. In some embodiments, the kit comprises a syringe for filling the port device with the pharmaceutical composition. In some embodiments, the container containing the pharmaceutical composition is a syringe for filling the port device.

[0218] As mentioned above, in the case of freeze-dried pharmaceutical compositions, in use, the freeze-dried pharmaceutical composition can be reconstituted by mixing with an aqueous diluent before administration.Therefore, also provided is a reconstitution kit comprising the freeze-dried pharmaceutical composition defined herein and an aqueous diluent.The aqueous diluent can be, for example, sterile water, for example, water for injection.

[0219] Combination therapy In some embodiments, the soluble VEGFR-3 capturing molecule may be administered as a monotherapy via a pharmaceutical composition of the present disclosure, while in other embodiments, it is administered as part of a combination therapy treatment regimen, e.g., in combination with an additional active agent, e.g., the additional active agent is useful for the treatment and / or prevention of a disease or disorder associated with abnormalities in angiogenesis, vasculogenesis, and / or lymphangiogenesis.

[0220] Pharmaceutical compositions containing soluble VEGFR-3 trapping molecules can be administered simultaneously, sequentially, or separately with additional active substances, for example. For example, a course of therapy can be defined for each patient, which includes the administration of soluble VEGFR-3 trapping molecules at a certain time point, and the administration of one or more additional active substances at a different time point.

[0221] The additional active agent can be, for example, an additional active agent useful for the treatment and / or prevention of an ocular disease or disorder, for example, an ocular disease or disorder selected from the group consisting of macular degeneration (e.g., wet age-related macular degeneration), diabetic retinopathy, macular edema (e.g., diabetic macular edema), retinal vein occlusion, and macular telangiectasia.

[0222] In some embodiments, the additional active agent is an anti-VEGF-A or anti-VEGF-B agent. Examples of anti-VEGF-A and / or anti-VEGF-B agents include ranibizumab (Lucentis®), aflibercept (Eylea®), bevacizumab (Avastin®), and brolucizumab (Beovu®).

[0223] In some embodiments, the additional active agent is pegaptanib (Macugen®). In some embodiments, the additional active agent is a steroid, such as triamcinolone acetonide, dexamethasone (Ozurdex®), or fluocinolone acetonide (Retisate®, Iluvien®).

[0224] In some embodiments, the pharmaceutical composition comprising the soluble VEGFR-3 trapping molecule is administered in combination with photodynamic therapy. Photodynamic therapy involves the administration of a photosensitive active substance (e.g., verteporfin (Visudyne®)) in combination with laser treatment. In some embodiments, the pharmaceutical composition comprising the soluble VEGFR-3 trapping molecule is administered in combination with laser coagulation therapy. Laser coagulation therapy involves directing a focused beam of high-energy laser light onto the retina to seal leaky blood vessels.

[0225] In some embodiments, the pharmaceutical composition comprising a soluble VEGFR-3 trapping molecule is administered in combination with focal-grid macular laser surgery. A pharmaceutical composition of the present disclosure may, for example, be packaged together with another pharmaceutical composition comprising an additional active agent, for example in a pack or kit containing both pharmaceuticals.

[0226] All publications and patents mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0227] Sequence Listing

[0228] [Table 2-1]

[0229] [Table 2-2]

[0230] [Table 2-3]

[0231] [Table 2-4]

[0232] [Table 2-5]

[0233] [Table 2-6]

[0234] The present disclosure is further illustrated by the following non-limiting examples. [Example]

[0235] Example 1: Initial formulation screening and DOE (design of experiments) testing Formulation testing was performed to identify a preferred formulation: pH, buffers, and excipients were screened and a design of experiments (DOE) was performed.

[0236] Briefly, initial high-throughput screening was performed using one or more of differential scanning calorimetry / fluorometry (DSC / F) and dynamic light scattering (DLS). Subsequent experiments focused on size exclusion chromatography (SEC) and ELISA (VEGF-D) to assess formulation stability.

[0237] The results of the pH / buffer screening showed an increase in the thermal denaturation temperature in buffers between pH 6.5 and 8.0, suggesting improved thermal stability. Results from initial excipient screening showed that sodium and trehalose conferred high thermal stability in citrate, phosphate, and Tris buffers.

[0238] In a one-week accelerated stability study in the same buffers with a panel of excipients, the least amount of degradation by SEC was found in Tris and phosphate buffers with mannitol, trehalose, and proline.

[0239] Surfactant studies showed that OPT-302, when formulated in 20 mM Tris buffer pH 8.0, had minimal total aggregate and dimer formation when trehalose and proline were the excipients, and that the inclusion of PS-20 (polysorbate 20) in the formulation had minimal impact on stability.

[0240] Results of the DOE solution stability study found that the stability of OPT-302 formulated in Tris buffer improved with increasing pH and trehalose concentration. From the planned and unplanned formulations in the DOE study, formulations were identified as potential candidates for further testing. - 40 mg / mL OPT-302 in 20 mM Tris pH 8.0, with or without 18.1% w / v trehalose, 0.01% w / v PS-20, and 100 mM proline 40 mg / mL OPT-302, 18.1% trehalose, and 0.01% w / v PS-20 in 20 mM phosphate buffer pH 7.5 [Example]

[0241] Example 2: Effect of Trehalose Content and pH on OPT-302 Formulation Stability Formulations of OPT-302 were prepared to analyze the tendency of the active ingredient to form dimers over time. The formulations contained 40 mg / mL OPT-302, water, 10 mM sodium phosphate buffer, and 0.01% polysorbate 20. The formulations were pH 7.50 or 7.20 and contained either 9.0%, 13.6%, or 18.1% w / v trehalose (prepared using 10%, 15%, and 20% w / v trehalose dihydrate, respectively).

[0242] The formulations were incubated at 37°C for 1 week and analyzed by SE-HPLC. The results showed a trend towards decreased dimerization with increasing trehalose content.

[0243] [Table 3] [Example]

[0244] Example 3: Effect of Trehalose Content and Sodium Chloride Content on OPT-302 Formulation Stability Formulations of OPT-302 were prepared and analyzed over time for the tendency of the active ingredient to form higher molecular weight species (e.g., dimers). The formulations contained 40 mg / mL OPT-302, water, and 10 mM sodium phosphate buffer at pH 7.4. The formulations contained either 4.5%, 6.8%, 9.0%, or 18.1% w / v trehalose (prepared using 5%, 7.5%, 10%, and 20% w / v trehalose dihydrate, respectively), and either 40 mM, 100 mM, or 140 mM sodium chloride.

[0245] The formulations were incubated at 37°C for up to 2 weeks and analyzed by SE-HPLC. The results are shown in Figure 1. The rate of dimer formation was highest in formulations containing lower concentrations of trehalose. [Example]

[0246] Example 4: Stability of additional formulations of OPT-302 The formulation was prepared starting with OPT-302 in 40 mM NaCl, 10 mM phosphate pH 7.2 and concentrated to 54.5 mg / mL using a 50 kDa membrane.

[0247] OPT-302 was purified using a Superdex S200 column, loaded at approximately 3.2% column volume (CV) and run at 0.5 CV / hr in 40 mM NaCl, 10 mM phosphate pH 7.2. S200 fractions with >97% monomer were pooled, and the purified pool of S200 was spiked with 7.5% trehalose using a 37.5% trehalose stock solution prior to TFF formulation. % monomer by SE-UPLC was 98.7%.

[0248] One-fifth of the stock solution was then spiked to obtain 6.8% trehalose, concentrated to 59 g / L, and then polysorbate 20 (0.03%) was added and diluted to 40 g / L in 6.85% w / v trehalose, 0.03% v / v PS20, 10 mM phosphate, 40 mM NaCl, pH 7.2.

[0249] Three-fifths of the stock solution was diafiltered against 9.0% w / v trehalose, 10 mM phosphate, pH 7.5 (>7 DV) and then split into three portions. The first portion was diluted to 40 mg / mL with 9.0% w / v trehalose, 10 mM phosphate, pH 7.5. The second portion was spiked with 40% trehalose to give 13.6% trehalose and then diluted with 40 mg / mL OPT-302 to give 13.6% w / v trehalose, 10 mM phosphate, pH 7.5, followed by the addition of PS-20 (0.01%). The third portion was spiked using 40% trehalose to give 18.1% trehalose and then diluted in 40 mg / mL OPT-302 to give 18.1% w / v trehalose, 10 mM phosphate pH 7.5, followed by addition of PS-20 (0.01%).

[0250] The final fifth of the stock solution was diafiltered against 13.6% w / v trehalose, 20 mM Tris pH 8.0 (>7 DV) and diluted to 40 mg / mL OPT-302 in the same buffer, followed by the addition of PS-20 (0.01%).

[0251] SE-UPLC was then performed on all formulations and the results are shown in the table below.

[0252] [Table 4]

[0253] As can be seen from the table, formulations concentrated in the presence of sodium chloride had the lowest monomer stability. Formulations that underwent diafiltration to remove sodium chloride showed improved stability, and formulations containing higher amounts of trehalose also had higher % monomer. [Example]

[0254] Example 5: Long-term accelerated stability study OPT-302 formulations were prepared as follows, all containing 40 mg / mL OPT-302. 1. Lyophilized formulation containing 6.8% w / v trehalose (prepared using 7.5% w / v trehalose dihydrate), 0.03% w / v PS20, 10 mM sodium phosphate, 40 mM NaCl, pH 7.2 2. Aqueous formulation containing 6.8% w / v trehalose (prepared using 7.5% w / v trehalose dihydrate), 0.03% PS20, 10 mM sodium phosphate, 40 mM NaCl, pH 7.2 3. Aqueous formulation containing 9.0% w / v trehalose (prepared using 10% w / v trehalose dihydrate), 0.01% PS20, 10 mM sodium phosphate, pH 7.5 4. Aqueous formulation containing 13.6% w / v trehalose (prepared using 15% w / v trehalose dihydrate), 0.01% nPS20, 10 mM sodium phosphate, pH 7.5 5. Aqueous formulation containing 18.1% w / v trehalose (prepared using 20% ​​w / v trehalose dihydrate), 0.01% PS20, 10 mM sodium phosphate, pH 7.5 6. Aqueous formulation containing 13.6% w / v trehalose (prepared using 15% w / v trehalose dihydrate), 0.01% PS20, 20 mM Tris, pH 8 Formulations were set at 25° C. (checked monthly for 3 months by SEC) and 5° C. (checked every 3 months for 24 months by SEC). Summary SEC results are presented in Figures 2 and 3.

[0255] The accelerated results at 25°C show that trehalose concentrations above 6.8% w / v substantially stabilize the monomer content, demonstrating that formulations with high trehalose concentrations and no added sodium chloride have the potential to be stable at 5°C.

[0256] Under real-time conditions (5°C, 24 months storage), all formulations containing trehalose above 6.8% w / v monomer content at 24 months were significantly more stable than the control formulation.

[0257] In summary, in liquid format, long-term refrigerated storage (15 months) and accelerated stability results demonstrate that OPT-302 can be reformulated in high trehalose concentrations resulting in excellent stability. [Example]

[0258] Example 6: OPT-302 Formulations A preferred aqueous formulation for OPT-302 was identified, containing the following components:

[0259] [Table 5]

[0260] Density of formulation = 1.041 kg / L Additional OPT-302 comparative formulations have the following components:

[0261] [Table 6]

[0262] The comparative formulation was found to form high levels of OPT-302 dimers upon storage, as demonstrated by Example 8 below. [Example]

[0263] Example 7: Preparation of OPT-302 formulations To the virus filtered pool containing OPT-302 (virus filtered pool), an aliquot of an aqueous solution containing a high concentration of trehalose (37.5% w / v trehalose dihydrate, 10 mM sodium phosphate, pH 7.5) is added (trehalose spike) to obtain a concentration of 12% w / v trehalose dihydrate and mixed for at least 10 minutes to obtain the mixture (adjusted virus filtered pool).

[0264] The mixture is filtered through a 0.2 μm filter and subjected to concentration using ultrafiltration-diafiltration and tangential flow filtration using an equilibration, diafiltration, and flush buffer containing 12% w / v trehalose dihydrate, 10 mM sodium phosphate, pH 7.5 to obtain a mixture (UFDF pool) with a target OPT-302 concentration of 46-55 mg / mL.

[0265] The concentration of OPT-302 in the UFDF pool is determined, and a fixed amount of dilution buffer (12% w / v trehalose dihydrate, 10 mM sodium phosphate, pH 7.5) is added to the UFDF pool to obtain an OPT-302 concentration of approximately 44 g / L (diluted UFDF pool). The diluted UFDF pool is mixed for at least 30 minutes using a Wave Mixer.

[0266] Calculate and add the target weight of formulation buffer (12% w / v trehalose dihydrate, 10 mM sodium phosphate, polyoxyethylene (20) sorbitan monolaurate, pH 7.5) required to the diluted UFDF pool to achieve a concentration of 0.01% (w / v) polyoxyethylene (20) sorbitan monolaurate. The formulated diluted UFDF pool is mixed for at least 15 minutes using a Wave Mixer.

[0267] The expected concentration of OPT-302 is in the range of 36-44 mg / mL. [Example]

[0268] Example 8: Stability of OPT-302 formulations Three OPT-302 formulations were prepared having the following components: Formulation 1: 41.5 mg / mL OPT-302, 10 mM sodium phosphate, 12% w / v trehalose dihydrate (10.9% w / v trehalose), 0.01% w / v polyoxyethylene(20)sorbitan monolaurate, WFI, pH 7.5 Comparative Formulation 1: 41 mg / mL OPT-302, 10 mM sodium phosphate, 40 mM sodium chloride, 7.5% w / v trehalose dihydrate (6.8% w / v trehalose), 0.03% w / v polyoxyethylene(2)sorbitan monolaurate, WFI, pH 7.2 Comparative Formulation 2: 42.4 mg / mL OPT-302, 10 mM sodium phosphate, 40 mM sodium chloride, 7.5% w / v trehalose dihydrate (6.8% w / v trehalose), 0.03% w / v polyoxyethylene(2)sorbitan monolaurate, pH 7.2, WFI The formulations were stored at various temperatures to determine the stability characteristics of the formulations. Data for Formulation 1 stored at -20°C, 5°C, 25°C, and 40°C are presented below, along with data for comparative formulations stored at 25°C and 40°C.

[0269] [Table 7]

[0270] [Table 8]

[0271] [Table 9]

[0272] [Table 10]

[0273] [Table 11]

[0274] [Table 12]

[0275] [Table 13]

[0276] [Table 14]

[0277] Formulation 1 formed lower levels of high molecular weight species over time compared to Comparative Formulations 1 and 2, especially at elevated temperature conditions. Formulation 1 also demonstrated good activity with respect to binding to VEGF-C and VEGF-D as determined by ELISA.

[0278] ELISA method Assay plates were coated overnight with 0.1 μg / mL VEGF-C ligand or 1.0 μg / mL VEGF-D ligand. OPT-302 reference standards and test samples were diluted to a starting concentration of 1500 ng / mL, followed by two-fold serial dilutions ranging from 1.5 ng / mL to 1500 ng / mL. The plates were incubated for 60 minutes at 25°C and then washed to remove unbound samples. Bound molecules were detected by adding HRP-conjugated rabbit anti-human IgG to the assay plate and incubating for 60 minutes at 25°C. TMB substrate was then added, and the assay plate was incubated for 10 minutes at room temperature in the dark. Color development was stopped by adding 1 M HCl stop solution. This color development was detected by absorbance at 450 nm. The intensity of the yellow color was proportional to the amount of OPT-302-bound molecules, which then reflected the activity of the OPT-302 reference standard or test sample. A four-parameter curve was then constructed by plotting the mean values ​​against the log 10 of the 2-fold serial dilutions. The value to be reported was the relative potency (%) of the test sample, i.e., the ratio of the EC50 of the reference standard to the EC50 of the test sample.

[0279] Upon storage at -20°C, Formulation 1 was found to have the following activity: 0 months, VEGF-C: 107%, VEGF-D: 102%; 0.5 months, VEGF-C: 126%, VEGF-D: 116%; 1 month, VEGF-C: 118%, VEGF-D: 100%; 2 months, VEGF-C: 108%, VEGF-D: 106%.

[0280] Upon storage at 5°C, Formulation 1 was found to have the following activity: 0 months, VEGF-C: 107%, VEGF-D: 102%; 0.5 months, VEGF-C: 114%, VEGF-D: 112%; 1 month, VEGF-C: 115%, VEGF-D: 101%; 2 months, VEGF-C: 107%, VEGF-D: 105%. [Example]

[0281] Example 9: Stability of OPT-302 formulations A batch of a preferred aqueous formulation for OPT-302 was prepared containing the following components:

[0282] [Table 15]

[0283] Portions of the formulation batch were stored long term at -20±5°C and 5±3°C to determine the stability characteristics of the formulation. When stored at either -20°C or 5°C for up to 24 months, key stability assays, e.g., SE-UPLC, showed minimal changes in monomer content, decreasing from the initial amount by 0.8% at -20°C and 3% at 5°C, while the higher molecular weight (dimer) increased by 0.7% at -20°C and 2.2% at 5°C compared to the start of the stability study. Similarly, binding activity measured by ELISA was minimally reduced by 9% to 19% for VEGF-C and VEGF-D.

[0284] The formulation had excellent storage stability properties at -20°C and 5°C over the specified period. An additional batch of OPT-302, formulated as described for the above batch, was similarly subjected to 24 months of storage at 5±3°C and then analyzed by mass spectrometry to characterize the isoform variants. It was found that deamidated variants increased by 9% over the period, and isomerized variants decreased by 9%, while there was negligible change in oxidized variants. Thus, changes in charge variants were minimal overall over long-term (24 months) storage, indicating that molecular structure was preserved in the formulation.

Claims

1. an active agent which is a soluble VEGFR-3 capture molecule, present at a concentration ranging from 5 mg / mL to 250 mg / mL; trehalose, buffering agents, and water An aqueous pharmaceutical composition comprising: the pH is in the range of 6.5 to 8.0; An aqueous pharmaceutical composition comprising trehalose at a concentration of at least 7.0% w / v and / or free of added sodium chloride.

2. 10. The aqueous pharmaceutical composition of claim 1, which is free of added sodium chloride.

3. 3. The aqueous pharmaceutical composition according to claim 1, comprising trehalose at a concentration of at least 7.0% w / v.

4. 4. The aqueous pharmaceutical composition according to claim 1, wherein trehalose is present in a concentration of up to 20% w / v.

5. 4. The aqueous pharmaceutical composition of claim 3, wherein the trehalose is present at a concentration of 8.5% w / v to 15% w / v.

6. 6. The aqueous pharmaceutical composition of claim 5, comprising about 10.9% w / v trehalose.

7. 7. The aqueous pharmaceutical composition of any one of claims 1 to 6, wherein the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand-binding polypeptide comprising immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain.

8. 8. The aqueous pharmaceutical composition of claim 7, wherein the ligand-binding polypeptide comprises an amino acid sequence defined by amino acids 25 to 329 of SEQ ID NO:1, with the proviso that the positions of the polypeptide corresponding to positions 104 to 106 of SEQ ID NO:1 are not identical to N-X-S or N-X-T, and the ligand-binding polypeptide retains four N-glycosylation sequon sites corresponding to positions 33 to 35 of SEQ ID NO:1, positions 166 to 168 of SEQ ID NO:1, positions 251 to 253 of SEQ ID NO:1, and positions 299 to 301 of SEQ ID NO:1, and is glycosylated at the four N-glycosylation sequon sites.

9. 9. The aqueous pharmaceutical composition according to claim 7 or 8, wherein the immunoglobulin constant domain fragment comprises an amino acid sequence defined by amino acids 99 to 330 of SEQ ID NO:

2.

10. The aqueous pharmaceutical composition according to claim 8 or 9, wherein the soluble VEGFR-3-trapping molecule has an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 6, or an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 3, or an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 4, or an amino acid sequence defined by positions 1 to 546 of SEQ ID NO: 5, or an amino acid sequence defined by positions 1 to 546 of SEQ ID NO:

6.

11. 8. The aqueous pharmaceutical composition of claim 7, wherein the ligand-binding polypeptide comprises an amino acid sequence defined by amino acids 25 to 329 of SEQ ID NO:1, and the ligand-binding polypeptide retains five N-glycosylation sequon sites corresponding to amino acids 33 to 35 of SEQ ID NO:1, amino acids 104 to 106 of SEQ ID NO:1, amino acids 166 to 168 of SEQ ID NO:1, amino acids 251 to 253 of SEQ ID NO:1, and amino acids 299 to 301 of SEQ ID NO:1, and is glycosylated at the five N-glycosylation sequon sites.

12. The aqueous pharmaceutical composition according to claim 7 or claim 11, wherein the immunoglobulin constant domain fragment comprises an amino acid sequence defined by amino acids 99 to 330 of SEQ ID NO:

2.

13. The aqueous pharmaceutical composition according to claim 12, wherein the soluble VEGFR-3-trapping molecule has the amino acid sequence set forth in SEQ ID NO: 7 or the amino acid sequence defined by amino acids 1 to 547 of SEQ ID NO:

7.

14. 14. The aqueous pharmaceutical composition of claim 1, wherein the active substance is present in a concentration of up to 120 mg / mL.

15. 15. The aqueous pharmaceutical composition of claim 1, wherein the active agent is present at a concentration of about 40 mg / mL or about 80 mg / mL or about 120 mg / mL.

16. 16. The aqueous pharmaceutical composition according to any one of claims 1 to 15, wherein the pH is in the range of 7.2 to 7.

8.

17. 17. The aqueous pharmaceutical composition of claim 16, wherein the pH is about 7.

5.

18. 18. The aqueous pharmaceutical composition of claim 1, wherein the buffering agent is sodium phosphate.

19. 19. The aqueous pharmaceutical composition according to any one of claims 1 to 18, wherein the buffering agent is present at a concentration ranging from 5 mM to 100 mM.

20. 20. The aqueous pharmaceutical composition of claim 1, wherein the buffering agent is present at a concentration ranging up to 50 mM.

21. 21. The aqueous pharmaceutical composition of claim 20, wherein the buffering agent is present at a concentration of about 10 mM.

22. 22. The aqueous pharmaceutical composition according to any one of claims 1 to 21, comprising a surfactant.

23. 23. The aqueous pharmaceutical composition of claim 22, wherein the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

24. 24. The aqueous pharmaceutical composition of claim 23, wherein the surfactant is present at a concentration ranging from 0.005% to 0.2% w / v.

25. 25. The aqueous pharmaceutical composition of claim 24, wherein the surfactant is present at a concentration of about 0.01% w / v.

26. 26. The aqueous pharmaceutical composition according to any one of claims 1 to 25, having an osmolality in the range of 300 mOsm / kg to 1000 mOsm / kg.

27. 27. The aqueous pharmaceutical composition of claim 26, having an osmolality in the range of 350 mOsm / kg or greater.

28. 28. The aqueous pharmaceutical composition of claim 27, having an osmolality in the range of 400 mOsm / kg or greater.

29. 29. The aqueous pharmaceutical composition according to claim 28, having an osmolality in the range of 400 mOsm / kg to 600 mOsm / kg.

30. 30. The aqueous pharmaceutical composition of any one of claims 1 to 29, which is substantially free of sodium chloride.

31. 31. The aqueous pharmaceutical composition of any one of claims 1 to 30, which does not contain an additional sugar.

32. 32. The aqueous pharmaceutical composition of any one of claims 1 to 31, which does not contain an additional osmolality adjusting agent.

33. an active agent at a concentration of about 40 mg / ml, which is a soluble VEGFR-3 capture molecule comprising a ligand-binding polypeptide fused to an immunoglobulin constant domain fragment, said ligand-binding polypeptide comprising immunoglobulin-like domains 1-3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of said extracellular domain; trehalose at a concentration of about 10.9% w / v, sodium phosphate at a concentration of about 10 mM, Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v, and water 33. The aqueous pharmaceutical composition of claim 1, consisting essentially of and having a pH of about 7.

5.

34. an active agent which is a soluble VEGFR-3 capture molecule; trehalose, and buffer 1. A lyophilized pharmaceutical composition for reconstitution comprising:

35. 35. The freeze-dried pharmaceutical composition of claim 34, wherein the weight ratio of trehalose to active substance is in the range of 1:1 to 7.5:1, 1:1 to 5:1, or 2.1:1 to 4.5:

1.

36. 36. The freeze-dried pharmaceutical composition of claim 35, wherein the weight ratio of trehalose to active agent is about 2.7:

1.

37. 37. The lyophilized pharmaceutical composition of any one of claims 34 to 36, wherein the buffering agent is sodium phosphate.

38. 38. The freeze-dried pharmaceutical composition of claim 37, wherein the buffering agent is sodium phosphate and the weight ratio of sodium phosphate to active substance ranges from 1:3 to 1:1000, or from 1:3 to 1:200, or from 1:5 to 1:

100.

39. 39. The lyophilized pharmaceutical composition of claim 38, wherein the buffer is sodium phosphate and the weight ratio of sodium phosphate to active agent is about 0.03:

1.

40. 40. The lyophilized pharmaceutical composition of any one of claims 34 to 39, comprising a surfactant.

41. 41. The freeze-dried pharmaceutical composition of claim 40, wherein the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

42. 42. A reconstituted pharmaceutical composition obtainable by mixing the lyophilized pharmaceutical composition of any one of claims 34 to 41 with an aqueous diluent.

43. 43. The pharmaceutical composition of any one of claims 1 to 33 or 42, formulated for intravitreal injection.

44. 44. A method of inhibiting angiogenesis in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 1 to 33, 42 or 43.

45. 44. A method for treating and / or preventing a disease or disorder associated with abnormalities in angiogenesis, vasculogenesis, and / or lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition described in any one of claims 1 to 33, 42, or 43.

46. Use of a VEGF-C trapping molecule and / or a VEGF-D trapping molecule or a salt thereof for the manufacture of a pharmaceutical composition defined in any one of claims 1 to 33, 42 or 43 for the treatment and / or prevention of a disease or disorder associated with abnormalities in angiogenesis, vasculogenesis and / or lymphangiogenesis.

47. 44. A pharmaceutical composition according to any one of claims 1 to 33, 42 or 43 for use in the treatment and / or prevention of diseases or disorders associated with abnormalities in angiogenesis, vasculogenesis and / or lymphangiogenesis.

48. 48. The method of claim 45, the use of claim 38 or the pharmaceutical composition for use of claim 47, wherein the disease or disorder is an ocular disease or disorder.

49. 49. The method, use or pharmaceutical composition for use of claim 48, wherein the ocular disease or disorder is selected from the group consisting of macular degeneration, diabetic retinopathy, macular edema, retinal vein occlusion, and macular telangiectasia.

50. 50. The method, use or pharmaceutical composition for use of claim 49, wherein the ocular disease or disorder is wet age-related macular degeneration.

51. 50. The method, use or pharmaceutical composition for use of claim 49, wherein the ocular disease or disorder is diabetic retinopathy.

52. 52. The method, use or pharmaceutical composition for use according to any one of claims 44 to 51, wherein the pharmaceutical composition is administered in combination with a further active substance.

53. 53. The method, use or pharmaceutical composition for use of any one of claims 44 to 52, wherein the further active agent is an anti-VEGF-A agent or an anti-VEGF-B agent.

54. 54. The method, use or pharmaceutical composition for use of claim 53, wherein the further active agent is selected from the group consisting of ranibizumab, aflibercept, bevacizumab, and brolucizumab.

55. 55. The method, use or pharmaceutical composition for use of any one of claims 44 to 54, wherein the pharmaceutical composition is administered intravitreally.

56. 56. The method, use or pharmaceutical composition for use of any one of claims 44 to 55, wherein the pharmaceutical composition is administered using a port device implanted in the eye, the port device containing a reservoir for the pharmaceutical composition and allowing controlled release of the active substance into the vitreous of the eye.

57. 44. A port device for intraocular implantation comprising a reservoir containing the pharmaceutical composition of any one of claims 1 to 33, 42 or 43, said port device allowing for controlled release of an active substance into the vitreous of the eye.

58. 58. The port device of claim 57, comprising a semipermeable membrane that allows passive diffusion of an active agent into the vitreous of the eye.

59. 59. The port device of claim 57 or 58, comprising a septum that allows the reservoir to be replenished with additional pharmaceutical composition using a needle.

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