OA22158A - Pharmaceutical composition and method of using same - Google Patents

Abstract

Provided herein are pharmaceutical compositions comprising an active agent which is a soluble VEGFR-3 trap molecule. Also provided herein are therapeutic methods and uses involving the pharmaceutical compositions, in particular ocular diseases and disorders, and port devices comprising the pharrnaceutical compositions.

Description

Pharmaceutical Composition and Method of Using Same

This patent application claims priority from US provisional patent application no. 63/374,366 fiied on l September 2022, the entire contents of which is incorporated herein by this reference.

Field

The present disclosure relates to pharmaceutical compositions comprising an active agent which is a soluble VEGFR-3 trap molécule. The disclosure also relates to therapeutic methods and uses involving the pharmaceutical compositions, in particular ocular diseases and disorders, and to port devices comprising the pharmaceutical compositions.

Background

Loss or détérioration of eyesight is an extremely debilitating condition that can hâve profound effects on an individual’s quality of life. Age-related maculardegeneration (AMD) is the leading cause of severe vision impairment in older people. The “wet” form of the disorder develops when abnormal blood vessels grow into the macular, which leak blood or fluid that leads to scarring of the macula and loss of vision. A further severe ocular disorder associated with leaking blood vessels is diabetic macular edema (DME), in which leaking fluid accumulâtes in the macula arising from damage to blood vessels in individuals having diabetic retinopathy.

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

Vascular endothélial growth factor (VEGF) proteins and their receptors play important rôles in both vasculogenesis, the development of the embryonic vasculature from early differentiating endothélial cells, angiogenesis, the process of forming new blood vessels from pre-existing ones, and lymphangiogenesis, the process of forming new lymph vessels. Dysfunction of the endothélial cell regulatory system is also a key feature of cancer and a number of other diseases associated with abnormal vasculogenesis, angiogenesis and lymphangiogenesis.

Thérapies directed to blockade of VEGF/PDGF signalling through their receptors hâve been approved for the therapy of eye conditions including AMD and DME, as well as for cancers. For example, aflibercept, referred to above, is an inhibitor of VEGF consisting of portions of human VEGF receptor l and 2 extracellular domains fused to the Fc portion 5 of human IgGl. It acts by binding to circulating VEGF-A and VEGF-B as well as placental growth factor (PIGF), which normally bind to VEGFR-1 and VEGFR-2, and thus is a VEGFR-l/VEGFR-2 trap molécule.

A further therapy in development for the treatment of eye conditions is OPT-302, a VEGFR-3 trap molécule containing portions of human VEGF receptor 3 extracellular 10 domain which is soluble in fluids such as blood and plasma, and binds circulating VEGF proteins that normally being bind to VEGFR-3, i.e. 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 phase 3 clinical trials for wet-AMD are ongoing. Soluble VEGFR-3 trap molécules such as OPT-302 are described in, for example, 15 WO2014/124487 and WO2015/123715, the entire contents of which are incorporated herein by reference.

However, drug discovery and development is a lengthy and complex process, and following identification of the therapeutic agent, there can often be significant obstacles to bringing the medicine to market and gaining approval to treat patients. For example, there 20 can be significant challenges in developing pharmaceutical formulations of active agents which provide the required properties, e.g. safety, having acceptable stability of the active, acceptable stability of the formulation, retaining sufficient activity over time, being convenient to administer and avoiding administration site reactions. For some therapeutic agents, despite efforts to identify a suitable formulation, spécial storage conditions may be 25 required to achieve an acceptable lifetime of the formulation, such as low or ultra-low température conditions.

There remains a need for further pharmaceutical products to treat conditions such as wet-AMD and DME. There also remains a need for formulations of soluble VEGFR-3 trap molécules such as OPT-302 which provide good properties, for example in relation to 30 storage stability.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

Summary of the Disclosure

OPT-302, a soluble VEGFR-3 trap molécule, has a tendency to form dimers when présent in some aqueous formulations, associated with a loss of purity and binding activity. For example, an aqueous composition of OPT-302 which was developed was found to form high levels of OPT-302 dimers rapidly, and needs to be stored at very low températures, e.g. -20°C, to achieve an acceptable lifetime.

Research has now identified a pharmaceutical formulation of OPT-302 which provides unexpectedly good stability properties, and does not require storage at -20°C in order to achieve an acceptable product lifetime.

Accordingly, in a first aspect, there is provided an aqueous pharmaceutical composition, comprising:

an active agent which is a soluble VEGFR-3 trap molécule, the active agent being présent at a concentration in the range of from 5 mg/mL to 250 mg/mL;

trehalose;

a buffer; and water;

wherein the pH ofthe aqueous pharmaceutical composition is in the range of from 6.5 to 8.0;

and wherein the pharmaceutical composition comprises trehalose in a concentration of at least 7.0% w/v and/or the pharmaceutical composition does not contain added sodium chloride.

In some embodiments, the pharmaceutical composition does not contain added sodium chloride.

In some embodiments, the pharmaceutical composition comprises trehalose in a concentration of at least 7.0% w/v.

In some embodiments, trehalose is présent at a concentration of up to 20% w/v. In some embodiments, trehalose is présent at a concentration of from 8.5% w/v to 15% w/v. In some embodiments, the composition comprises about 10.9% w/v trehalose.

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand binding polypeptide comprising immunoglobulin-like domains 1-3 ofthe extracellular domain of human VEGFR-3 and optionally having one or more modifications in an N-glycan région of the extracellular domain.

In some embodiments, the ligand binding polypeptide comprises the amino acid sequence defîned by positions 25-329 of SEQ ID NO: l, with the proviso that positions of 5 the polypeptide corresponding to positions 104-106 of SEQ ID NO: I are not identical to NX-S or N-X-T; wherein the ligand binding polypeptide retains four N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO: l, positions 166-168 of SEQ ID NO: l, positions 251-253 of SEQ ID NO: 1, and positions 299-301 of SEQ ID NO: 1, and is glycosylated at said four N-glycosylation sequon sites.

In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defîned by positions 99-330 of SEQ ID NO: 2.

In some embodiments, the soluble VEGFR-3 trap molécule has the amino acid sequence set forth in any of SEQ ID NOs: 3-6, or has an amino acid sequence as defîned by positions 1-536 of SEQ ID NO: 3, or has an amino acid sequence as defîned by positions 115 536 of SEQ ID NO: 4, or has an amino acid sequence as defîned by positions I -546 of SEQ

ID NO: 5, or has an amino acid sequence as defîned by positions 1 -546 of SEQ ID NO: 6.

In some embodiments, the ligand binding polypeptide comprises the amino acid sequence defîned by positions 25-329 of SEQ ID NO: 1; wherein the ligand binding polypeptide retains five N-glycosylation sequon sites corresponding to positions 33-35 of 20 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 said five N-glycosylation sequon sites.

In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defîned by positions 99-330 of SEQ ID NO: 2.

In some embodiments, the soluble VEGFR-3 trap molécule has the amino acid sequence set forth in SEQ ID NO: 7, or has an amino acid sequence as defîned by positions 1-547 ofSEQ IDNO:7.

In some embodiments, the active agent is présent at a concentration of up to 120 mg/mL. In some embodiments, the active agent is présent at a concentration of about 40 30 mg/mL, or about 80 mg/mL, or about 120 mg/mL.

In some embodiments the pH of the composition is in the range of from 7.2 to 7.8.

In some embodiments, the pH of the composition is about 7.5.

In some embodiments, the buffer is a sodium phosphate. In some embodiments, the buffer is présent in a concentration in the range of from 5 mM to 100 mM. In some embodiments, the buffer is présent in a concentration in the range of up to 50 mM. In some embodiments, the buffer is présent in a concentration of about 10 mM.

In some embodiments, the composition comprises a surfactant. In some embodiments, the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate. In some embodiments, the surfactant is présent at a concentration in the range of from 0.005% to 0.2% w/v. In some embodiments, the surfactant is présent at a concentration of about 0.01% w/v.

In some embodiments, the composition has an osmolality in the range of from 300 mOsm/kg to 1000 mOsm/kg. In some embodiments, the composition has an osmolality in the range of from 350 mOsm/kg. In some embodiments, the composition has an osmolality in the range of from 400 mOsm/kg. In some embodiments, the composition has an osmolality in the range of from 400 mOsm/kg to 600 mOsm/kg.

In some embodiments, the composition is substantially free of sodium chloride.

In some embodiments, the composition does not contain an additional sugar.

In some embodiments, the composition does not contain an additional tonicity modifier.

In some embodiments, the composition essentially consists of an active agent in a concentration of about 40 mg/ml, which is a soluble VEGFR-3 trap molécule that 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 an N-glycan région of the extracellular domain;

trehalose in a concentration of about 10.9% w/v;

sodium phosphate in a concentration of about 10 mM;

polyoxyethylene (20) sorbitan monolaurate in a concentration of about 0.01% w/v; and water;

wherein the pH of the aqueous pharmaceutical composition is about 7.5.

In a further aspect, there is provided a lyophilised pharmaceutical composition for reconstitution, comprising:

an active agent which is a soluble VEGFR-3 trap molécule;

trehalose; and a buffer;

wherein the weight ratio of trehaiose to active agent is in the range of from l :3 to 40:1.

In some embodiments, the weight ratio of trehaiose to active agent is in the range of from I : I to 7.5:1, from I :l to 5:1, or from 2.1:1 to 4.5:1. In some embodiments, the weight 5 ratio of trehaiose to active agent is about 2.7:1.

In some embodiments, the buffer is a sodium phosphate. In some embodiments, the buffer is a sodium phosphate, and the weight ratio of sodium phosphate to active agent is in the range of from 1:3 to 1:1000, or from 1:3 to 1:200, or from 1:5 to 1:100. In some embodiments, the buffer is a sodium phosphate, and the weight ratio of sodium phosphate 10 to active agent is about 0.03:1.

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

In a further aspect, there is also provided a reconstituted pharmaceuticai composition, 15 wherein the pharmaceuticai composition is obtained by admixing a lyophilised pharmaceuticai composition as defined herein with an aqueous diluent.

In some embodiments of the aspects defined above, the pharmaceuticai composition is formulated for intravitreal injection.

In a further aspect, there is provided a method of inhibiting neovascularisation in a 20 subject, comprising administering to the subject an effective amount of a pharmaceuticai composition as defined herein.

in a further aspect there is provided a method of treating and/or preventing a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesis in a subject, comprising administering to the subject an effective amount 25 of a pharmaceuticai composition as defined herein.

In a further aspect there is provided use of a VEGF-C and/or VEGF-D trap molécule or a sait thereof for the manufacture of a pharmaceuticai composition as defined herein, for the treatment and/or prévention of a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesis.

In a further aspect there is provided a pharmaceuticai composition as defined herein, for use in the treatment and/or prévention of a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesis.

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 agerelated 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 a further active agent. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the further active agent is an anti-VEGF-A agent or an anti-VEGF-B agent. In some embodiments of the methods, uses, and pharmaceutical compositions for use, the further active agent is selected from the group consisting of ranibizumab, aflibercept, bevacizumab and brolucizumab.

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 which is implanted in an eye, which port device comprises a réservoir for the pharmaceutical composition, and permits controlled release of active agent into the vitreous of the eye.

In a further aspect, there is also provided a port device for implantation in an eye, the port device comprising a réservoir containing a pharmaceutical composition as defined herein, and wherein the port device permits controlled release of active agent into the vitreous of the eye.

In some embodiments, the port device comprises a semipermeable membrane that permits passive diffusion of active agent into the vitreous of the eye.

In some embodiments, the port device comprises a septum which permits refilling of the réservoir with additional pharmaceutical composition using a needle.

Brief Description of the Drawings

Figure l shows a chart showing the results of stability studies for OPT-302 compositions according to the présent disclosure and comparative compositions. The % level formation of high molecular weight species of active agent over time was determined for compositions at 37°C.

Figure 2 shows a chart showing the results of stability studies for OPT-302 compositions according to the présent disclosure. The % level formation of monomer of active agent over time was determined for compositions at 25°C.

Figure 3 shows a chart showing the results of stability studies for OPT-302 compositions according to the présent disclosure. The % level formation of monomer of active agent over time was determined for compositions at 5°C.

Detailed Description

Définitions

Throughoutthis spécification the word comprise, or variations such as comprises or comprising, will be understood to imply the inclusion of a stated element. integer or step, or group of éléments, integers or steps, but not the exclusion of any other element, integer or step, or group of éléments, integers or steps. unless otherwise required by context.

As used herein, the term “and/or”, e.g. “X and/or Y”, shall be understood to mean either or both of X and Y and X or Y and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, of the designated value.

As used herein, the terms “a”, “an” and “the” include both singular and plural aspects, unless the context clearly indicates otherwise.

As used herein, the phrase “at least one of, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed.

As used herein, the term “subject” refers to any organism that is susceptible to a disease or condition. For example, the subject can be an animal, a mammal, a primate, a livestock animal (e.g., sheep, cow, horse, pig), a companion animal (e.g., dog. cat), or a laboratory animal (e.g., tnouse, rabbit, rat, guinea pig. hamster). In some embodiments, the

subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject is a non-human animal.

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, S preventing or slowing progression of a disease or disorder, and alleviation of symptoms associated with a disease or disorder.

As used herein, the terms “preventing” and “prévention” 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 prophylaxis of a disorder or condition.

The term “therapeutically effective amount”, as used herein, refers to a pharmaceutical composition comprising a soluble VEGFR-3 trap molécule being administered in an amount sufficient to treat or prevent the disorder or condition being treated.

The term identity, as known in the art, refers to a relationship between the 15 sequences of two or more polypeptide molécules or two or more nucleic acid molécules, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness of nucleic acid molécules or polypeptides sequences, as the case may be, as determined by the match between strings of two or more nucléotide or two or more amino acid sequences. Identity measures the percent of identical matches between the 20 smaller of two or more sequences with gap alignments (if any) addressed by particular a mathematical model of computer program (i.e., algorithms). Appropriate algorithms for determining the percent identities of the présent disclosure include BLASTP and BLASTN, using the most common and accepted default parameters.

The term component domain as used herein to refers to a domain within a ligand 25 binding molécule which is derived from or based on a protein domain within the extracellular portion of a receptor protein. For example, each Ig-domain of VEGFR-3 (D1-D7) constitues a component domain. Reference herein to a component domain includes both the complété native wildtype domain and also insertions, délétions and/or substitutional variants thereof which substantially retain the functional characteristics of the intact domain. It will be 30 readily apparent to one of ski 11 in the art that numerous variants of the above domains (e.g. Ig-domains) can be obtained which will retain substantially the same functional characteristics as the wild-type domain.

ΙΟ

Active Agent

The pharmaceutical compositions of the présent disclosure contain an active agent, which is a soluble VEGFR-3 trap molécule.

Vascular endothélial growth factor receptor 3 (VEGFR-3; previously known as Flt4) 5 is the receptor for VEGF-C and VEGF-D ligands and is found primarily on vascular and lymphatic endothélial cells. It is primarily involved in angiogenesis and iymphangiogenesis. Soluble VEGFR-3 trap molécules find utility in the treatment of disease and disorders associated with neovascularization and/or vascular permeability, aberrant angiogenesis and/or Iymphangiogenesis, such as a number of ocular disorders including wet age-related 10 macular degeneration and diabetic macular edema. They also hâve application in other disease indications associated with aberrant angiogenesis and/or Iymphangiogenesis (e.g. cancer).

The term “soluble” as used herein in relation to the active agent, means that the active agent has high enough solubility in biological fluid e.g. blood, plasma, and/or vitreous 15 humour, such that it is available for binding circulating VEGF-C. In some embodiments, the soluble VEGFR-3 trap molécule has a solubility in plasma of at least 1 mg/mL, or at least 2 mg/mL, or at least 5mg/mL, or at least lOmg/mL, or at least 20 mg/mL, or at least 30 mg/mL, or at least 40 mg/mL.

The active agent is a VEGFR-3 trap molécule. As referred to herein, a VEGFR-3 20 trap molécule is a molécule which is capable of binding to circulating VEGF-C and/or VEGF-D. In some embodiments, the soluble VEGFR-3 trap molécule binds human VEGFC with a KD of about InM 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 trap molécule binds 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 25 pM, 100 pM, 50 pM, 10 pM or less).

Binding affinity to VEGF-C and VEGF-D may be determined by any suitable assay. For example binding affinity may be determined using ELI SA, or using surface plasmon résonance. Such techniques are described in, for example, WO 2014/124487 Al, the entire contents of which is incorporated herein by référencé.

In some embodiments, the soluble VEGFR-3 trap molécule is or comprises a polypeptide.

In some embodiments. the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide. For example, the ligand binding polypeptide may be or may comprise

a fragment of a growth receptor tyrosine kinase extracellular domain (ECD). The fragment may in some embodiments vary from the wildtype sequence in ways that do not eliminate growth factor binding, and the fragment preferably is engineered in ways described herein to improve its properties as a therapeutic for administration to subjects/patients in need.

VEGF-C and D bind with high affinity to, and stimulate phosphorylation of, at least one VEGF receptor (or receptor heterodimer) selected from VEGFR-2 and VEGFR-3. Preferred ligand binding polypeptides do more than simply bind their target growth factors. A preferred ligand binding polypeptide also inhibits the growth factor(s) to which it binds from stimulating phosphorylation of at least one (and preferably ail) ofthe receptor tyrosine kinases to which the growth factor(s) bind. Stimulation of tyrosine phosphorylation is readily measured using in vitro cell-based assays and antibodies.

Ligand binding polypeptides that are spécifie for a particular growth factor are ligand binding molécules that speciflcally recognize an active form of the growth factor (e.g., a form found circulating in the body). Preferably, the ligand binding polypeptide specifically binds other forms of the growth factors as well. By way of example, VEGF-C (and VEGFD) is translated as a prepro-molecule with extensive amino-terminal and carboxy-terminal propeptides that are cleaved to yield a fully processed form of VEGF-C (or VEGF-D) that binds and stimulâtes VEGFR-2 and VEGFR-3. Ligand binding polypeptides spécifie for VEGF-C (or VEGF-D) bind to at least the fully processed form of VEGF-C (or VEGF-D), 20 and preferably also bind to partially processed forms and unprocessed forms.

SEQ ID NO: 1 contains an arnino acid sequence for human VEGFR-3, with positions 1-24 of SEQ ID NO: 1 corresponding to a putative signa! peptide and position 25 onwards of SEQ ID NO: l corresponding to a putative mature form of the receptor lacking a putative signal peptide.

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

The complété ECD of VEGFRs is not required for ligand (growth factor) binding.

The ECD of VEGFR-3 has six intact Ig-like domains and one cleaved Ig-like domain - D5 of VEGFR-3 is cleaved post-translationally into disulfïde linked subunits leaving VEGFR3 (Veikkola, T., et al., Cancer Res. 60:203-212 (2000)). In some embodiments, receptor fragments comprising at least the first three Ig-like domains for this family are sufficient to bind ligand. Soluble receptors capable of binding VEGF-C and VEGF-D, thereby inhibiting VEGF-C or VEGF-D activity or signaling via VEGFR-3, are also disclosed in W02000/023565, W02000/021560, W02002/060950 and W02005/087808, the S disclosures of which are incorporated herein by reference in their entireties. Those soluble receptors, optionally with modifications described herein, are contemplated as soluble VEGFR-3 trap molécules of the présent disclosure.

The table below defines approximate boundaries of the Ig-like domains for human VEGFR-3. These boundaries are significant as the boundaries chosen can be used to form 10 ligand binding molécules, and so can influence the binding properties of the resulting constructs.

	VEGFR-3 SEQ 1DNO: 1 positions
DI	47-115
D2	154-210
D3	248-314
D4	351-403
D5	441-538
D6	574-657
D7	695-752

In some embodiments, the ligand binding polypeptide comprises a portion of the amino acid sequence of at least one of immunoglobulin-like domains 1, 2 and 3 of the ECD 15 of human VEGFR-3.

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

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand 20 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 1D NO: 1 or positions 25-1 15 of SEQ 1D NO: 1. The foregoing segments of SEQ ID NO: 1 roughly correspond to or include the first immunoglobulin-like domain of the extracellular domain (ECD) of huinan VEGFR-3 (DI of VEGFR-3).

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide which is a purified or isolated ligand binding polypeptide comprising 5 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- l 15 of SEQ ID NO: l or positions 25115 of SEQ ID NO: l, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: l are not identical to N-X-S or N-X-T (X representing 10 any amino acid).

Ig-like domains 1-3 of VEGFR-3 comprises five putative N-glycosylation sites (referred to herein as NI, N2, N3, N4 and N5 sequons of VEGFR-3, respectively). NI corresponds to amino acids 33-35 of SEQ ID NO: 1; N2 corresponds to amino acids 104106 of SEQ ID NO: 1; N3 corresponds to amino acids 166-168 of SEQ ID NO: 1; N4 15 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, a ligand binding molécule described herein comprises a modification in the N2 sequon of the molécule.

In some embodiments, said putative glycosylation sequon at positions 104-106 is eliminated from the amino acid sequence of the ligand binding polypeptide. The term 20 “eliminated” as used in this context means an alteration of the primary amino acid sequence in at least one position (by substitution, délétion or insertion) to destroy the N-X-T 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 (such as glutamine, aspartate, glutamate, arginine and lysine).

For example, in some embodiments, the amino acid in the ligand binding molécule 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 ofSEQ 1DNO: 1 is deleted and replaced with an amino acid selected from the group consisting of glutamine, aspartate, glutamate, arginine and lysine.

In embodiments where the N2 sequon ofSEQ ID NO: 1 is modified as described above, the NI, N3, N4 and N5 sequons ofSEQ ID NO: 1 are preferably unaltered in terms of amino acid sequence.

In some embodiments, the soluble VEGFR-3 trap molécule 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% 5 identical to the sequence of amino acids defined by positions 154-210 of SEQ ID NO: l. The sequence of amino acids defined by positions of the polypeptide corresponding to positions 154-210 roughly corresponds to or includes the second immunoglobuiin-like domain of the ECD of human VEGFR-3 (“D2 of VEGFR-3”).

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand 10 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% identical to the sequence of amino acids defined by positions 248-314 of SEQ ID NO:1. The sequence of amino acids defined by positions of the polypeptide corresponding to positions 15 248-314 roughly corresponds to or includes the third immunoglobulin-like domain of the

ECD of human VEGFR-3 (“D3 of VEGFR-3”).

In some embodiments, the ligand binding polypeptide comprises substantially ail or ail of the amino acid sequence of immunoglobulin-like domains 1 and 2 of the ECD of human VEGFR-3. In some embodiments, the ligand binding polypeptide comprises 20 substantially ail or ail of the amino acid sequence of immunoglobulin-like domains 2 and 3 of the ECD of human VEGFR-3. In some embodiments, the ligand binding polypeptide comprises substantially ail or ail of the amino acid sequence of immunoglobulin-like domains 1, 2 and 3 of the ECD of human VEGFR-3.

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand 25 binding polypeptide comprising immunoglobulin-like domains 1-3 of the extracellular domain of human VEGFR-3 and optionally having one or more modifications in an N-glycan région ofthe extraceliular domain.

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide which is a purified or isolated ligand binding polypeptide sequence 30 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% identical, or which is identical to the sequence of amino acids defined by positions 25-329 ofSEQ 1DNO:1.

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide comprising the amino acid sequence defined by positions 25-329 of SEQ ID NO: l; wherein the ligand binding polypeptide retains five N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO: l, positions 104-106 of SEQ ID NO: l, positions 166-168 of SEQ ID NO: l, positions 251 -253 of SEQ ID NO: l, and positions 299-301 of SEQ ID NO: 1, and is glycosylated at said flve N-glycosylation sequon sites.

In some embodiments, the soluble VEGFR-3 trap molécule 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% identical, or which is identical to the sequence of amino acids defined by positions 25-329 of SEQ ID NO:1, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 1 are not identical to N-X-S or N-X-T (X representing any amino acid).

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide comprising the amino acid sequence defined by positions 25-329 of SEQ ID NO: 1, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 1 are not identical to N-X-S or N-X-T; wherein the ligand binding polypeptide retains four N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO: 1, positions 166-168 ofSEQ ID NO: 1, positions 251-253 of SEQ ID NO: 1, and positions 299-301 ofSEQ ID NO: 1, and is glycosylated at said four N-glycosylation sequon sites.

In some embodiments, said putative glycosylation sequon at positions 104-106 is eliminated from the amino acid sequence of the ligand binding polypeptide. The term “eliminated” as used in this context means an alteration of the primary amino acid sequence in at least one position (by substitution, délétion or insertion) to destroy the N-X-T sequon motif. In one variation, the amino acid corresponding to position 104 ofSEQ ID NO: 1 may be deleted and replaced with another amino acid (such as glutamine, aspartate, glutamate, arginine and lysine).

Constructs that comprise additional Ig-like domains of VEGFR-3, attached in a manner that resuit in a ligand binding polypeptide, are specifically contemplated. For example, the soluble VEGFR-3 trap molécule may contain portions of, or substantially ail or ail of, seven Ig-like domains of VEGFR-3.

In embodîments where the ligand binding polypeptide comprises amino acid sequences roughly corresponding to two or more component domains of VEGFR-3, the component domains may be connected directly to each other or may be connected via one or more spacers. Preferably, the component domains are connected by one or more spacers.

For example, the ligand binding polypeptide may optionally include sequence before the most N-terminally positioned Ig-like domain, between the Ig-like domains, and/or after the most C-terminally positioned Ig-like domain.

In one embodiment, the spacer comprises one or more peptide sequences between the component domains which is (are) between 1-100 amino acids, preferably l-50 amino acids in length. In one embodiment, the spacer between two component domains substantially consists of peptide sequences naturelly connected to the component domain in native VEGFR-3.

In embodîments where the ligand binding polypeptide comprises amino acid sequences roughly corresponding to or including contiguous component domains of VEGFR-3 (for example, D1-D2 or D1-D2-D3), the component domains may be connected via one or more spacers comprising one or more peptide sequences between the component domains which is (are) between I-IOO amino acids, preferably l -50 amino acids in length.

In some embodîments, the spacer between two component domains substantially consists of peptide sequences corresponding to those connecting the respective contiguous component domains in the native VEGFR-3. In some embodîments, the spacer between two contiguous component domains comprises an amino acid sequence 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, or which is identical, to the sequence of amino acids that connects the contiguous domains in the native VEGFR-3.

In embodîments where the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide comprises multiple component domains, for example component domains Dl, D2 and D3 of VEGFR-3, the component domains may be connected directly to each other or may be connected via one or more spacers. Generally, the term “spacer” means one or more molécules, for example nucleic acids or amino acids, or non-peptide moieties, such as polyethylene glycol or disulfide bridges, which may be inserted between one or more component domains forming a covalent bond. Spacer sequences may be used to provide a désirable site of interest between components for ease of manipulation. A spacer may also be provided to enhance expression of the ligand binding polypeptide from a host cell, to decrease steric hindrance such that the component or group of components may assume its/their optimal tertiary structure and/or interact appropriately with its/their target molécule. For spacers and methods of identifying désirable spacers, see, for example, George et al. (2003) Protein Engineering 15:871 -879, herein specifically incorporated by reference. A spacer sequence may include one or more amino acids naturally connected to a receptor component. or may be an added sequence used to enhance expression of the ligand binding polypeptides, provide specifically desired sites of interest, allow component domains to form optimal tertiary structures and/or to enhance the interaction of a component or group of components with its/their target molécule. In one embodiment, the spacer comprises one or more peptide sequences between one or more components which is (are) between I- 100 amino acids, preferably 1-50 amino acids in length. In a preferred embodiment, the spacer between two component domains substantially consists of amino acids naturally connected to the receptor component in the wildtype receptor. In the case of a ligand binding polypeptide comprising multiple component domains from the same receptor which domains are adjacent each other in the native receptor, such as for example Dl, D2 and D3 of VEGFR-3, in one embodiment, the domains are connected to each other (e.g. Dl to D2 and D2 to D3) using spacers corresponding to the naturaliy-occurring amino acid linking sequences.

In some variations, each ligand binding polypeptide is expressed as a fusion with a fusion partner protein, such as an immunoglobulin constant région, and the heterologous fusion partners are linked to form the ligand binding molécule.

In some embodiments, the ligand binding molécule is a polypeptide that comprises a portion of a human VEGFR-3 ECD, wherein the portion binds to one or both of human VEGF-C and human VEGF-D, and comprises at least the first, second and third Ig-like domains of the VEGFR-3 ECD, wherein the amino acid sequence of the ECD fragment of VEGFR-3 is modified from wildtype VEGFR-3 to eliminate the second putative N-linked glycosylation sequon of wildtype VEGFR-3, and wherein the polypeptide lacks VEGFR-3 Ig-like domains 4-7 and preferably any transmembrane domain and preferably any intracellular domain.

Alongside monomeric constructs, the présent disclosure also includes multimeric ligand binding constructs comprising two or more ligand binding molécules as described herein, covalently or non-covalently attached to each other to form a dimeric or multimeric structure. In some variations, the attachaient occurs between the VEGFR.-3-like sequences of the ligand binding polypeptides; in other variations, the attachment occurs between heterologous polypeptides attached to one or both of the VEGFR-3 like sequences.

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 avariant.

VEGFR-3, from which ligand binding polypeptides may be derived, include splice variants and naturally-occurring allelic variations. Allelic variants are well known in the art, and represent alternative forms or a nucleic acid sequence that comprise substitution, délétion or addition of one or more nucléotides, but which do not resuit in any substantial functional alteration of the encoded polypeptide. Exemplary allelic variants of VEGFR-3 hâve been reported in the literature, e.g., at http://www.uniprot.org/uniprot/P359l6, and include positions 149, 378, 494, 527, and 641 within the ECD. Standard methods can readily be used to generate such polypeptides including site-directed mutagenesis of polynucleotides, or spécifie enzymatic cleavage and ligation. Similarly, use of peptidomimetic compounds or compounds in which one or more amino acid residues are replaced by a non-naturally-occurring amino acid or an amino acid analog that retain binding activity is contemplated.

Preferably, where amino acid substitution is used, the substitution is conservative, i.e. an amino acid is replaced by one of similar size and with similar charge properties. As used herein, the term “conservative substitution” dénotés the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobie residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norîeucine or méthionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine. The term “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted amino acid.

Alternat!vely, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY, pp. 71-77 ( 1975)) as setout in the following:

Non-polar (hydrophobie)

A. Aliphatic: A, L, l, V, P,

B. Aromatic: F, W,

C. Sulfur-containing: 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.

Negatively Charged (Acidic): D, E.

The soluble VEGFR-3 trap molécule may for example contain a fusion partner (such as, for example, a heterologous peptide), e.g. to impart desired characteristics (such as, for example, increase the sérum half-life, increase the solubility in an aqueous medium and/or 20 enable targeting to a spécifie cell population, e.g., tumor cells or retinal cells).

In some embodiments, the fusion partner is any heterologous component that enhances the functionality of the ligand binding polypeptide. Thus, for example, a fusion partner may increase the solubility, modulate the clearance, facilitate targeting of particular cell or tissue types, enhance the biological activity, aid the production and/or recovery, 25 enhance a pharmacological property or enhance a pharmacokinetic (PK) profile of the ligand binding polypeptide. With regards to enhancing the PK profile, this may be achieved by, for example, enhancing the sérum half-life, tissue penetrability, lack of immunogenicity or stability of the ligand binding molécule. In some embodiments, a fusion partner is selected from the group consisting of a multimerizing component, a sérum protein or a molécule 30 capable of binding a sérum protein.

In some embodiments, the fusion component comprises an immunoglobulin-derived domain from, for example, human IgG, 1g or IgA.

In some variations, the soluble VEGFR-3 trap molécule comprises an immunoglobulin constant domain or fragment thereof. In some embodiments, the soluble VEGFR-3 trap molécule comprises a human immunoglobulin G domain or fragment thereof. The amino acid sequence of human immunoglobulin l heavy chain constant domain is set 5 out in SEQ ID NO: 2. In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defïned by positions 99-330 of SEQ ID NO: 2.

In some embodiments, the soluble VEGFR-3 trap molécule comprises an immunoglobulin constant domain fragment which 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 10 96%, or at least 97%, or at least 98%, or at least 99% identical, or which is identical to the sequence of amino acids defïned by positions 99-330 of SEQ ID NO:2.

In some embodiments, the immunoglobulin constant domain fragment may hâve a C-terminal amino acid residue omitted. For example, in some embodiments, the immunoglobulin constant domain fragment has the amino acid sequence defïned by 15 positions 99-329 of SEQ ID NO: 2.

The amino acid sequence derived from the immunoglobulin may be linked to the Cterminus or to the N-terminus of the ligand binding polypeptide, preferably to the Cterminus. Cells transfected with DNA encoding the immunoglobulin light chain fusion protein and the immunoglobulin heavy chain fusion protein express heavy chain/lîght chain 20 heterodimers containing each a ligand binding polypeptide. Both ligand binding polypeptides advantageously comprise a native or heterologous signal peptide when inïtially synthesized, to promote sécrétion from the cell, but the signal sequence ma for example be cleaved upon sécrétion. Variations of any of the foregoing embodiments that include the signal peptide are contemplated. The native signal peptide of human VEGFR-3 comprises 25 residues 1-24 of SEQ ID NO: 1. Numerous other signal peptide proteins are taught in the 1 iterature.

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide which is fused (as a single polypeptide chain) to the Fc portion of human immunoglobulin G (IgG).

In some embodiments, the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide which is fused (as a single polypeptide chain) to the Fc portion of human immunoglobulin G (IgG), in which there is a single amino acid substitution in the second 1glike domain to reinove an N-glycosylation site.

In some embodiments, a ligand binding polypeptide as described herein optionally comprises a linker connecting the fusion partner, such as. for example, a heterologous peptide to the ligand binding polypeptide, such as the factor Xa linker sequence DPIEGRGGGGG (SEQ ID NO: 8). In other embodiments, the ligand binding molécule comprises a polypeptide in which a C-terminal amino acid of the ligand binding polypeptide is directly attached to an 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 attached (directly or through a linker polypeptide) by amide bonding to form a single polypeptide chain.

In some embodiments, the soluble VEGFR-3 trap molécule is OPT-302. OPT-302 has the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the soluble VEGFR-3 trap molécule comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the soluble VEGFR-3 trap molécule has an amino acid sequence consisting of the amino acid sequence set forth in SEQ 15 ID NO: 3. In some embodiments, the soluble VEGFR-3 trap molécule has an amino acid sequence deftned by positions 1-536 of SEQ ID NO: 3.

In some embodiments, the soluble VEGFR-3 trap molécule comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the soluble VEGFR-3 trap molécule has an amino acid sequence consisting ofthe amino acid sequence set forth in SEQ 20 ID NO: 4. In some embodiments, the soluble VEGFR-3 trap molécule has an amino acid sequence deftned by positions 1-536 of SEQ ID NO: 4.

In some embodiments, the soluble VEGFR-3 trap molécule comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the soluble VEGFR-3 trap molécule 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 trap molécule has an amino acid sequence deftned by positions 1 -546 of SEQ ID NO: 5.

In some embodiments, the soluble VEGFR-3 trap molécule comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the soluble VEGFR-3 trap molécule 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 trap molécule has an amino acid sequence deftned by positions 1-546 of SEQ ID NO: 6.

In some embodiments, the soluble VEGFR-3 trap molécule is VGX-300. VGX-300 has the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the soluble

VEGFR-3 trap molécule comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the soluble VEGFR-3 trap molécule has an amino acid sequence consïsting of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the soluble VEGFR-3 trap molécule has an amino acid sequence defîned by positions 1-546 5 ofSEQ!DNO:7.

If needed, soluble VEGFR-3 trap molécules described herein may comprise a functional région facilitating purification or production. Spécifie examples of such additional amino acid sequences include a GST sequence or a His tag sequence. In some variations, the région facilitating purification is removed for formulation of a composition 10 for pharmaceutical use.

Soluble VEGFR-3 trap molécules can be chemically modified (e.g., glycosylation, PEGylation, etc.) to impart desired characteristics. Such modifications preferably do not substantially reduce the growth factor binding affinities or specificities of the ligand binding molécule.

Polypeptides can be modified. for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the création of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl dérivatives.

In some embodiments, a soluble VEGFR-3 trap molécule described herein optionally comprises at least one PEG (polyethylene glycol) moiety attached to the molécule. For 20 example, in some embodiments, PEG of about 20-40 kDa is attached to the amino terminus of the ligand binding molécule. As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that can be used to derivatize other proteins, such as mono-(C 1-C10) alkoxy- or aryloxy-polyethylene glycol. PEG is a linear or branched neutral polyether, availabié in a 25 broad range of molecular weights, and is soluble in water and most organic solvents. PEG is effective at excluding other polymers or peptides when présent in water, primarily through its high dynamic chain mobility and hydrophilic nature, thus creating a water shell or hydration sphere when attached to other proteins or polymer surfaces. PEG is nontoxic, nonimmunogenic, and approved by the Food and Drug Administration for internai consumption.

Polypeptides can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a calorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). Examples of analogs are described in WO 98/28621 and in Olofsson, et al., Proc. Nat'l. Acad. Sci. USA. 95:1 70922158 l 714 (1998), U.S. Patent Nos. 5,51 2,545, and 5,474,982; U.S. Patent Application Nos. 20020164687 and 20020164710, the entire contents of each of which are incorporated herein by reference.

In some variations, the ligand binding molécule comprises a signal peptide that 5 directs sécrétion of the molécule from a cell that expresses the molécule.

The soluble VEGFR-3 trap molécule may be prepared by any suitable process. For example, a cell line (e.g. a eukaryotic cell line. Chines Hamster Ovary cell line) may be transfected with a vector comprising a polynucleotide sequence encoding the amino acid sequence of the soluble VEGFR-3 trap molécule, and which is cultured to express the trap 10 molécule. Methods for producing and purifying soluble VEGFR-3 trap molécules are described in WO2014/124487 A1 WO2015/123715 A1 and W02002/060950 A1, the entire contents of each of which are incorporated herein by reference.

The soluble VEGFR-3 trap molécule is présent in the aqueous pharmaceutical composition at a concentration in the range of from 5 mg/mL to 250 mg/mL.

In some embodiments, the soluble VEGFR-3 trap molécule is présent at a concentration of from 10 mg/mL, offrom 20 mg/mL, of from 30 mg/mL, of from 40 mg/mL, of from 50 mg/mL, of from 60 mg/mL, of from 70 mg/mL, of from 80 mg/mL, of from 90 mg/mL, offrom 100 mg/mL, of from 110 mg/mL, of from 120 mg/mL, offrom 130mg/mL, offrom 140 mg/mL, offrom 150 mg/mL, offrom 160 mg/mL, offrom 170 mg/mL, offrom 20 180 mg/mL of from 190 mg/mL, of from 200 mg/mL, of from 210 mg/mL, of from 220 mg/mL, of from 230 mg/mL, or of from 240 mg/mL. In some embodiments, the soluble VEGFR-3 trap molécule is présent 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, 25 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 rng/mL, up to 30 mg/mL, up to 20 mg/mL, or up to 10 mg/mL. Preferably, the soluble VEGFR-3 trap molécule is présent a concentration of up to 120 mg/mL, more preferably up to 100 mg/mL, still more preferably up to 80 mg/mL, yet more preferably up to 60 mg/mL, further more preferably up to 40 rng/mL.

In some embodiments, the soluble VEGFR3 trap molécule is présent at a concentration of from 5 mg/mL up to 120 mg/mL, of from 5 mg/mL up to 100 mg/mL, of from 5 mg/mL up to 80 mg/mL, of from 5 mg/mL up to 60 mg/mL, of from 5 mg/mL up to mg/mL, of from 5 mg/mL up to 40 mg/mL, of from 10 mg/mL up to 120 mg/mL, of from 10 mg/mL up to 100 mg/mL, of from 10 mg/mL up to 80 mg/mL, of from 10 mg/mL up to 60 mg/rnL, of from 10 mg/mL up to 50 mg/mL, of from 10 mg/mL up to 40 mg/mL, of from 20 mg/mL up to 120 mg/mL, of from 20 mg/rnL up to 100 mg/mL, of from 20 mg/mL up to 80 mg/mL, of from 20 mg/mL up to 60 mg/mL, of from 20 mg/mL up to 50 mg/mL, of from 20 mg/mL up to 40 mg/mL, of from 30 mg/rnL up to 120 mg/mL, of from 30 mg/mL up to 100 mg/mL, of from 30 mg/mL up to 80 mg/mL, of from 30 mg/mL up to 60 mg/mL, of from 30 mg/mL up to 50 mg/mL, or of from 30 mg/mL up to 40 mg/mL.

In some embodiments, the soluble VEGFR-3 trap molécule is présent 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.

Excipients

The aqueous pharmaceutical compositions of the présent diselosure contain trehalose. Trehalose is a disaccharide consisting of two glucose units joined by a l,lglycosidic bond. Typically, the glucose units présent in trehalose are α-glucose units.

Trehalose also has the names α,α-trehalose, a-D-glucopyranosyl-( l—>l)-a-Dglucopyranoside, α-D-glucopyranosyl-a-D-glucopyranoside, and D-(+)-trehalose.

Trehalose has the CAS no. 6138-23-4.

Trehalose has the Chemical structure:

Forms of trehalose include the 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. Trehalose can be obtained from a variety of suppliers, for example Pfanstiel (www.pfanstiehl.com). Trehalose dihydrate is also available from Sigma Aldrich, Merck, Fisher Scientific, Acros, and Alfa Aesar.

In some embodiments, the aqueous pharmaceutical compositions of the présent diselosure contain trehalose in a concentration of at least 7.0% w/v. Regardless of the form of trehaiose used to produce the formulation (e.g. anhydrous trehaiose, or trehaiose dihydrate), the % w/v of trehaiose relates to the percentage by weight of trehaiose, and for example does not include any associated solvaté (e.g. dihydrate). For example, a 10% w/v solution of trehaiose may be prepared using 10g of anhydrous trehaiose and making up to 5 lOOml with water. Alternatively, a 10% w/v solution of trehaiose may be prepared using 11.1 g of trehaiose dihydrate and making up to 100ml with water.

In some embodiments, trehaiose is présent 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 I2% w/v, or up to 11% w/v. In some embodiments, trehaiose is présent at a concentration of at least 7.5% w/v, at least 10 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, trehaiose is présent at a concentration of from 7.0% w/v to 20% w/v, or from 7.0% w/v to 15% w/v, or from 7.0% w/v to 14% w/v, or from 7.0% w/v to 13% w/v, or from 7.0% w/v to 12% w/v, or from 7.5% w/v to 20% w/v, or from 7.5% w/v to 15% w/v, or from 7.5% w/v to 14% w/v, or from 7.5% w/v to 13% w/v, or from 7.5% w/v to 12% w/v, or from 8.0% w/v to 20% w/v, or from 8.0% w/v to 15% w/v, or from 8.0% w/v to 14% w/v, or from 8.0% w/v to 13% w/v, or from 8.0% w/v to 12% w/v, or from 8.5% w/v to 20% w/v, or from 8.5% w/v to 15% w/v, or from 8.5% w/v to 14% w/v, or from 8.5% w/v to 13% w/v, or from 8.5% w/v to 12% w/v, or from 9% w/v to 20% w/v, or from 9% w/v to 15% w/v, or from 9% w/v to 14% w/v, or from 9% w/v to 13% w/v, or from 9% w/v to 12% 20 w/v, or from 9.5% w/v to 20% w/v, or from 9.5% w/v to 15% w/v, or from 9.5% w/v to 14% w/v, or from 9.5% w/v to 13% w/v, or from 9.5% w/v to 12% w/v, or from 10% w/v to 20% w/v, or from 10% w/v to 15% w/v, or from 10% w/v to 14% w/v, or from 10% w/v to 13% w/v, or from 10% w/v to 12% w/v, or from 10.5% w/v to 20% w/v, or from 10.5% w/v to 15% w/v, or from 10.5% w/v to 14% w/v, or from 10.5% w/v to 13% w/v, or from 10.5% 25 w/v to 12% w/v. In some embodiments, trehaiose is présent at a concentration of 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% 30 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.

The aqueous pharmaceutical composition contains water. Typically stérile, high purity water is used, such as for example water for injection.

The aqueous pharmaceutical composition contains a buffer, e.g. for maintaining the pH in the desired range. Any suitable buffer may be utilised. Examples of buffers include 5 a phosphate buffer (such as sodium dihydrogen phosphate, disodium phosphate), an amino acid such as a histidine buffer (e.g. histidine hydrochloride), a citrate buffer (e.g. sodium citrate), tris (2-Amino-2-(hydroxymethyi) propane-1,3-diol), and an acetate buffer (e.g. sodium acetate). In some embodiments, the buffer is tris or a phosphate buffer. In some embodiments, the buffer is a phosphate buffer, for example it may be a mixture of acidic and 10 basic forms of phosphate. In some embodiments, the buffer is a sodium phosphate, for example a mixture of sodium dihydrogen phosphate and disodium phosphate.

The buffer is included at a suitable concentration, for example it may be présent 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, 15 the buffer is présent at a concentration of at least 5 mM, or at least 10 mM. In some embodiments, the buffer is présent at a concentration in the range of from 5 mM to 100 mM, of from 5 mM to 80 mM, of from 5 mM to 70 mM, of from 5 mM to 60 mM, of from 5 mM to 50 mM, of from 5 mM to 40 mM, of from 5 mM to 30 mM, or of from 5 mM to 20 mM. In some embodiments, the buffer is included at a concentration of about 5 mM, about 10 20 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.

It has been found that aqueous pharmaceutical compositions comprising a soluble VEGFR-3 trap molécule having higher pH hâve improved stability with regard to dimer 25 formation.

The aqueous pharmaceutical composition has a pH in the range of from 6.5 to 8.0. In some embodiments, the pH of the composition is in the range of from 6.5 to 7.0, from 7.0 to 7.5, from 7.5 to 8.0, from 7.2 to 7.8, from 7.3 to 7.7, or from 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 30 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.

In some embodiments, the aqueous pharmaceutical composition comprises a surfactant. Where a surfactant is included, it may for example be an ionic surfactant (e.g.

cationic, anionic or zwitterionic) or non-ionic (e.g. neutral) surfactant. Examples of surfactants include polyoxyethylene (20) sorbitan monolaurate (e.g. sold under the brand names polysorbate 20®, Tween 20®), polyoxyethylene (20) sorbitan monooleate (e.g. sold under the brand names polysorbate 80® and Tween 80®), polyethylene glycol, and a 5 poloxamer (e.g. a copolymer of poly(propylene oxide) and poly(ethylene oxide)) such as that sold under the brand name Pluronic F68®). In some embodiments, the aqueous pharmaceutical composition comprises a surfactant which is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

Where a surfactant is included, it may be présent in a concentration in the range, for 10 example, of from 0.005% to 0.2% w/v. In some embodiments, a surfactant is présent at a concentration of from 0.005% to 0.1% w/v, or from 0.005% to 0.05% w/v, or from 0.005% to 0.02% w/v. In some embodiments, a surfactant is présent 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 15 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.

In some other embodiments, the aqueous pharmaceutical composition does not comprise a surfactant.

It has been found that aqueous pharmaceutical compositions containing a soluble 20 VEGFR-3 trap molécule, and having relatively high osmolality, provide good stability properties. Osmolality relates to the concentration of osmotically active particles in solution, and is typically defined in units of mOsm/kg.

Accordingly, 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. In 25 some embodiments, the aqueous pharmaceutical composition has an osmolality of up to 1000 mOsm/kg, or up to 900 mOsm/kg, or up to 800 mOsm/kg, or up to 700 mOsm/kg, or up to 600 mOsm/kg, or up to 500 mOsm/kg. In some embodiments, the aqueous pharmaceutical composition has an osmolality in the range of from 300 mOsm/kg to 1000 mOsm/kg, or from 350 mOsm/kg to 1000 mOsm/kg, or from 400 mOsm/kg to 1000 30 mOsm/kg. or from 300 mOsm/kg to 800 mOsm/kg, or from 350 mOsm/kg to 800 mOsm/kg, or from 400 mOsm/kg to 800 mOsm/kg, or from 300 mOsm/kg to 600 mOsm/kg, or from 350 mOsm/kg to 600 mOsm/kg, or from 400 mOsm/kg to 600 mOsm/kg.

The aqueous pharmaceutical compositions can contain relatively few components, but still provides good stability properties.

As defined herein, a tonicity agent is a substance which affects the osmolality of the pharmaceutical composition. Tonicity agents are typically încluded to adjust the osmolality 5 of a composition to a desired value.

Whilst in some embodiments, the aqueous pharmaceutical composition comprises an additional tonicity agent, in other embodiments, the aqueous pharmaceutical composition does not contain an additional tonicity agent. The term additional tonicity agent refers to a substance which materially affects the osmolality of the pharmaceutical composition and 10 which is other than the active agent, trehalose, buffer, water, and surfactant (if présent). Examples of tonicity agents include sugars (e.g. sucrose, dextrose), certain salts (e.g. sodium chloride, potassium chloride), polyols (e.g. mannitol, sorbitol, glycerin).

In some embodiments, the pharmaceutical composition contains less than 50mM sodium chloride, or less than 25mM sodium chloride, or less than lOmM sodium chloride, 15 or less than 5 mM sodium chloride

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 no sodium chloride is added during préparation of the pharmaceutical composition. It will be understood that the pharmaceutical composition may nevertheless include minor 20 amounts of sodium chloride, for example if the pH of the formulation is adjusted by addition of hydrochloric acid and sodium hydroxide, some small quantity of sodium chloride may form.

In some embodiments, the aqueous pharmaceutical composition is substantially free of sodium chloride. In some embodiments, the composition contains less than 1 mM sodium 25 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 someembodiments, the aqueous pharmaceutical composition contains no détectable sodium chloride.

In some embodiments, the aqueous pharmaceutical composition is substantially free of additional sugar (i.e. it is substantially free of sugars (e.g. monosaccharides or 30 disaccharides) other than trehalose and any sugars forming part of the active ingrédient). In some embodiments, the aqueous pharmaceutical composition contains less than ImM additional sugar, less than 0.5 mM additional sugar, less than 0.2 mM additional sugar, less than 0.1 mM additional sugar, or less than 0.05 mM addîtional sugar. In some embodiments, the aqueous pharmaceutical composition contains no détectable additional sugar.

In some embodiments, the aqueous pharmaceutical composition does not contain any constituents beyond water, soluble VEGFR-3 trap molécule, a phosphate buffer, and surfactant (other than impurities which may be present in those constituents).

In some embodiments, the aqueous pharmaceutical composition comprises an active agent which is a soluble VEGFR-3 trap molécule, the active agent being present at a concentration in the range of from 5 mg/rnL to 120 mg/mL, trehalose in a concentration in the range of from 8.5% w/v to 20% w/v;

a buffer at a concentration in the range of from 5 mM to 20 mM;

optional surfactant; and water;

wherein the pH of the aqueous pharmaceutical composition is in the range of from 6.5 to 8.0.

In some embodiments, the aqueous pharmaceutical composition comprises an active agent which is a soluble VEGFR-3 trap molécule, the active agent being present at a concentration in the range of from 20 mg/mL to 80 mg/mL, trehalose in a concentration in the range of from 9% w/v to 13% w/v;

a buffer at a concentration in the range of from 5 mM to 20 mM, wherein the buffer is a sodium phosphate;

a surfactant which is polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w/v; and water;

wherein the pH of the aqueous pharmaceutical composition is in the range of from 7.0 to 8.0.

In some embodiments, the aqueous pharmaceutical composition consists or consists essentially of active agent, trehalose, buffer and surfactant.

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

In some embodiments, the aqueous pharmaceutical composition consists or consists essentially of:

an active agent in a concentration of about 40 mg/ml, which is a soluble VEGFR-3 trap molécule that comprises a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand binding polypeptide comprising immunoglobulin-like domains l-3 of the extracellular domain of human VEGFR-3 and optionally having one or more modifications in an N-glycan région of the extracellular domain;

trehalose in a concentration of about 10.9% w/v;

a buffer in a concentration of about 10 mM, wherein the buffer is a sodium phosphate;

polyoxyethylene (20) sorbitan monolaurate in a concentration of about 0.01% w/v; and water;

wherein the pH of the aqueous pharmaceutical composition is about 7.5.

In some embodiments, the aqueous pharmaceutical composition consists or consists essentially of:

an active agent in a concentration of about 40 mg/ml, which is OPT-302 or VGX-300;

trehalose in a concentration of about 10.9% w/v;

a buffer in a concentration of about 10 mM, wherein the buffer is a sodium phosphate; polyoxyethylene (20) sorbitan monolaurate in a concentration of about 0.01% w/v; and water;

wherein the pH of the aqueous pharmaceutical composition is about 7.5.

In some embodiments, the aqueous pharmaceutical composition consists or consists essentially of:

an active agent in a concentration of about 40 mg/ml, which comprises or consists of the amino acid sequence of any one of SEQ ID NOs.: 3, 4, 5 and 6;

trehalose in a concentration of about 10.9% w/v;

a buffer in a concentration of about 10 mM, wherein the buffer is a sodium phosphate; polyoxyethylene (20) sorbitan monolaurate in a concentration of about 0.01% w/v; and water;

wherein the pH of the aqueous pharmaceutical composition is about 7.5.

In some embodiments, the aqueous pharmaceutical composition consists or consists essentially of:

an active agent in a concentration of about 40 mg/ml. which comprises or consists of the amino acid sequence of SEQ ID NO: 7;

trehalose in a concentration of about 10,9% w/v;

a buffer in a concentration of about 10 mM, wherein the buffer is a sodium phosphate;

polyoxyethylene (20) sorbitan monolaurate in a concentration of about 0.01% w/v; and water;

wherein the pH of the aqueous pharmaceutical composition is about 7.5.

The aqueous pharmaceutical compositions of the may be prepared in advance and provided to hospitals, surgeries and the like as a pre-prepared aqueous pharmaceutical formulation, Alternatively, they may be provided as a solid composition, e.g. a lyophilised pharmaceutical composition, which is for reconstitution with water prior to use.

Accordingly, there is also provided a lyophilised pharmaceutical composition for reconstitution, comprising:

an active agent which is a soluble VEGFR-3 trap molécule;

trehalose; and a buffer;

wherein the weight ratio of trehalose to active agent is in the range of from 1:3 to 40:1.

There is also provided a reconstituted pharmaceutical composition, wherein the pharmaceutical composition is obtained by admixing a lyophilised pharmaceutical composition as deflned herein with an aqueous diluent.

In use, an appropriate quantity of aqueous diluent is added to the lyophilised pharmaceutical composition to achieve the desired concentration of the constituents (e.g. the desired concentration of active agent, trehalose and/or buffer).

A lyophilised pharmaceutical composition containing trehalose and active agent in a weight ratio range of from 1:3 to 40:1 corresponds to, following reconstitution with an appropriate quantity of aqueous diluent, a reconstituted pharmaceutical composition containing 5-250 mg/mL active agent, and 8.5% w/v to 20% w/v trehalose.

In some embodiments, the weight ratio of trehalose to active agent in the lyophilised pharmaceutical composition is in the range of from 1:1 to 7.5:1, from 1:1 to 5:1, or from 2.1:1 to4.5:l.

In some embodiments, the weight ratio of trehalose to active agent in the lyophilised pharmaceutical composition is in the range offrom 0.7:1 to 3.3:1, or from 0.8:1 to 3:1.

In some embodiments, the weight ratio oftrehalose to active agent in the lyophilised pharmaceutical composition is about 2.7:1, or about 1.4:1, or about 0.9; 1.

As described above, trehalose is a disaccharide consisting of two glucose units joined by a 1,1-glycosidic bond. Typically, the glucose units présent in trehalose are a-glucose units. Trehalose also has the names α,α-trehalose, a-D-glucopyranosyl-(l—>1 )-a-D22158 glucopyranoside, α-D-glucopyranosyl-a-D-glucopyranoside, and D-(+)-trehalose.

Trehalose has the CAS no. 6138-23-4. Trehalose has the Chemical structure:

ÔH OH

Forms of trehalose include the 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.

Trehalose can be obtained from a variety of suppliers, for example Pfanstiel (www.pfanstiehl.com). Trehalose dihydrate is also available from Sigma Aldrich, Merck, Fisher Scientific, Acros, and Alfa Aesar.

The lyophilised pharmaceutical composition contains a buffer. Any suitable buffer may be utilised. Examples of buffers include a phosphate buffer(such as sodium dihydrogen phosphate, disodium phosphate), an amino acid such as a histidine buffer (e.g. histidine hydrochloride), a citrate buffer (e.g. sodium citrate), tris (2-Amino-2-(hydroxymethyl) propane-l,3-diol), and an acetate buffer (e.g. sodium acetate). In some embodîments, the buffer is tris or a phosphate buffer. In some embodîments, the buffer is a phosphate buffer, for example it may be a mixture of acidic and basic forms of phosphate, in some embodîments, the buffer is a sodium phosphate, for example a mixture of sodium dihydrogen phosphate and disodium phosphate.

In some embodîments, the weight range of buffer to active agent is in the range of from l :420 to 3 : l, or from l :200 to l : l .8, or from l :26 to l :40.

In some embodîments, the buffer is a sodium phosphate, and the weight ratio of sodium phosphate to active agent is in the range of from l :3 to l : 1000, or from l :3 to 1:200, or from l :5 to l : 100.

In some embodîments, the buffer is a sodium phosphate, and the weight ratio of sodium phosphate to active agent is about 0.03:1.

In some embodîments, the lyophilised pharmaceutical composition comprises a surfactant. Where a surfactant is included, it may for example be an ionic surfactant (e.g. cationic, anionic or zwitterionic) or non-ionic (e.g. neutral) surfactant. Examples of surfactants include polyoxyethylene (20) sorbitan monolaurate (e.g. sold under the brand names polysorbate 20®, Tween 20®), polyoxyethylene (20) sorbitan monooleate (e.g. sold under the brand naines polysorbate 80® and Tween 80®), polyethylene glycol, and a poloxamer (e.g. a copolymer of poly(propylene oxide) and poly(ethylene oxide)) such as that sold under the brand name Pluronic F68®). In some embodiments, the lyophilised 5 pharmaceutical composition comprises a surfactant which is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

The process of lyophilisation typically involves multiple steps, for example a freezing step, and one or more drying steps (e.g. a primary drying step involving sublimation, and a secondary drying step involving desorption).

The lyophilised pharmaceutical composition may for example contain one or more lyophilisation excipients. Examples of lyophilisation excipients include cryoprotectants an lyoprotectants. Examples of lyophilisation excipients include sugars such as sucrose, mannitol and dextrose, polymeric excipients such as polyvinylpyrrolidone, and amino acids such as glycine.

In some embodiments, the lyophilised pharmaceutical composition does not include any additional lyophilisation excipients (i.e. other than the active agent, trehalose, buffer, and surfactant, if présent).

As discussed above, in use an appropriate quantity of aqueous diluent is added to the lyophilised pharmaceutical composition to achieve the desired concentration of the 20 constituents (e.g. the desired concentration of active agent, trehalose and/or buffer). The aqueous diluent may for example be water. Typically stérile, high purity water is used, such as for example water for injection.

A quantity of aqueous diluent may be added such that the concentration of active agent in the reconstituted pharmaceutical composition is typically in the range of from 5 25 mg/tnL to 250 mg/mL, or for example from 20 mg/mL to 120 mg/mL, or about 40 mg/mL, or about 80 mg/mL, or about 120 mg/mL.

As a further example, a quantity 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 from 8.5% w/v to 20% w/v, or from 8.5% w/v 30 to 15% w/v, or from 9% w/v to 13% w/v, or from 10% w/v to 12% w/v, or about 10.9% w/v.

As a still further example a quantity of aqueous diluent may be added such that the concentration of buffer is typically up to 100 mM, for example up to 50 mM. or up to 20 mM, or at least 5 mM, or in the range of from 5 mM to 50 mM, or in the range of from 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.

Typically, following reconstitution, the pH of the reconstituted lyophilised pharmaceuticai composition is in the range of from 6.5 to 8.0, for example in the range of 5 from 6.5 to 7.0, from 7.0 to 7.5, from 7.5 to 8.0, from 7.2 to 7.8, from 7.3 to 7.7, or from 7.4 to 7.6. In some embodiments, the pH of the reconstituted lyophilised pharmaceuticai 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.

The discussion above in relation to the aqueous pharmaceuticai composition and the nature and amount of the required and optional features, such as the active agent, trehalose, buffer, surfactant, pH, osmolality, and tonicity agent, also applies to the lyophilised pharmaceuticai composition and the reconstituted lyophilised pharmaceuticai composition as appropriate.

Composition Préparation

The pharmaceuticai compositions of the présent disclosure may be prepared by any suitable method. For example, an aqueous solution of purified soluble VEGFR-3 trap molécule may be subjected to buffer exchange processing steps, filtration, and/or admixing 20 with other excipients as required.

In some embodiments, aqueous pharmaceuticai compositions according to the présent disclosure may be prepared by admixing an aqueous solution of soluble VEGFR-3 trap molécule with an aqueous trehalose solution, followed by subjecting the resulting mixture to ultrafiltration-diafiltration with buffer containing trehalose and sodium 25 phosphate, followed by admixing with surfactant (e.g. polyoxyethylene (20) monolaurate).

In the case of lyophilised formulations, an aqueous pharmaceuticai composition as described above may be prepared, and then subjected to lyophilisation. The aqueous composition may for example be subjected to low température conditions such that the mixture freezes, and subjected to low pressure conditions such that water is removed by 30 sublimation.

Composition Properties

Soluble VEGFR-3 trap molécules hâve poor stability properties and tend to form dimers or other high molecular weight aggregates on storage in aqueous pharmaceutical compositions, resulting in loss of purity, activity and reduced shelf life, or otherwise a need 5 for low température storage conditions.

However, as demonstrated by the examples below, the aqueous pharmaceutical compositions of the présent disclosure hâve demonstrated unexpectedly improved stability properties, with reduced dimer formation, and maintaining good levels of binding activity for VEGF-C and VEGF-D over time.

In some embodiments, the pharmaceutical composition forms less than 6%, or iess than 5%, or less than 4% dimerised active agent following 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% dimerised active agent, following storage at 25°C 15 for a period of 3 months.

In some embodiments, the pharmaceutical composition forms less than less than 5%, or less than 4%, or less than 3%, or less than 2% dimerised active agent, following storage at 5°C for a period of 2 months.

!n some embodiments, the pharmaceutical composition forms less than 6%, or less 20 than 5%, or less than 4%, or less than 3%, or less than 2% dimerised active agent, following storage at 5°C for a period of 3 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% dimerised active agent, following 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% dimerised active agent, following storage at 5°C for a period of 12 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% dimerised active 30 agent, following storage at 5°C for a period of 18 months.

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% dimerised active agent, following storage at 5°C for a period of 24 months.

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%, dimerised active agent, following storage at 5°C for a period of 30 months.

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

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% dimerised active agent, following storage at -20°C for a period of 6 months.

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% 15 dimerised active agent, following storage at -20°C for a period of 12 months.

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% dimerised active agent, following storage at -20°C for a period of 18 months.

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

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% dimerised active agent, following storage at -20°C for a period of 30 months.

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

In some embodiments, the pharmaceutical composition retains at least 70%, or at 30 least 80%, or at least 90% binding activity against VEGF-C and/or VEGF-D following storage at 5°C for a period of 3 months, compared with the binding activity of the composition at 0 months.

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

In some embodiments, the pharmaceutical composition retains at least 80% binding activity against VEGF-C and/or VEGF-D following storage at 5°C for a period of 12 months, compared with the binding activity of the composition at 0 months.

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

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

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

In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% binding activity against VEGF-C and/or VEGF-D following storage at -2Û°C for a period of 2 months, compared with the binding activity of the composition at 0 months.

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

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

In some embodiments, the pharmaceutical composition retains at least 80% binding activity against VEGF-C and/or VEGF-D following storage at -20°C for a period of I2 months, compared with the binding activity of the composition at 0 months.

In some embodiments, the pharmaceutical composition retains at least 80% binding activity against VEGF-C and/or VEGF-D following storage at -20°C for a period of 18 months, compared with the binding activity of the composition at 0 months.

In some embodiments, the pharmaceutical composition retains at least 80% binding activity against VEGF-C and/or VEGF-D following storage at -20°C for a period of 24 months, compared with the binding activity of the composition at 0 months.

In some embodiments, the pharmaceutical composition retains at least 80% binding activity against VEGF-C and/or VEGF-D following storage at -20°C for a period of 30 months, compared with the binding activity of the composition at 0 months.

As defined herein, the shelf-life ofa pharmaceutical composition is the period oftime in months during which, on storage, the extent of dimerised active agent is less than 10%, and the binding activity against VEGF-C and/or VEGF-D remains at least 70% of that achieved at 0 months.

In some embodiments, the pharmaceutical composition has at least a 2 month shelf 15 life, or at least a 3 month shelf life, on storage at 25°C.

In some embodiments, the pharmaceutical composition has at least a 3 month shelf life, or at least a 6 month shelf life, or at least a 12 month shelf life, or at least an 18 month shelf life, or at least a 24 month shelf life, or at least a 30 month shelf life, on storage at 5°C.

In some embodiments, the pharmaceutical composition has at least a 3 month shelf life, or at least a 6 month shelf life, or at least a 12 month shelf life, or at least an 18 month shelf life, or at least a 24 month shelf life, or at least a 30 month shelf life, on storage at 20°C.

In some embodiments, the pharmaceutical composition remains physically stable, i.e, there is no significant phase séparation or précipitation of solid material, for at least 3 25 months, on storage at 25°C.

in some embodiments, the pharmaceutical composition remains physically stable, i.e. there is no significant phase séparation or précipitation of solid material, for at least 3 months, at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months, on storage at 5°C.

In some embodiments, the pharmaceutical composition remains physically stable, i .e. there is no significant phase séparation or précipitation of solid material, for at least 3 months, at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months, on storage at -20°C.

Therapeutic Uses and Methods

The pharmaceutical compositions of the présent disclosure find use in the therapy of diseases and/or disorders for which inhibition of the interaction of VEGF-C and/or VEGF5 D with the VEGFR-3 receptor provides a therapeutic response. For example, the pharmaceutical compositions of the présent disclosure are useful in inhibiting neovascularisation, and find use in the therapy of diseases and/or disorders associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesîs.

Neovascularisation is the formation of new blood vessels. Angiogenesis is the 10 formation of new blood vessels from existing blood vessels, and plays a rôle in range of conditions including cancers, and ocular disorders such as age-related macular degeneration. Lymphangiogenesîs is the formation of lymphatic vessels from pre-existing lymphatic vessels, and excessive lymphatic vessel formation has been associated with a range of conditions including edema, neoplasm metastasis, and lymphangiomatosis.

Accordingly, the présent disclosure also provides a method of inhibiting neovascularisation in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defîned herein.

The présent disclosure also provides a method of treating and/or preventing a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or 20 lymphangiogenesîs in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defîned herein. Also provided is use of a soluble VEGFR-3 trap molécule for the manufacture of a pharmaceutical composition as defîned herein, for the treatment and/or prévention of a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesîs. Also provided herein is a 25 pharmaceutical composition as defîned herein for use in the treatment and/or prévention of a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesîs.

In some embodiments, the disease or disorder is an ocular disease or disorder. In some embodiments, the ocular disease of disorder is selected from the group consisting of 30 macular degeneration, diabetic retinopathy, macular edema, retinal vein occlusion and macular telangiectasia. In some embodiments, the ocular disease or disorder is wet agerelated macular degeneration. In some embodiments, the ocular disease or disorder is diabetic macular edema.

The pharmaceutical composition comprising a soluble VEGFR-3 trap molécule may also find use in the therapy of other diseases and/or disorders associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesis. In some embodiments, the disease or disorder is a cancer, for example in colorectal cancer, lung cancer, breast cancer, 5 glioblastoma, ovarian cancer, cervical cancer, and rénal cancer.

Administration

The pharmaceutical composition may be administered by any suitabie route, e.g. which is compatible with the disease or disorder to be treated.

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 involves administration into the vitreous humor of an eye.

In some embodiments, the pharmaceutical composition is administered using an 15 implant device which is implanted into an eye, and which permits controlled release ofactive agent into the vitreous of the eye.

In some embodiments, the pharmaceutical composition is administered using a port device which is implanted in an eye, which port device comprises a réservoir for the pharmaceutical composition, and permits controlled release of active agent into the vitreous 20 of the eye.

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 25 be administered intrathecally.

In some embodiments, the pharmaceutical composition is administered by injection. In some embodiments, the pharmaceutical composition is administered by infusion.

The pharmaceutical composition may accordingly be formulated for injection (e.g. for intravitreal injection, subcutaneous objection, intravenous injection, intramuscular 30 injection, or intrathecal injection), or for administration using an ocular implant device.

In use, a suitabie dosage amount and dosing régime of the pharmaceutical composition comprising the soluble VEGFR-3 trap molécule is utilised. The amount and frequency of dosing may dépend on factors including the type of disease or disorder, whether the active agent is being administered for prévention or treatment purpose, the âge, weight, sex and health of the person to be treated, the route of administration, and whetherthe soluble VEGFR-3 trap molécule is administered in combination with other active agents. Additionally, pharmacogenomic information (the effect ofgénotype on the pharmacokinetic, 5 pharmacodynamie or efficacy profile of a therapeutic) about a particular patient may affect dosage the used.

Dosage Forms

The pharmaceutical compositions may be administered in a range of dosage forms. 10 The présent disclosure accordingly also includes a container containing the pharmaceutical composition. The présent disclosure also includes a kit comprising a container containing the pharmaceutical composition, and optionally comprising instructions for administering the pharmaceutical composition to a subject.

For example, in the case of the aqueous pharmaceutical formulation, the 15 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. Accordingly, in some embodiments, the kit comprises a device for administering the pharmaceutical composition.

Where multiple doses are to be delivered (e.g. over time to the same subject, or to 20 different subjects), for convenience a container containing multiple separate sections each containing a unit dosage of the pharmaceutical composition may be used for example (e.g. a divided bottle or container having multiple wells). As an alternative, a kit comprising multiple containers each containing a unit dosage of the pharmaceutical composition may be utilised.

As described above, in some embodiments the pharmaceutical composition may be administered utilising a port device which may be implanted in an eye. Accordingly, there is also provided a port device for implantation in an eye, the port device comprising a réservoir containing a pharmaceutical composition as defmed herein, and wherein the port device permits controlled release of active agent into the vïtreous of the eye.

In use, the port device may for example be implanted through the surface of the eye,

e.g. through the sciera, such that it can release active agent into the vïtreous, but at least a portion of the device can be accessible for re-filling of the réservoir.

In some embodiments, the port device may comprise a réservoir chamber coupled to a membrane, an opening, a diffusion barrier, a diffusion mechanism and/or porous structure for controlled release of the active agent. For example it may contain, e.g., a semipermeable membrane that permits passive diffusion of the active agent into the vitreous of the eye, such 5 as a titanium-containing semipermeable membrane.

The port device will typically contain one or more rétention éléments for retaining the device in position, e.g. through the sciera. The port device may for example extend through the sciera but be covered by the conjunctiva.

The port device may be refillable, for example it may contain a re-filling element, 10 such as a septum (e.g. a silicone septum) which permits refilling of the réservoir with additional pharmaceutical composition using a needle.

In some embodiments, the port device is configured to receive an amount of pharmaceutical composition sufficient to deliver a therapeutic dosage of 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 15 to 4 months, up to 5 months, or up to 6 months.

Examples of port devices are disclosed in W02012/019176, WO2012/065006 and WO2014/152959, the contents of each of which are incorporated herein by reference in their entirety.

Also provided herein is a kit comprising (i) a port device for implantation in an eye, 20 the port device comprising a réservoir for containing a pharmaceutical composition as defined herein. wherein the port device permits controlled release of active agent into the vitreous of the eye; and (ii) a container comprising an amount of pharmaceutical composition as defined herein, for filling of the port device. In some embodiments, the kit comprises a syringe for filling the port device with the pharmaceutical composition. In some 25 embodiments, the container comprising the pharmaceutical composition is a syringe for filling of the port device.

As described above, in the case of a lyophilised pharmaceutical composition, in use the lyophilised pharmaceutical composition may be reconstituted by admixing with an aqueous diluent prior to administration. Accordingly, there is also provided a kit for 30 reconstitution comprising a lyophilised pharmaceutical composition as defined herein, and an aqueous diluent. The aqueous diluent may for example be stérile water, e.g. water for injection.

Combination Therapy

Whilst in some embodiments, the soluble VEGFR-3 trap molécule may be administered via the pharmaceutical compositions of the present disclosure as monotherapy, in other embodiments it is administered as part of a combination therapy treatment régime, 5 e.g. in combination with a further active agent, for example a further active agent useful for the treatment and/or prévention of a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesis.

The pharmaceutical composition comprising the soluble VEGFR-3 trap molécule may for example be administered with a further active agent simultaneously, sequentially, 10 or separately. For example, a course of therapy may be prescribed for a patient involving administration of the soluble VEGFR-3 trap molécule at certain timepoints, and involving administration of one or more further active agents at different timepoints.

The further active agent may, for example, be a further active agent useful for the treatment and/or prévention of an ocular disease or disorder, e.g. an ocular disease or 15 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.

In some embodiments, the further active agent is an anti-VEGF-A agent or an antVEGF-B agent. Examples of anti-VEGF-A and/or anti-VEGF-B agents include 20 ranibizumab (Lucentis®), aflibercept (Eylea®), bevacizumab (Avastin®) and brolucizumab (Beovu®).

In some embodiments, the further active agent is pegaptanib (Macugen®).

In some embodiments the further active agent is a steroid, for example triamcinolone acetonide, dexamethasone (Ozurdex®), or fluocinolone acetonide (Retisert®, Iluvien®).

In some embodiments, the pharmaceutical composition comprising the soluble

VEGFR-3 trap molécule is administered in combination with photodynamic therapy. Photodynamic therapy ïnvolves administration of a photosensitive active agent (e.g. verteporfin (Visudyne®) in conjonction with laser treatment. In some embodiments, the pharmaceutical composition comprising the soluble VEGFR-3 trap molécule is administered in combination with laser photocoagulation therapy. Laser photocoagulation therapy involves direction of a concentrated beam of high energy laser light directed on to the retina, to seal leaky blood vessels.

In some embodiments, the pharmaceutical composition comprising the soluble VEGFR-3 trap molécule is administered in combination with focal-grid macular laser surgery.

The pharmaceutical composition of the présent disclosure may, for example, be 5 packaged together with another pharmaceutical composition containing further active agent, e.g. in a pack or kit containing both médications.

AU publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specîfically and individually indicated to be incorporated by reference. In case of conflict, the présent application, 10 including any définitions herein. will control.

Sequence Listing

Sequences

Sequence Number [ID]

Molécule Type

Length

Features Location/Quali fiers

NonEnglishQualifier Value

AA

764 source I..764 mol_type= protein organism= Homo sapiens

Residues
MQRGAALCLR	LWLCLGLLDG	LVSGYSMTPP
WPGAQEAPAT	GDKDSEDTGV	VRDCEGTDAR
EGTTAASSYV	FVRDFEQPFI	NKPDTLLVNR
QEVVWDDRRG	MLVSTPLLHD	ALYLQCETTW
ELLVGEKLVL	NCTVWAEFNS	GVTFDWDYPG
SQHDLGSYVC	KANNGIQRFR	ESTEVIVHEN
YPPPEFQWYK	dgkalsgrhs	PHALVLKEVT
PPQIHEKEAS	SPSIYSRHSR	QALTCTAYGV
DLMPQCRDWR	AVTTQDAVNP	IESLDTWTEF
GQDERLIYFŸ	VTTIPDGFTI	ESKPSEELLE
AHGNPLLLDC	KNVHLFATPL	AASLEEVAPG
HDKHCHKKYL	SVQALEAPRL	TQNLTDLLVN
KSGVDLADSN	QKLSIQRVRE	EDAGRYLCSV

TLNITEESHV	IDTGDSLSIS	CRGQHPLEWA	60
PYCKVLLLHE	VHANDTGSYV	CYYKYIKARI	120
KDAMWVPCLV	SIPGLNVTLR	SQSSVLWPDG	180
GDQDFLSNPF	LVHITGKELY	DIQLLPRKSL	240
KQAERGKWVP	ERRSQQTHTE	LSSILTIHNV	300
PFISVEWLKG	PILEATAGDE	LVKLPVKLAA	360
EASTGTYTLA	LWNSAAGLRR	NISLELWNV	420
PLPLSIQWHW	RPWTPCKMFA	QRSLRRRQQQ	480
VEGKNKTVSK	LVIQNANVSA	MYKCVVSNKV	540
GQPVLLSCQA	DSYKYEHLRW	YRLNLSTLHD	600
ARHATLSLSI	PRVAPEHEGH	YVCEVQDRRS	660
VSDSLEMQCL	VAGAHAPSIV	WYKDERLLEE	720
CNAKGCVNSS	AS VA		764

Sequences
Sequence Number [ID]	2
Molécule Type	AA
Length	330
Features Location/Qualifiers	source 1.330 mol_type= protein organism= Homo sapiens
NonEnglishQualifier Value

Residues

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 120 PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 180 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 240 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHÏT QKSLSLSPGK 330
Sequences Sequence Number [ID] Molécule Type Length Features Location/Qualifiers NonEnglishQualifter Value Features Location/Qualifiers NonEnglishQualifter Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Residues YSMTPPTLNI TEESHVIDTG DSLSISCRGQ HPLEWAWPGA EGTDARPYCK VLLLHEVHAQ DTGSYVCYYK YIKARIEGTT TLLVNRKDAM WVPCLVSIPG LNVTLRSQSS VLWPDGQEVV QCETTWGDQD FLSNPFLVHI TGNELYDIQL LPRKSLELLV DWDYPGKQAE RGKWVPERRS QQTHTELSSI LTIHNVSQHD VIVHEEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE	3 AA 537 source 1..537 mol_type= protein organism= synthetic construct CARBOHYD 9.11 note= N-GIycosylation CARBOHYD 142 144 note= N-Glycosylation CARBOHYD 227. 229 note= N-Glycosylation CARBOHYD 275. 277 note= N-GIycosylation CARBOHYD 387. 389 note= N-Glycosylation DOMAIN 1..305 note= VEGFR-3 ECD 1 -3 Portion DOMAIN 306 .537 note= Fc Portion QEAPATGDKD SEDTGWRDC 60 AASSYVFVRD FEQPFINKPD 120 WDDRRGMLVS TPLLHDALYL 180 GEKLVLNCTV WAEFNSGVTF 240 LGSYVCKANN GIQRFRESTE 300 LMISRTPEVT CWVDVSHED 360 QDWLNGKEYK CKVSNKALPA 420 GFYPSDIAVE WESNGQPENN 480 ALHNHYTQKS LSLSPGK 537
Sequences Sequence Number [ID]	4

Molécule Type Length Features Location/Qualifiers NunEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Residues YSMTPPTLNI TEESHVIDTG DSLSISCRGQ HPLEWAWPGA EGTDARPYCK VLLLHËVHAQ DTGSYVCYYK YIKARIEGTT TLLVNRKDAM WVPCLVSIPG LNVTLRSQSS VLWPDGQEVV QCETTWGDQD FLSNPFLVHI TGNELYDIQL LPRKSLELLV DWDYPGKQAE RGKWVPERRS QQTHTELSSI LTIHNVSQHD VIVHEEPKS3 DKTHTCPPCP APELLGGPSV FLFPPKPKDT PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

AA 537 source 1..537 mol_type= protein organism- synthetic construct CARBOHYD 9. Il note= N-Glycosylation CARBOHYD 142. .144 note= N-Glycosylation CARBOHYD 227. 229 note= N-Glycosylation CARBOHYD 275..277 note= N-Glycosylation CARBOHYD 387. 389 note= N-Glycosylation DOMAIN 1 .305 note= VEGFR-3 ECD 1-3 Portion DOMAIN 306..537 note= Fc Portion SITE 310 note= Substitution of C with S QEAPATGDKD SEDTGVVRDC 60 AASSYVFVRD FEQPFINKPD 120 WDDRRGMLVS TPLLHDALYL 1Θ0 GEKLVLNCTV WAEFNSGVTF 240 LGSYVCKANN GIQRFRESTE 300 LMISRTPEVT CWVDVSHED 360 QDWLNGKEYK CKVSNKALPA 420 GFYPSDIAVE WESNGQPENN 480 ALHNHYTQKS LSLSPGK 537

Sequences Sequence Number [ID] Molécule Type Length Features Location/Qualifiers

5 AA 547 source 1..547 mol_type= protein organism= synthetic construct

NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Residues YSMTPPTLNI TEESHVIDTG DSLSISCRGO HPLEWAWPGA EGTDARPYCK VLLLHEVHAQ DTGSYVCYYK YIKARIEGTT TLLVNRKDAM WVPCLVSIPG LNVTLRSQSS VLWPDGQEVV QCETTWGDQD FLSNPFLVHI TGNELYDIQL LPRKSLELLV DWDYPGKQAE RGKWVPERRS QQTHTELSSI LTIHNVSQHD VIVHENPFIS VEWLKEPKSC DKTHTCPPCP APELLGGPSV CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY CKVSNKALPA ΡΙΕΚΤΙΞΚΑΚ GQPREPQVYT LPPSRDELTK WESNGQPENN YKTTPPVLDS DGSFFLYSKL· TVDKSRWQQG LSLSPGK

CARBOHYD 9. Il note- N-Glycosylation CARBOHYD 142..144 note= N-Glycosylation CARBOHYD 227. 229 note= N-Glycosylation CARBOHYD 275 .277 note= N-Glycosylation CARBOHYD 397 399 note= N-Glycosylation DOMAIN l .305 note= VEGFR-3 ECD l-3 Portion DOMAIN 3I6..547 note- Fc Portion OEAPATGDKD SEDTGWRDC 60 AASSYVFVRD FEQPFINKPD 120 WDDRRGMLVS TPLLHDALYL 180 GEKLVLNCTV WAEFNSGVTF 240 LGSYVCKANN GIQRFRESTE 300 FLFPPKPKDT LMISRTPEVT 360 RWSVLTVLH QDWLNGKEYK 420 NQVSLTCLVK GFYPSDIAVE 480 NVFSCSVMHE ALHNHYTQKS 540 547

Sequences Sequence Number [ID] Molécule Type Length Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value

6 AA 547 source 1 .547 mol_type= protein organisrn- synthetic construct CARBOHYD 9 11 note= N-Glycosylation CARBOHYD 142 144 note= N-Glycosylation

Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Residues YSMTPPTLNI TEESHVIDTG DSLSISCRGQ HPLEWAWPGA EGTDARPYCK VLLLHEVHAQ DTGSYVCYYK YIKARIEGTT TLLVNRKDAM WVPCLVSIPG LNVTLRSQSS VLWPDGQEVV QCETTWGDQD FLSNPFLVHI TGNELYDIQL LPRKSLELLV DWDYPGKQAE RGKWVPERRS QQTHTELSSI LTIHNVSQHD VIVHENPFIS VEWLKEPKSS DKTHTCPPCP APELLGGPSV CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY CKVSNKALPA ΡΙΕΚΤΙΞΚΑΚ GQPREPQVYT LPPSRDELTK WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG LSLSPGK

CARBOHYD 227 .229 note- N-Giycosylation CARBOHYD 275 .277 note- N-Glycosylation CARBOHYD 397 399 note= N-Glycosylation DOMAIN I..305 note= VEGFR-3 ECD l-3 Portion DOMAIN 316..547 note= Fc Portion SITE 320 note= Substitution of C with S QEAPATGDKD SEDTGWRDC 60 AASSYVFVRD FEQPFINKPD 120 WDDRRGMLVS TPLLHDALYL 180 GEKLVLNCTV WAEFNSGVTF 240 LGSYVCKANN GIQRFRESTE 300 FLFPPKPKDT LMISRTPEVT 360 RVVSVLTVLH QDWLNGKEYK 420 NQVSLTCLVK GFYPEDIAVE 480 NVFSCSVMHE ALHNHYTQKS 540 547

Sequences Sequence Number [ID] Molécule Type Length Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value Features Location/Qualifiers NonEnglishQualifier Value

7 AA 548 source 1 .548 mol_type= protein organisme synthetic construct CARBOHYD 9. 11 note= N-Glycosylation CARBOHYD 80..82 note= N-Glycosylation CARBOHYD 142.144 note- N-Glycosylation

Features Location/Qualifiers			CARBOHYD 227. .229 note= N-Glycosyiation
NonEnglishQualifier Value
Features Location/Qualifiers			CARBOHYD 275 .277 note= N-Glycosylalion
NonEnglishQualifler Value
Features Location/Qualifiers			CARBOHYD 398 400 note= N-Glycosyiation
NonEnglishQualifler Value
Features Location/Qualifiers			DOMAIN 1.305 note= VEGFR-3 ECD 1-3 Portion
NonEnglishQualifier Value
Features Location/Qualifiers			DOMAIN 306..316 note= Factor Xa linker
NonEnglishQualifier Value
Features Location/Qualifiers			DOMAIN 317. 548 note= Fc Portion
NonEnglishQualifier Value
Residues YSMTPPTLNI TEESHVIDTG	DSLSISCRGQ	HPLEWAWPGA	QEAPATGDKD SEDTGWRDC	60
EGTDARPYCK VLLLHEVHAN	DTGSYVCYYK	YIKARIEGTT	AASSYVFVRD FEQPFINKPD	120
TLLVNRKDAM WVPCLVSIPG	LNVTLRSQSS	VLWPDGQEVV	WDDRRGMLVS TPLLHDALYL	180
QCETTWGDQD FLSNPFLVHI	TGNELYDIQL	LPRKSLELLV	GEKLVLNCTV WAEFNSGVTF	240
DWDYPGKQAE RGKWVPERRS	QQTHTELSSI	LTIHNVSQHD	LGSYVCKANN GIQRFRESTE	300
VIVHEDPIEG RGGGGGDPKS	CDKPHTCPLC	PAPELLGGPS	VFLFPPKPKD TLMISRTPEV	360
TCVVVDVSHE DPEVKFNWYV	DGVEVHNAKT	KPREEQYNST	YRWSVLTVL HQDWLNGKEY	420
KCKVSNKALP ΑΡΙΕΚΤΙΞΚΑ	KGQPREPQVY	TLPPSRDELT	KNQVSLTCLV KGFYPSDIAV	480
EWESNGQPEN NYKATPPVLD	SDGSFFLYSK	LTVDKSRWQQ	GNVFSCSVMH EALHNHYTQK	540
SLSLSPGK				548
Seq	uences
Sequence Number [1D]			8
Molécule Type			AA
Length			11
			source 1.11
Features Location/Qualifiers			mol_type= protein organism= synthetic construct
NonEnglishQualifier Value
Residues DPIEGRGGGG G				11

Examples

The présent disclosure is further exemplified by the following non-limiting 5 examples.

Example 1: Initial Formulation Screening and DOE (Design of Experiments) Study

A formulation study was undertaken to identify preferred formulations. pH, buffers and excipients were screened and a Design of Experiments (DOE) was conducted.

Briefly, initial high-throughput screens were performed using one or more of 5 Differential Scanning Calorimetry/Fluorimetry (DSC/F) and Dynamic Light Scattering (DLS). Subséquent experiments focused on Size Exclusion Chromatography (SEC) and ELISA (VEGF-D) to assess formulation stability.

The results from pH/buffer screens showed an increase in thermal unfolding températures in buffers from pH 6.5 to 8.0, indicative of increased thermal stability.

Results from an initial excipient screen found that sodium and trehalose impart higher thermal stability in citrate, phosphate, and tris buffer.

A 1 -week accelerated stability study, in the same buffers with a panel of excipients, found that tris and phosphate buffer with mannitol, trehalose and proline showed the least amount of dégradation by SEC.

A surfactant study showed that OPT-302 when formulated in 20 mM tris buffer at pH 8.0 had the least amount of total aggregate and dimer formation, when trehalose and proline were excipients, and that the inclusion of PS-20 (polysorbate 20) into the formulations had minimal impact on stability.

The results of the DOE solution stability study found that the stability of the OPT20 302 formulated in tris buffer increased with increasing pH and increasing concentration of trehalose.

From the in-design and off-design formulations from the DOE study, formulations were identified as possible candidates for additional study:

mg/mL OPT-302 in 20 mM tris, pH 8.0, 18.1 % w/v trehalose, 0.01 % w/v PS-20, 2S with or without 100 mM proline;

mg/mL OPT-302 in 20 mM phosphate buffer at pH 7.5, 18.1% trehalose, and 0.01% w/v PS-20.

Example 2: Efïect of trehalose content and pH on OPT-302 formulation stability

Formulations of OPT-302 were prepared and their tendency to form dimers of active ingrédient over time was analysed. The formulations contained 40 mg/mL OPT-302, water, ΙΟιηΜ sodium phosphate buffer, and 0.01% polysorbate 20. The formulations were at pH

7.50 or 7.20, and contained either 9.0% w/v, 13.6% w/v or 18.1% w/v trehalose (prepared respectively using 10% w/v, 15 %w/v and 20 % w/v trehalose dihydrate).

Formulations were incubated for 1 week at 37°C, and analysed by SE-HPLC. The results showed a trend towards decreased dimerization with increasing trehalose content.

Formulation (10 mM phosphate, 0.01% PS-20)	% Dimer
Trehalose (% w/v)	pH	T=0	1 week at 37°C
9.0	7.50	3.8	4.3
13.6	7.50	3.4	4.0
18.1	7.50	4.1	2.9
9.0	7.20	4.3	6.5
13.6	7.20	3.8	5.1
18.1	7.20	4.4	4.4

Example 3: Effect of trehalose content and sodium chloride content on OPT-302 formulation stability

Formulations of OPT-302 were prepared and their tendency to form high molecular weight species (e.g. dimers) of active ingrédient over time was analysed. The formulations contained 40 mg/mL OPT-302, water, and lOmM sodium phosphate buffer, at pH 7.4, The formulations contained either 4.5% w/v, 6.8% w/v, 9.0% w/v or 18.1% w/v trehalose (prepared respectively using 5% w/v, 7.5 %w/v, 10% w/v and 20 % w/v trehalose dihydrate), and either 40mM, 100 mM or 140 mM sodium chloride.

Formulations were incubated for up to 2 weeks at 37°C, and analysed by SE-HPLC.

The results are shown in Figure 1. The rate of dimerization formation was highest for formulations containing lower concentrations of trehalose.

Example 4: Stability of further OPT-302 formulations

Formulations were prepared commencing with OPT-302 in 40 mM NaCl, 10 mM phosphate pH 7.2, concentrated to 54.5 mg/mL using a50 kDa membrane.

OPT-302 was purified using aSuperdex S200 column loaded at --3.2% of the 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, 7.5% Trehalose spiked into the S200 purified pool with 37.5% Trehalose stock prior to pre-formulation TFF. The % monomer by SE-UPLC was 5 98.7%

One-fifth of the stock was then spiked to 6.8% trehalose and concentrated to 59 g/L before adding polysorbate 20 (0.03%) and diluted to 40 g/L with 6.85% w/v Trehalose, 0.03% v/v PS20, 10 mM Phos, 40 mM NaCl, pH 7.2.

Three-fifths of the stock was diafiltered with 9.0% w/v Trehalose, 10 mM Phos, pH 10 7.5, (>7DV) 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 to 13.6% trehalose using 40% trehalose, and then diluted to 40 mg/mL OPT-302 to achieve 13.6% w/v Trehalose, 10 mM Phos, pH 7.5, before adding PS-20 (0.01%). The third portion was spiked to 18.1% trehalose using 40% trehalose, and then diluted to 40 mg/mL OPT-302 15 to achieve 18.1% w/v Trehalose, 10 mM Phos, pH 7.5, before adding PS-20 (0.01%).

The final one-fifth of the stock was diafiltered with 13.6% w/v Trehalose, 20 mM Tris, pH 8.0, (>7DV), diluted to 40 mg/mL OPT-302 in the same buffer before adding PS20 (0.01%).

SE-UPLC was then performed on ali formulations, with the results being shown in 20 the table below.

Formulation no.	Contents	SE-UPLC Monomer %
Pre-UFDF stock	40mM NaCl, 10 mM Phosphate, pi! 7.2	98.7%
1	40 mM NaCl, 10 mM Phosphate, 6.8% w/v Trehalose, 0.03% v/v PS20, pH 7.2	95.5%
2	10 mM Phosphate, 9.0% w/v Trehalose, 0.01% v/v PS20, pH 7.5	97.5%
3	10 mM Phosphate, 13.6% w/v Trehalose. 0.01% v/v PS20, pH 7.5	98.2%

4	10 mM Phosphate. 18.1% w/v Trehalose, 0.01% v/v PS20, pH 7.5	98.3%
5	20 mM Tris, 13.6% w/v Trehalose, 0.01% v/v PS20. pH 8	98.1%

As can be seen from the table, the formulation which was concentrated in the presence of sodium chloride had the lowest monomer stability. Those formulations which underwent diafiltration to remove sodium chloride had improved stability, with % monomer also being higher for those formulations containing higher amounts of trehalose.

Example 5: Long Term and Accelerated Stability Study

OPT-302 formulations were prepared as follows, ail containing40 mg/mL OPT-302: l. Lyophilised formulation containing 6.8% w/v trehalose (prepared using 7.5% w/v trehalose dihydrate), 0.03% w/v PS 20, 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% PS 20, 10 mM sodium phosphate, 40 mM NaCl, pH 7.2.

3. Aqueous formulation containing 9.0% w/v trehalose (prepared using !0%w/v trehalose dihydrate), 0.01% PS 20, 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% PS 20, 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% PS 20, 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% PS 20, 20 mM Tris, pH 8.

The formulations were set-up at 25°C (testing monthly for 3 months, SEC ), and 5°C (testing quarterly for 24 months by SEC). Summary results for SEC are provided in Figures 2 and 3.

The accelerated results at 25°C indicate that trehalose concentrations of more than 6.8% w/v substantially stabilize monomer content, and demonstrate that formulations containing high trehalose concentrations without added sodium chloride are likely to be stable at 5°C.

For the real time conditions (5°C, 24 months storage), monomer content for formulations containing more than 6.8% w/v trehalose at 24 months ail hâve significantly greater stability than the comparator formulation.

(n summary, in liquid format, long term refrigerated storage (15 month) and 5 accelerated stability results demonstrate that OPT-302 can be refonnulated at higher trehalose concentrations which results in excellent stability.

Example 6: OPT-302 Formulation

A preferred aqueous formulation for OPT-302 was identified, containing the 10 following constituents:

Constituent	Amount
OPT-302 (active ingrédient)	36-44 mg/mL (preferably 40 mg/mL)
Trehalose dihydrate	10.9% w/v trehalose
Sodium phosphate ( 1:7.8 weight ratio mixture of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate. 71hO)	10mM
Polyoxyethylene (20) sorbitan monolaurate (Polysorbate 20. Tween 20)	0.01% w/v
Water For Injection	To volume
PH	7.5

Density of formulation = 1.041 kg/L

A further comparative OPT-302 formulation has the following constituents:

Constituent	Amount
OPT-302 {active ingrédient)	36-44 mg/mL (preferably 40 mg/mL)
Trehalose dihydrate	6.8% w/v trehalose (7.5% w/v trehalose dihydrate)
Sodium phosphate (Mixture of sodium dihydrogen phosphate dihydrate and di-sodium hydrogen phosphate.7HiO)	lOmM
Sodium chloride	40mM
Polyoxyethylene (20) sorbitan monolaurate (Polysorbate 20. Tween 20)	0.03% w/v
Water For Injection	To volume
pH	7.2

The comparative formulation was found to form high levels of OPT-302 dimer on storage, as demonstrated by Example 8 below.

Example 7: Préparation of OPT-302 Formulation

To a virus-filtered pool containing OPT-302 (Virus Filtered Pool) is added an aliquot of aqueous solution containing trehalose at high concentration (37.5% w/v trehalose 5 dihydrate, lOmM sodium phosphate, pH 7.5) (Trehalose Spike) in order to achieve a concentration of 12% w/v trehalose dihydrate and mixed for at least 10 minutes to provide a mixture (the Adjusted Virus Filtered Pool).

The mixture is filtered through a 0.2gm filter and subjected to ultrafiltrationdiafiltration and concentration using tangential flow filtration, using an Equilibration, 10 Diafiltration and Flush Buffer containing 12% w/v trehalose dihydrate, 10 mM sodium phosphate at pH 7.5, to provide a mixture having a target OPT-302 concentration of 46-55 mg/mL (UFDF Pool).

The concentration of OPT-302 in the UFDF Pool is determined, and an amount of Dilution Buffer (12% w/v trehalose dihydrate, 10mM sodium phosphate, pH 7.5) is added 15 to the UFDF Pool to obtain an OPT-302 concentration of around 44 g/L (Diluted UFDF Pool). The Diluted UFDF Pool is mixed using a Wave Mixer for at least 30 minutes.

The target weight of Formulation Buffer (12% w/v trehalose dihydrate, 10mM sodium phosphate, polyoxyethylene (20) sorbitan monolaurate, pH 7.5) required to be added to the diluted UFDF Pool to obtain a concentration of polyoxyethylene (20) sorbitan 20 monolaurate of 0.01% (w/v) is calculated and added. The formulated, diluted UFDF Pool is mixed using a wave mixer for at least 15 minutes.

The expected concentration of OPT-302 is in the range of from 36-44 mg/mL.

Example 8: Stability of OPT-302 Formulations

Three OPT-302 formulations were prepared having the following constituents:

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, WF1, pH 7.5.

Comparative Formulation L 41 mg/mL OPT-302, 10mM sodium phosphate, 40mM sodium chloride, 7.5% w/v trehalose dihydrate (6.8% w/v trehalose), 0.03% w/v polyoxyethylene (2) sorbitan monolaurate, WF1, pH 7.2;

Comparative Formulation 2; 42,4 mg/mL OPT-302, lOmM sodium phosphate, 40mM 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 températures, and stability properties of the formulations were determined. Data for Formulation l stored at -20, 5, 25 and 40 °C is presented below, together with data for the Comparative Formulations stored at 25 and 40 °C.

Formulation 1 at-20°C

Criteria	0 months	0.5 months	1 month	2 months
Appearance Visual inspection (Colour, clarity and visible particles)	Clear Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer: 99% High MWT: 1% Low MWT: 0%	Monomer: 98% High MWT: 1% Low MWT: 0%	Monomer: 99% High MWT; 1% Low MWT: 0%	Monomer; 98% High MWT: 1% Low MWT; 0%
Concentration OD280	41 mg/mL	41 mg/mL	42 mg/mL	42 mg/mL

Formulation 1 at 5°C

Criteria	0 months	0.5 months	1 month	2 months
Appearance Visual inspection (Colour, clarity and visible particles)	Clear Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very' slightly opalescent Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer; 99% High MWT: 1% Low MWT: 0%	Monomer: 98% High MWT: 1% Low MWT: 0%	Monomer: 98% High MWT: 2% Low MWT: 0%	Monomer: 98% High MWT: 2% Low MWT: 0%
Concentration OD280	41 mg/mL	42 mg/mL	41 mg/mL	42 mg/mL

Formulation 1 at 25°C

Criteria	0 months	0.5 months	1 month	2 months
Appearance Visual inspection (Colour, clarity and visible particles)	Clear Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer: 99% High MWT: 1%	Monomer: 98% High MWT: 2%	Monomer: 98% High MWT; 2%	Monomer: 97% High MWT: 2%

	Low MWT: 0%	Low MWT: 0%	Low MWT: 0%	Low MWT: 1%
Concentration OD280	41 mg/mL	43 mg/mL	41 mg/mL	42 mg/mL

Formulation 1 at 40°C

Criteria	0 months	0.5 months	1 month
Appearance Visual inspection (Colour, clarity and visible particles)	Clear Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid One extrinsic particle	Very slightly opalescent Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer: 99% High MWT: 1% Low MWT: 0%	Monomer: 92% High MWT: 6% Low MWT: 2%	Monomer: 88% High MWT: 9% Low MWT: 3%
Concentration OD280	4! mg/mL	41 mg/mL	42 mg/mL

Comparative Formulation 1 at 25°C

Criteria	0 months	0.5 months	1 month	2 months
Appearance Visual inspection (Colour, clarity and visible particles)	Very slightly opalescent Slightly yellowish liquid Few particles	Very·1 slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer: 95% High MWT: 5% Low MWT: 0%	Monomer: 93% High MWT: 6% Low MWT: 0%	Monomer: 90% High MWT: 9% Low MWT: 1%	Monomer: 86% High MWT: 13% Low MWT: 1%
Concentration OD280	43 mg/mL	42 mg/mL	43 mg/mL	43 mg/mL

Comparative Formulation 1 at 4Û°C

Criteria	0 months	0.5 months	1 month
Appearance Visual inspection (Colour, clarity and visible particles)	Very slightly opalescent Slightly yellowish liquid Few particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer: 95% High MWT: 5% Low MWT: 0%	Monomer: 69% High MWT: 29% Low MWT: 2%	Monomer: 65% High MWT: 33% Low MWT: 3%
Concentration OD280	43 mg/mL	42 mg/mL	44 mg/mL

Comparative Formulation 2 at 25°C

Criteria	0 months	0.5 months	1 month	2 months
Appearance	Clear Slightly yellowish liquid	Very slightly opalescent	Very slightly opalescent	Very slightly opalescent

Visual inspection (Colour, clarity and visible particles)	No particles	Slightly yellowish liquid No particles	Slightly yellowish liquid No particles	Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer: 97% High MWT: 3% Low MWT: 0%	Monomer: 95% High MWT: 5% Low MWT: 0%	Monomer: 93% High MWT: 6% Low MWT: 0%	Monomer: 89% High MWT: 10% Low MWT: 1%
Concentration OD280	41 mg/mL	40 mg/mL	39 mg/mL	43 mg/mL

Comparative Formulation 2 at 40°C

Criteria	0 months	0.5 months	1 month
Appearance Visual inspection (Colour, clarity and visible particles)	Clear Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles	Very slightly opalescent Slightly yellowish liquid No particles
Purity SEC-UPLC	Monomer: 97% High MWT: 3% Low MWT: 0%	Monomer: 73% High MW 1: 26% Low MWT: 2%	Monomer: 67% High MWT: 30% Low MWT: 2%
Concentration OD280	41 mg/mL	40 mg/mL	38 mg/mL

Formulation l had lower levels of high molecular weight species formation over 5 time, compared with comparative formulations l and 2, particularly at higher température conditions.

Formulation l was also demonstrated to hâve good activity in respect of binding to VEGF-C and VEGF-D, as determined by ELISA.

ELISA Method

The assay plate was coated overnight with VEGF-C ligand at 0.1 pg /mL or VEGFD ligand at LO pg/mL. OPT-302 reference standard and test samples were diluted to a starting concentration of 1500 ng/mL and serial 2-fold dilutions are performed covering from 1.5 ng/mL to 1500 ng/mL. The plate was incubated for 60 min at 25°C and then washed 15 to remove unbound sample. The bound molécules were detected by adding HRP conjugated rabbit anti-human IgG to the assay plate and incubated for 60 min at 25°C. This was followed by the addition of TMB substrate and the assay plates were incubated in the dark for 10 minutes at room température. Colour development was stopped by adding IM HCl stop solution, which was detected by absorbance at 450 nm. The intensity of the yellow colour 20 was proportional to the amount of bound OPT-302 molécules, which in tum reflected the activity of the OPT-302 reference standard or test samples. 4-parameter curves were then generated by plotting the mean values against logIO of the serial 2-fold dilution concentrations. The reportable value was relative potency (%) of the test sample, i.e. the ratio of the EC5Û of the reference standard to the EC50 of the test sample.

On storage at -20°C, Formulation l was found to hâve 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%.

On storage at 5°C, Formulation 1 was found to hâve the followingactivity: 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 9: Stability of OPT-302 Formulations

A batch of a preferred aqueous formulation for OPT-302 was prepared, containing the following constituents:

Constituent	Amount
OPT-302 (active ingrédient)	36-44 mg/mL
Trehalose dîhydrate	10.9% w/v trehalose
Sodium phosphate (1:7.8 weight ratio mixture of sodium di hydrogen phosphate di hydrate and disodium hydrogen phosphate.7ΗιΟ)	lOmM
Polyoxyethylene (20) sorbitan monolaurate (Polysorbate 20. Tween 20)	0.01% w/v
Water For Injection	To volume
PH	7.5

Portions of the formulation batch were stored at -20 ±5°C and 5 ±3°C for prolonged periods oftime, and stability properties of the formulations were determined.

When stored at either -20°C or 5°C for up to 24 months, key stability assays such as SE-UPLC showed minimal changes in monomer content, declining from the initial amount by 0.8% at -20°C, and by 3% at 5°C, while high-molecular weight (dimer) increased by 0.7% at -20°C, and by 2.2% at 5°C compared to initiation of the stability study. Similarly, binding activity measured by ELISA decreased minimally by 9% - 19% for VEGF-C and VEGF-D.

The formulation had excellent storage stability properties at -20 and 5°C over the specified time periods.

A further batch of OPT-302 formulated as described for the batch above, was similarly subjected to 24 months storage at 5 ±3°C, followed by analysis by mass spectrometry to characterise isoform variants. It was found that deamidation variants increased by 9% over the period, isomerised variants decreased by 9%, while there were 5 negligible changes in oxidised variants. Therefore, charged variant changes were overall minimal across long-term (24 months) storage, indicating préservation of molecular structure in the formulation.

Claims (10)

1. l. An aqueous pharmaceutical composition, comprising:

   an active agent which is a soluble VEGFR-3 trap molécule, the active agent being présent at a concentration in the range of from 5 mg/rnL to 250 mg/mL, wherein the VEGFR3 trap molécule is or comprises a polypeptide;

   trehalose;

   a buffer; and water;

   wherein the pH of the aqueous pharmaceutical composition is in the range of from 6.5 to 8.0;

   and wherein the pharmaceutical composition does not contain added sodium chloride.
2. 2. An aqueous pharmaceutical composition as claimed in claim l, wherein one of the following applies:

   i) the pharmaceutical composition comprises trehalose in a concentration of at least 7.0% w/v, optionally wherein trehalose is présent at a concentration of from 8.5% w/v to 15% w/v, or wherein the composition comprises about 10.9% w/v trehalose; or ii) the pharmaceutical composition comprises trehalose at a concentration of up to 20% w/v.
3. 3. An aqueous pharmaceutical composition as claimed in claim l, wherein the soluble VEGFR-3 trap molécule comprises a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand binding polypeptide comprising immunoglobulin-like domains l-3 of the extracellular domain of human VEGFR-3 and optionally having one or more modifications in an N-glycan région of the extracellular domain, optionally wherein one of the following applies:

   i) the ligand binding polypeptide comprises the amino acid sequence defined by positions 25-329 of SEQ ID NO: I, with the proviso that positions of the polypeptide corresponding to positions I04-106 of SEQ ID NO: l are not identical to N-X-S or N-X-T; wherein the ligand binding polypeptide retains four N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO: l, positions 166-168 of SEQ ID NO: l, positions 251-253 of SEQ ID NO: 1, and positions 299-301 of SEQ ID NO: 1, and is glycosylated at said four N-glycosylation sequon sites;

   ii) the immunoglobulin constant domain fragment comprises the amino acid sequence defined by positions 99-330 of SEQ ID NO: 2;

   iii) the ligand binding polypeptide comprises the amino acid sequence defined by positions 25-329 of SEQ ID NO: 1; wherein the ligand binding polypeptide retains five N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO: I, positions 104-106 ofSEQIDNO: 1, positions 166-168 ofSEQIDNO: 1, positions 251-253 of SEQ ID NO: 1, and positions 299-301 of SEQ ID NO: 1, and is glycosylated at said five Nglycosylation sequon sites; or iv) the immunoglobulin constant domain fragment comprises the amino acid sequence defined by positions 99-330 of SEQ ID NO: 2, optionally wherein the soluble VEGFR-3 trap molécule has the amino acid sequence set forth in SEQ ID NO: 7, or has an amino acid sequence as defined by positions 1-547 of SEQ ID NO: 7.
4. 4. An aqueous pharmaceutical composition as claimed in claim 1, wherein one of the following applies:

   i) the composition is substantially free of sodium chloride;

   ii) the composition does not contain an additional sugar; or iii) the composition does not contain an additional tonicity modifier.
5. 5. An aqueous pharmaceutical composition as claimed in claim 1, wherein the composition essentially consists of:

   an active agent in a concentration of about 40 mg/ml, which is a soluble VEGFR-3 trap molécule that 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 an N-glycan région of the extracellular domain;

   trehalose in a concentration of about 10.9% w/v;

   sodium phosphate in a concentration of about 10 mM;

   polyoxyethylene (20) sorbitan monolaurate in a concentration of about 0.01% w/v; and water;

   wherein the pH of the aqueous pharmaceutical composition is about 7.5.
6. 6. A lyophilised pharmaceutical composition for reconstitution, comprising:

   an active agent which is a soluble VEGFR-3 trap molécule, wherein the VEGFR-3 trap molécule is or comprises a polypeptide;

   trehalose; and a buffer;

   wherein the weight ratio of trehalose to active agent is in the range of from l :3 to 40: l.
7. 7. A lyophilised pharmaceutical composition as claimed in claim 6, wherein the weight ratio of trehalose to active agent is in the range of from l:l to 7.5:1, from 1:1 to 5:1, or from 2.1:1 to 4.5:1, optionally wherein the weight ratio of trehalose to active agent is about 2.7:1.
8. 8. A lyophilised pharmaceutical composition as claimed in claim 7, wherein the buffer is a sodium phosphate, and the weight ratio of sodium phosphate to active agent is in the range of from 1:3 to 1:1000, or from 1:3 to 1:200, or from 1:5 to 1:100, optionally wherein the weight ratio of sodium phosphate to active agent is about 0.03:1.
9. 9. A pharmaceutical composition as claimed in claim 1, for use in the treatment and/or prévention of a disease or disorder associated with aberrant neovascularisation, angiogenesis and/or lymphangiogenesis, optionally wherein the disease or disorder is an ocular disease or disorder.
10. 10. A port device for implantation in an eye, the port device comprising a réservoir containing a pharmaceutical composition as claimed in claim 1, and wherein the port device permits controlled release of active agent into the vitreous of the eye, optionally wherein one or both of the following applies:

    i) the port device comprises a semipermeable membrane that permits passive diffusion of active agent into the vitreous of the eye; and ii) the port device comprises a septum which permits refilling of the réservoir with additional pharmaceutical composition using a needie.