阅读说明：本技术 治疗眼部疾病或病症的方法 (Methods of treating ocular diseases or disorders ) 是由 K·E·希利 E·I·阿尔蒂奥克 D·V·谢弗 W·M·杰克逊 于 2016-12-08 设计创作，主要内容包括：本公开提供治疗眼部疾病或病症的方法。所述方法涉及将包含生物活性多肽和生物相容性聚合物的缀合物直接施用到眼中。(The present disclosure provides methods of treating ocular diseases or disorders. The methods involve administering a conjugate comprising a biologically active polypeptide and a biocompatible polymer directly into the eye.)

1. A method of treating an ocular disease or disorder in an individual, the method comprising administering to the individual an effective amount of a conjugate comprising:

a) a biologically active polypeptide having a molecular weight of about 5kDa to about 2000 kDa; and

b) a biocompatible polymer having a molecular weight of at least about 50,000 daltons,

wherein the polypeptide is covalently linked to the polymer, either directly or through a linker, and wherein the molar ratio of the biologically active polypeptide to the polymer is at least about 10:1,

wherein the administration is by intravitreal administration.

2. The method of claim 1, wherein the biologically active polypeptide is: i) a receptor; ii) a ligand for a receptor; iii) an antibody; or iv) an enzyme.

3. The method of claim 1, wherein the polymer is a linear polymer comprising a plurality of subunits selected from the group consisting of: hyaluronic acid, acrylic acid, ethylene glycol, methacrylic acid, acrylamide, hydroxyethyl methacrylate, mannitol, maltose, glucose, arabinose, taurine, betaine, modified cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, modified starch, hydrophobically modified starch, hydroxyethyl starch, hydroxypropyl starch, amylose, amylopectin, oxidized starch, heprosan, heparin, chondroitin sulfate, heparin sulfate and copolymers thereof.

4. The method of claim 1, wherein the polymer is linear poly (acrylic acid) or carboxymethyl cellulose.

5. The method of claim 1, wherein the polymer is hyaluronic acid.

6. The method of claim 1, wherein the molar ratio of the biologically active polypeptide to the polymer is about 10:1 to about 25: 1.

7. The method of claim 1, wherein the molar ratio of the biologically active polypeptide to the polymer is about 25:1 to about 50: 1.

8. The method of any one of claims 1-7, wherein the biologically active polypeptide is an angiogenesis inhibitor.

9. The method of claim 1, wherein the biologically active polypeptide is a soluble Vascular Endothelial Growth Factor (VEGF) receptor, angiostatin, endostatin, angiostatin, or a VEGF-specific antibody.

10. The method of claim 1, wherein: a) the biologically active polypeptide is a soluble Vascular Endothelial Growth Factor (VEGF) receptor and the polymer is hyaluronic acid; b) the biologically active polypeptide is a VEGF-specific antibody and the polymer is carboxymethylcellulose; or c) the biologically active polypeptide is a VEGF-specific antibody and the polymer is hyaluronic acid.

11. The method of claim 10, wherein the hyaluronic acid has a molecular weight of about 600kDa to about 700kDa, or about 750kDa to about 1 MDa.

12. The method of claim 10 or claim 11, wherein the molar ratio of the VEGF receptor to the hyaluronic acid is about 20: 1.

13. The method of any one of claims 1-12, wherein the vitreal half-life of the conjugate is at least 7 days.

14. The method of any one of claims 1-13, wherein the individual is a human.

15. The method of any one of claims 1-14, wherein the ocular disorder is macular degeneration, choroidal neovascularization, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, or ocular inflammation.

16. The method of any one of claims 1-15, wherein the conjugate is administered once every two months, once every three months, once every 6 months, or once a year.

17. The method of claim 1, wherein the vitreous half-life of the conjugate is at least 5-fold greater than the half-life of the biologically active polypeptide not conjugated to the biocompatible polymer.

Disclosure of Invention

The present disclosure provides methods of treating ocular diseases or disorders. The methods involve administering a conjugate comprising a biologically active polypeptide and a biocompatible polymer directly into the eye.

The present disclosure provides a method of treating an ocular disease or disorder in an individual, the method comprising administering to the individual an effective amount of a conjugate comprising: a) a biologically active polypeptide having a molecular weight of about 5kDa to about 2000 kDa; and b) a biocompatible polymer having a molecular weight of at least about 50,000 daltons, wherein the polypeptide is covalently attached to the polymer, either directly or through a linker, and wherein the molar ratio of the biologically active polypeptide to the polymer is at least about 10:1, wherein the administration is by intravitreal administration. In some cases, the biologically active polypeptide is: i) a receptor; ii) a ligand for a receptor; iii) an antibody; or iv) an enzyme. In some cases, the polymer is a linear polymer comprising a plurality of subunits selected from the group consisting of: hyaluronic acid, acrylic acid, ethylene glycol, methacrylic acid, acrylamide, hydroxyethyl methacrylate, mannitol, maltose, glucose, arabinose, taurine, betaine, modified cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, modified starch, hydrophobically modified starch, hydroxyethyl starch, hydroxypropyl starch, amylose, amylopectin, oxidized starch, heprosan, heparin, chondroitin sulfate, heparin sulfate and copolymers thereof. In some cases, the polymer is a linear poly (acrylic acid). In some cases, a transparent acid. In some cases, the molar ratio of the biologically active polypeptide to the polymer is about 10:1 to about 25: 1. In some cases, the molar ratio of the biologically active polypeptide to the polymer is about 25:1 to about 50: 1. In some cases, the biologically active polypeptide is an angiogenesis inhibitor. In some cases, the biologically active polypeptide is a soluble Vascular Endothelial Growth Factor (VEGF) receptor, angiostatin, endostatin, angiostatin, or a VEGF-specific antibody. In some cases, the biologically active polypeptide is a soluble Vascular Endothelial Growth Factor (VEGF) receptor, and wherein the polymer is hyaluronic acid. In some cases, the hyaluronic acid has a molecular weight of about 600kDa to about 700 kDa. In some cases, the molar ratio of the VEGF receptor to the hyaluronic acid is about 20: 1. In some cases, the vitreal half-life of the conjugate is at least 7 days. In some cases, the individual is a human. In some cases, the ocular disorder is macular degeneration, choroidal neovascularization, macular edema, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, or ocular inflammation. In some cases, the conjugate is administered once every two months, once every three months, once every 6 months, or once a year. In some cases, the vitreous half-life of the conjugate is at least 5-fold greater than the half-life of the biologically active polypeptide not conjugated to the biocompatible polymer.

Drawings

FIG. 1 is a schematic depiction of the synthesis of the conjugate mvsFlt.

FIGS. 2A-2C depict inhibition of corneal angiogenesis by sFlt and mvsFlt.

FIGS. 3A-3B depict the residence time of mvsFlt in rat vitreous.

FIGS. 4A-4C depict the inhibition of retinal angiogenesis by mvsFlt.

FIGS. 5A-5B provide schematic depictions of proposed mechanisms of action of mvsFlt.

Figure 6 depicts the in vivo residence time of higher molecular weight dextran.

FIGS. 7A-7D depict multivalent sFlt synthesis and schematic.

FIGS. 8A-8D depict characterization of mvsFlt conjugation efficiency and size.

FIGS. 9A-9B depict VEGF₁₆₅ELISA and VEGF₁₆₅mvsFlt bioconjugates to VEGF in a dependent HUVEC viability assay₁₆₅Effects of dependent Activity.

FIGS. 10A-10E depict the effect of mvsFlt on HUVEC tube formation.

FIGS. 11A-11B depict mvsFlt versus VEGF₁₆₅Influence of driven HUVEC migration.

FIGS. 12A-12C depict the effect of sFlt conjugation to HyA on migration and diffusion of mvsFlt in HyA gels.

FIGS. 13A-13B depict data showing that conjugation to HyA reduces the susceptibility of proteases to degradation by MMP-7.

Fig. 14A-14B depict the characterization of HyA hydrogels.

Figures 15A-15E depict data from gel release data fitting Fickian diffusion.

FIGS. 16A-16C depict amino acid sequences of biologically active polypeptides.

FIG. 17 depicts the amino acid sequence of an exemplary biologically active polypeptide, sFlt.

FIG. 18 depicts the amino acid sequence of the scFv anti-VEGF antibody (SEQ ID NO: 5).

FIG. 19 depicts the amino acid sequence of a VHH anti-VEGF antibody (SEQ ID NO: 6).

FIGS. 20A-20C depict unconjugated and conjugated anti-VEGF antibodies withVEGF-A₁₆₅In combination with (1).

Figure 21 depicts the half-life of conjugated multivalent VHH anti-VEGF antibodies compared to unconjugated VHH anti-VEGF antibodies.

Figures 22A-22B depict the in vivo residence time and percentage of protein recovered for conjugated and unconjugated VHH anti-VEGF antibodies after injection into rat eyes.

FIGS. 23A-23B binding of conjugated multivalent VHH anti-VEGF antibodies to VEGF-A using an ELISA assay₁₆₅The capabilities of (a) were compared.

Definition of

The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term "polypeptide" includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; an immunolabeling protein; and similar proteins. The term "polypeptide" includes polypeptides comprising one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety. The term "polypeptide" includes post-translationally modified polypeptides.

The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including, but not limited to, Fab, Fv, single chain Fv (scfv), and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, single domain antibodies (VHH and VANR), and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

An "antibody fragment" includes a portion of an intact antibody, such as the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (Zapata et al, Protein Eng.8(10):1057-1062 (1995)) ); a single chain antibody molecule; single domain antibodies (e.g., camelid antibodies or "VHH" fragments (see, e.g., Harmsen and De Haard (2007) appl. Microbiol. Biotechnol.77: 13); VNARs; and nanobodies; see, e.g., Wesolowski et al (2009) Med. Microbiol. Immunol.198: 157); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the designation of which reflects the ability to crystallize readily. Pepsin treatment produces F (ab') which has two antigen binding sites and is still capable of cross-linking antigens₂And (3) fragment.

"Single chain Fv" or "sFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide is further at V_HDomains with V_LPolypeptide linkers are included between the domains to enable the sFv to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315 (1994).

As used herein, the term "affinity" refers to the equilibrium constant for reversible binding of two reagents and is expressed as the dissociation constant (Kd). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater or greater than the affinity of the antibody for an unrelated amino acid sequence. The affinity of an antibody for a target protein can be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactivity" and "preferential binding" are used interchangeably herein with respect to antibodies and/or antigen binding fragments.

The term "binding" refers to a direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Non-specific binding means at less than about 10^-7Binding with affinity for M, e.g. at 10^-6M、10^-5M、10^-4M, etc.

As used herein, the term "copolymer" describes a polymer containing more than one type of subunit. The term encompasses polymers comprising two, three, four, five or six types of subunits.

The terms "subject," "individual," "host," and "patient" are used interchangeably herein for one or more members of any mammalian or non-mammalian species. Thus, subjects and patients include, but are not limited to, humans, non-human primates, canines, felines, ungulates (e.g., equines, bovines, porcines (e.g., swine)), avians, rodents (e.g., rats, mice), and other subjects. Non-human animal models, particularly mammals, such as non-human primates, muroids (e.g., mice, rats), lagomorphs, and the like, are useful for experimental studies.

"Treating" or "treatment" of a condition or disease includes: (1) preventing at least one symptom of the condition, i.e., causing clinical symptoms to not significantly develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or exhibit symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or symptoms thereof, or (3) alleviating the disease, i.e., causing regression of the disease or clinical symptoms thereof.

By "therapeutically effective amount" or "effective amount" is meant an amount of conjugate that, when administered to a mammal or other subject to treat a disease, is administered alone, in combination with another agent or in one or more doses, sufficient to effect such treatment of the disease. The "therapeutically effective amount" may vary depending on the conjugate and on one or more other factors, such as the disease and its severity, the age, weight, etc., of the subject to be treated.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the conjugate in association with a pharmaceutically-acceptable diluent, carrier or vehicle calculated to produce the desired effect.

"pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", "pharmaceutically acceptable carrier" and "pharmaceutically acceptable adjuvant" means excipients, diluents, carriers and adjuvants that are generally safe, non-toxic and neither biologically nor otherwise undesirable and that can be used to prepare pharmaceutical compositions, and include excipients, diluents, carriers and adjuvants that are acceptable for veterinary use as well as human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipients, diluents, carriers and adjuvants" includes one and more than one such excipient, diluent, carrier and adjuvant.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the stated limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and/or materials to which the publications refer.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide-polymer conjugate" includes a plurality of such conjugates and reference to "the ocular disorder" includes reference to one or more ocular disorders and equivalents thereof known to those skilled in the art, and so forth. It is also noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," etc., or use of a "negative" limitation in reciting claim elements.

It is to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations that are embodiments of the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically contemplated in the present invention and disclosed herein as if each and every such subcombination was individually and specifically disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication date provided may be different from the actual publication date that may need to be independently confirmed.

Detailed Description

The present disclosure provides a method of treating an ocular disease or disorder in a subject. The methods generally involve administering to an individual in need thereof an effective amount of a conjugate comprising a biologically active polypeptide and a biocompatible polymer, wherein the administration is direct administration into the eye, e.g., the administration is intravitreal administration.

The intravitreal injection route is the most effective method of delivering drug products to intraocular structures located in the posterior chamber. It is therefore the preferred method of delivering drugs that act on the retina. However, each intravitreal injection risks causing retinal detachment due to interference with the integrity of the eye. Increasing the volume to the posterior chamber leads to elevated intraocular pressure and there are risks associated with ocular discomfort relieving the increased pressure. There is also a risk of introducing infection into the eye. These complications all have the risk of impaired vision, which must be balanced against the potential benefits of using intravitreal routes to administer drugs.

Therefore, there is a strong need to improve the residence time of drugs designed to be conventionally injected into the vitreous via the intravitreal route. Alternatively, in some cases, chemical modification of existing drugs may be practical in order to increase their residence time in the posterior chamber. This strategy has the potential to reduce the frequency of drug administration and thus the overall risk of complications due to drug administration over time. Increasing the duration of biological activity may also produce enhanced drug treatment results.

Drugs that exhibit greater intravitreal residence times may be preferred for patients relative to drug products that must be administered more frequently to achieve equivalent therapeutic function. Although intravitreal injections are performed under local anesthesia and are not generally considered painful, they place a burden on the patient. It must be performed by a clinician and therefore requires a clinic visit for each administration of the drug. Due to increased tearing, there is often short-term irritation and blurred vision. Transient tear changes may also be present in the appearance of the eye near the injection site. Thus, the patient may show a preference for equivalent treatments that require fewer intravitreal injections.

The need for less frequent injections is also preferred from the perspective of the physician. Intravitreal injections must be performed by the ophthalmologist and therefore this procedure can account for a significant portion of its clinical time. The number of patients receiving intravitreal treatment in practice may be limited by the frequency with which each patient must receive intravitreal injections. Less frequent injections will increase the number of patients that can receive a treatment regimen. Longer acting drugs are also preferred for depot or long-term drug delivery devices, as these typically require longer implantation procedures and access to the operating room, which may offset the benefits of less frequent administration for the clinician.

The conjugate comprising a biologically active polypeptide and a biocompatible polymer exhibits a greater half-life in the vitreous than the half-life in the vitreous of a biologically active polypeptide not conjugated to a biocompatible polymer. The increased half-life of the conjugate in the vitreous confers certain advantages, including, for example, reduced patient burden; reducing the number and/or frequency of administrations; the safety is increased; the incidence of infection is reduced; increasing patient compliance; and increase the therapeutic effect. In addition, conjugates as described herein allow for the use of polypeptides to treat ocular disorders that will not remain in unconjugated form in the eye for a period of time suitable for treatment.

In some cases, an effective amount of the conjugate is an amount effective to inhibit pathological angiogenesis in the eye of the subject. For example, in some cases, an effective amount of the conjugate is an amount effective to inhibit pathological angiogenesis in the eye of the subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, or more than 80%, when administered in one or more doses, as compared to the extent of pathological angiogenesis in the eye without treatment with the conjugate or prior to treatment with the conjugate.

In some cases, an effective amount of the conjugate is an amount effective to reduce intraocular pressure in an eye of the subject. For example, in some cases, an effective amount of the conjugate is an amount effective to reduce intraocular pressure by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% or more than 80% when administered in one or more doses as compared to intraocular pressure in the eye without treatment with the conjugate or prior to treatment with the conjugate.

In some cases, an effective amount of the conjugate is an amount effective to reduce macular edema in an eye of the subject. For example, in some cases, an effective amount of the conjugate is an amount effective to reduce macular edema by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% or more than 80% when administered at one or more doses as compared to the level of macular edema in the eye without treatment with the conjugate or prior to treatment with the conjugate.

In some cases, an effective amount of the conjugate is an amount effective to increase visual acuity in the eye of the subject. For example, in some cases, an effective amount of the conjugate is an amount effective to increase visual acuity in an eye of the subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, or at least 10-fold or more than 10-fold when administered at one or more doses compared to visual acuity in the eye in the absence of treatment with the conjugate or prior to treatment with the conjugate.

In some cases, an effective amount of the conjugate is an amount effective to inhibit progression of an ocular disease in the subject. For example, in some cases, an effective amount of the conjugate is an amount effective to inhibit progression of an ocular disease in an individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more when administered at one or more doses compared to progression in the absence of or prior to treatment with the conjugate.

For example, in some cases, an effective amount of a conjugate is an amount that is effective to inhibit the progression of non-exudative ARMD to exudative ARMD or to inhibit the progression of non-exudative ARMD to a more severe form when administered in one or more doses. In some embodiments, the effective amount of the conjugate is an amount effective to inhibit progression of early stage ARMD (AREDS 2) to intermediate stage ARMD (AREDS 3) or to late stage ARMD (AREDS 4). In some embodiments, the effective amount of the conjugate is an amount effective to inhibit progression of intermediate arm md (arms 3) to late arm md (arms 4).

In some cases, an effective amount of the conjugate is an amount effective to enhance the bioactivity of a retinal cell, e.g., wherein the retinal cell is a photoreceptor, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell.

Conjugates

In some embodiments, polypeptide-polymer conjugates (also referred to herein as "conjugates" for simplicity) suitable for use in the methods of the present disclosure have the formula:

X-(Y)_n-Z，

wherein X is a biologically active polypeptide;

y is an optional linker moiety such that n is 0 or an integer from 1 to about 10; and is

Z is a biocompatible polymer comprising about 50 subunits to 100,000 subunits and/or having a molecular weight of 10kDa to 500 kDa.

A conjugate comprising a biologically active polypeptide and a biocompatible polymer exhibits a half-life in the vitreous that is at least about 25%, at least about 50%, at least about 75%, at least about 2 fold, at least about 5 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 500 fold, or at least about 1000 fold, or more than 1000 fold greater than the half-life of the biologically active polypeptide in the vitreous that is not conjugated to the biocompatible polymer. The conjugate comprising a biologically active polypeptide and a biocompatible polymer exhibits a half-life in the vitreous that is 5-fold to 10-fold greater than the half-life in the vitreous of the biologically active polypeptide that is not conjugated to the biocompatible polymer.

In some cases, a conjugate comprising a biologically active polypeptide and a biocompatible polymer exhibits a half-life in the vitreous of about 12 hours to about 24 hours, about 1 day to about 3 days, about 3 days to about 7 days, one week to about 2 weeks, about 2 weeks to about 4weeks, or about 1 month to about 6 months.

In some cases, a conjugate comprising a biologically active polypeptide and a biocompatible polymer exhibits a therapeutically effective residence time in the vitreous of about 12 hours to about 24 hours, about 1 day to about 3 days, about 3 days to about 7 days, one week to about 2 weeks, about 2 weeks to about 4weeks, about 1 month to about 3 months, or about 3 months to about 6 months.

The biological activity of a polypeptide conjugated to a polymeric substrate is enhanced relative to the activity of a polypeptide in soluble form, e.g., as compared to the activity of a polypeptide not conjugated to a polymer. In some embodiments, the biological activity of the polypeptide in the polypeptide-polymer conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold or more than 1000-fold greater than the biological activity of the polypeptide in a soluble (unfugated) form.

In some embodiments, the bioactivity of a polypeptide in a suitable polypeptide-polymer conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2 fold, at least about 5 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 500 fold, or at least about 1000 fold or more than 1000 fold greater than the bioactivity of a polypeptide conjugated to a polymer at a 1:1 molar ratio.

In some embodiments, the biological activity of the polypeptide in a suitable polypeptide-polymer conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold or more than 1000-fold greater than the biological activity of the polypeptide when present in admixture with the polymer.

In some cases, the half maximal Effective Concentration (EC) of the polypeptide in the subject polypeptide-polymer conjugates₅₀) Specific EC for soluble (unconjugated form) polypeptides₅₀At least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold or more than 1000-fold lower.

In some cases, the half maximal Inhibitory Concentration (IC) of the polypeptide in the subject polypeptide-polymer conjugates₅₀) IC of a more soluble (unconjugated form) polypeptide₅₀At least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold or more than 1000-fold lower.

Whether the biological activity of a polypeptide in a polypeptide-polymer conjugate is increased relative to the biological activity of the polypeptide in soluble (unconjugated) form is readily determined using one or more appropriate assays for biological activity.

The molar ratio of polypeptide to polymer may vary from about 5:1 to about 100:1, such as from about 5:1 to about 7:1, from about 7:1 to about 10:1, from about 10:1 to about 12:1, from about 12:1 to about 15:1, from about 15:1 to about 20:1, from about 20:1 to about 25:1, from about 25:1 to about 30:1, from about 30:1 to about 35:1, from about 35:1 to about 40:1, from about 40:1 to about 45:1, from about 45:1 to about 50:1, from about 50:1 to about 60:1, from about 60:1 to about 70:1, from about 70:1 to about 80:1, from about 80:1 to about 90:1, or from about 90:1 to about 100: 1.

For example, where the polypeptide polymer conjugate comprises an angiogenesis-inhibiting polypeptide (e.g., the polypeptide is an anti-angiogenic polypeptide), in some embodiments, the anti-angiogenic polypeptide of the polypeptide-polymer conjugate inhibits angiogenesis by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, compared to the extent of inhibition of angiogenesis by the anti-angiogenic polypeptide conjugated to the polymer in admixture with the polymer, in soluble (unconjugated) form, or in a 1:1 molar ratio, At least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times, or more than 1000 times or more.

Polymer and method of making same

Suitable polymers for conjugation to a biologically active polypeptide include biocompatible polymers comprising from about 50 to about 100,000 subunits, e.g., from about 50 to about 100 subunits, from about 100 to about 500 subunits, from about 500 to about 1,000 subunits, from about 1,000 to about 5,000 subunits, from about 5,000 to about 10,000 subunits, from about 10,000 to about 25,000 subunits, from about 25,000 to about 50,000 subunits, or from about 50,000 to about 100,000 subunits. In some embodiments, the linear polymer comprises more than 100,000 subunits.

Suitable polymers for conjugation with the biologically active polypeptide include biocompatible polymers having a molecular weight of 10 kilodaltons (kDa) to 500 kDa. For example, suitable polymers conjugated to the bioactive polypeptide include biocompatible polymers having a molecular weight of 10kDa to 15kDa, 15kDa to 20kDa, 20kDa to 25kDa, 25kDa to 50kDa, 50kDa to 75kDa, 75kDa to 100kDa, 100kDa to 125kDa, 125kDa to 150kDa, 150kDa to 200kDa, 200kDa to 250kDa, 250kDa to 300kDa, 300kDa to 350kDa, 350kDa to 400kDa, 400kDa to 450kDa, or 450kDa to 500 kDa. Suitable polymers for conjugation with the biologically active polypeptide include biocompatible polymers having a molecular weight of greater than 500 kDa. Suitable polymers for conjugation with the biologically active polypeptide include biocompatible polymers having a molecular weight of 500kDa to 2 million daltons (MDa). For example, suitable polymers conjugated to a biologically active polypeptide include biocompatible polymers having a molecular weight of 500kDa to 750kDa, 750kDa to 1MDa, 1MDa to 1.5MDa, 1.5MDa to 2MDa, or 2MDa to 3 MD.

In some cases, the subunits are all the same, e.g., the polymer is a homopolymer. In other cases, more than one subunit is present, e.g., the polymer is a heteropolymer or copolymer. In some cases, the polymer is a linear polymer. In other cases, the polymer may include one or more branches.

Suitable polymers include natural polymers, semi-synthetic polymers, and synthetic polymers.

Suitable natural polymers include hyaluronic acid, collagen, glycosaminoglycans, cellulose, polysaccharides, and the like.

Suitable semi-synthetic polymers include, but are not limited to, collagen or its precursors crosslinked with aldehydes, dicarboxylic acids or their halides, diamines, cellulose derivatives, hyaluronic acid, chitin, chitosan, gellan gum, xanthan gum, pectin or pectic acid, polysaccharides, polymannan, agar, agarose, natural gums and glycosaminoglycans.

Suitable synthetic polymers include, but are not limited to, polymers or copolymers obtained from: polydioxanone, polyphosphazene, polysulfone resins, poly (acrylic acid), poly (butyl acrylate), poly (ethylene glycol), poly (propylene), polyurethane resins, poly (methacrylic acid) -methyl ester, poly (methacrylic acid) -N-butyl ester, poly (methacrylic acid) -t-butyl ester, polytetrafluoroethylene, polyperfluoropropylene, poly N-vinylcarbazole, poly (methyl isopropenyl ketone), poly alpha-methylstyrene, polyvinyl acetate, poly (formaldehyde), poly (ethylene-co-vinyl acetate), polyurethane, poly (vinyl alcohol), and polyethylene terephthalate; ethylene vinyl alcohol copolymers (commonly known under the generic name EVOH or EVAL); poly (butyl methacrylate); poly (hydroxyvalerate); poly (L-lactic acid); polycaprolactone; poly (lactide-co-glycolide); poly (hydroxybutyrate); poly (hydroxybutyrate-co-valerate); polydioxanone; a polyorthoester; a polyanhydride; poly (glycolic acid) (PGA); poly (D, L-lactic acid) (PLA); copolymers of PGA and PLA; poly (glycolic acid-co-trimethylene carbonate); polyphosphate ester; polyphosphate polyurethane; poly (amino acids); a cyanoacrylate; poly (trimethylene carbonate); poly (imino carbonates); copoly (ether-ester) (e.g., PEO/PLA); polyalkylene oxalates; polyphosphazene; a polyurethane; a siloxane; a polyester; a polyolefin; polyisobutylene and ethylene-alpha olefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; a polyvinyl ketone; polyvinyl aromatic compounds such as polystyrene; polyvinyl esters such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, and ethylene-vinyl acetate copolymer; polyamides such as nylon 66 and polycaprolactam; an alkyd resin; a polycarbonate; polyformaldehyde; a polyimide; a polyether; an epoxy resin; a polyurethane; artificial silk; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; a cellulose ether; amorphous teflon; and carboxymethyl cellulose.

The polymer conjugated to the biologically active polypeptide may comprise a plurality of subunits selected from the group consisting of: hyaluronic acid, acrylic acid, ethylene glycol, vinyl, propylene, methyl methacrylate, methacrylic acid, acrylamide, hydroxyethyl methacrylate, tetrafluoroethylene, formaldehyde, sugars (e.g., glucose, mannitol, maltose, arabinose, etc.), taurine, betaine, modified cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, modified starch, hydrophobically modified starch, hydroxyethyl starch, hydroxypropyl starch, amylose, amylopectin, oxidized starch, amino acids, and copolymers of any of the foregoing. In some embodiments, the polymer does not comprise an amino acid. In some cases, the polymer conjugated to the biologically active polypeptide comprises heprosan, heparin, chondroitin sulfate, or heparin sulfate.

In some embodiments, the polymer comprises hyaluronic acid or a hyaluronic acid derivative. Transparent acid derivatives include, for example, hyaluronic acid esters, in which some or all of the carboxylic acid functions are esterified with alcohols of aliphatic, aromatic, araliphatic, cycloaliphatic or heterocyclic series; a heavy metal salt of a half-ester of succinic acid with hyaluronic acid or with a partial or full ester of hyaluronic acid or a half-ester of succinic acid with hyaluronic acid or with a partial or full ester of hyaluronic acid; sulfated or N-sulfated hyaluronic acid. In some embodiments, the polymer is hyaluronic acid. In some embodiments, the polymer is a hyaluronic acid derivative.

Biologically active polypeptides

The size of the polypeptide may range from 2kDa to about 2000kDa, e.g., from about 2kDa to about 5kDa, from about 5kDa to about 10kDa, from about 10kDa to about 25kDa, from about 25kDa to about 50kDa, from about 50kDa to about 100kDa, from about 100kDa to about 250kDa, from about 250kDa to about 500kDa, from about 500kDa to about 1000kDa, from about 1000kDa to about 2000 kDa.

Biologically active polypeptides suitable for inclusion in conjugates for use in the methods of the present disclosure include, but are not limited to, neuroprotective polypeptides, anti-angiogenic polypeptides, anti-apoptotic factors, and polypeptides that enhance retinal cell function.

Biologically active polypeptides suitable for inclusion in conjugates for use in the methods of the present disclosure include, but are not limited to, neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., soluble Vascular Endothelial Growth Factor (VEGF) receptor; VEGF-binding antibodies; VEGF-binding antibody fragments (e.g., single chain anti-VEGF antibodies); endostatin; tumstatin; angiostatin; soluble Flt polypeptide (Lai et al (2005) mol. Ther.12: 659); Fc fusion proteins comprising soluble Flt polypeptide (see, e.g., Pechan et al (2009) Gene Ther.16: 10); Pigment Epithelium Derived Factor (PEDF); soluble Tie-2 receptor; etc.); tissue inhibitors of metalloproteinase-3 (TIMP-3); light-responsive opsins, such as rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl); and similar polypeptides. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF); fibroblast growth factor 2; nerve growth factor (NTN); ciliary neurotrophic factor (CNTF); nerve Growth Factor (NGF); neurotrophin-4 (NT 4); brain Derived Neurotrophic Factor (BDNF); an epidermal growth factor; rhodopsin; an X-linked apoptosis inhibitor; and Sonic hedgehog (Sonic hedgehog).

Biologically active polypeptides suitable for inclusion in conjugates for use in the methods of the present disclosure include, but are not limited to, soluble Vascular Endothelial Growth Factor (VEGF) receptors; angiostatin, endostatin; angiostatin; the retinal pigment epithelium specific protein 65kDa (RPE 65); and compactin (compstatin). In some cases, the biologically active polypeptide is a soluble fms-like tyrosine kinase-1 (sFlt-1) polypeptide. In some cases, the biologically active polypeptide is a single domain camelid (VHH) anti-VEGF antibody (VHH anti-VEGF antibody). In some cases, the biologically active polypeptide is a single chain Fv anti-VEGF antibody (scFv anti-VEGF antibody).

Biologically active polypeptides suitable for inclusion in conjugates for use in the methods of the present disclosure include, but are not limited to, glial-derived neurotrophic factor, fibroblast growth factor 2, nerve growth factor, ciliary neurotrophic factor, nerve growth factor, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked apoptosis inhibitors, retinoschisin, RPE65, retinitis pigmentosa gtpase interacting protein-1, peripherin-2, rhodopsin, and sonic hedgehog.

Suitable polypeptides also include retinolytic proteins. Suitable polypeptides include, for example, retinitis pigmentosa gtpase modulator (RGPR) interacting protein-1 (see, e.g., GenBan k accession nos. Q96KN7, Q9EPQ2, and Q9GLM 3); peripherin-2 (Prph2) (see, e.g., GenBank accession NP-000313; and Travis et al (1991) Geno mics 10: 733); a peripherin protein; retinal pigment epithelium-specific protein (RPE65) (see, e.g., GenBank AAC 39660; and Morimura et al (1998) Proc. Natl. Aca d. Sci. USA 95: 3088); and similar proteins.

Suitable polypeptides also include: CHM (choroideremia (Rab convoluting protein 1)), polypeptides that cause choroideremia when defective or absent (see, e.g., Donnelly et al (1994) hum. mol. Genet.3: 1017; and van Bokhoven et al (1994) hum. mol. Genet.3: 1041); and clastic homolog 1(CRB1), polypeptides that cause Leber congenital melasma and retinitis pigmentosa when defective or absent (see, e.g., den Hollander et al (1999) nat. Genet.23: 217; and GenBank accession number CAM 23328).

Suitable polypeptides also include polypeptides that cause color blindness when defective or absent, where such polypeptides comprise, for example, cone cGMP-regulated channel subunit α (CNGA3) (see, e.g., GenBank accession No. NP _ 001289; and gaij et al (2011) Ophthalmology 118: 160-; cone cGMP-regulated cation channel β -subunit (CNGB3) (see, e.g., Kohl et al (2005) Eur J Hum genet.13(3): 302); guanine nucleotide binding protein (G protein), alpha transduction active polypeptide 2(GNAT2) (ACHM 4); and ACHM 5; and polypeptides that cause various forms of color blindness when defective or absent (e.g., L-opsin, M-opsin, and S-opsin). See Mancuso et al (2009) Nature 461(7265):784- & 787.

Biologically active polypeptides suitable for inclusion in conjugates for use in the methods of the present disclosure include antibodies. Suitable antibodies include, for example, VEGF-specific antibodies; antibodies specific for tumor necrosis factor-alpha (TNF-alpha); and similar antibodies.

Suitable antibodies include, but are not limited to, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, palivizumab, brodadamumab, canamumab, certolizumab, claakizumab, dallizumab, denosumab, efuzumab, epratuzumab, edalizumab, not zanuzumab, rituximab, aryltuzumab, gavoruzumab, golimumab, infliximab, nanobubuzumab, natalizumab, nextromab, oxcarbazepine, ofatumumab, oxutalizumab, oblizumab, palivizumab, prilizumab, ranibizumab, rituximab, secukinumab, chikunmumab, schicurizumab, matuzumab, tacitumumab, tuzumab, tollizumab, eculizumab, rivaluzumab, vallizumab, dolizumab, and the like, Vituzumab, vesizumab, volastuzumab and ziprasumab.

In some cases, the biologically active polypeptide is a soluble fms-like tyrosine kinase-1 (sFlt-1) polypeptide. In some cases, the biologically active polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a contiguous extension of 100 amino acids (aa) to 200 aa, 200 aa to 300 aa, 300 aa to 400 aa, 400 aa to 500 aa, 500 aa to 600 aa, 600 aa to 700 aa, or 700 aa to 755 aa of the amino acid sequence depicted in fig. 16A. In some cases, a biologically active polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in figure 16B. In some cases, a biologically active polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in figure 16C. In some cases, a biologically active polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in figure 17. In some cases, the biologically active polypeptide comprises the amino acid sequence depicted in fig. 17. An enterokinase cleavage site (DDDDK; SEQ ID NO:7) and a poly (His) sequence (HHHHHHHH; SEQ ID NO:8) are present at the carboxy terminus of the amino acid sequence depicted in FIG. 17. In some cases, the sFlt polypeptide does not comprise an enterokinase cleavage site or a poly (His) sequence.

In some cases, the biologically active polypeptide is an sFlt-1 polypeptide having a length of 150 amino acids to 200 amino acids, 200 amino acids to 250 amino acids, 250 amino acids to 300 amino acids, 300 amino acids to 350 amino acids, or 350 amino acids to 400 amino acids.

In some cases, the biologically active polypeptide is a scFv anti-VEGF antibody. Any suitable scFv anti-VEGF antibody may be used. A non-limiting example of the amino acid sequence of the scFv anti-VEGF antibody is provided in fig. 18. An enterokinase cleavage site (DDDDK; SEQ ID NO:7) and a poly (His) sequence (HHHHHHHH; SEQ ID NO:8) were present at the carboxy terminus of the scFv anti-VEGF antibody depicted in FIG. 18. In some cases, the scFv anti-VEGF antibody does not comprise an enterokinase cleavage site or a poly (His) sequence.

In some cases, the biologically active polypeptide is a single domain camelid (VHH) anti-VEGF antibody. Any suitable VHH anti-VEGF antibody may be used. Non-limiting examples of amino acid sequences of VHH anti-VEGF antibodies are provided in fig. 19. An enterokinase cleavage site (DDDDK; SEQ ID NO:7) and poly (His) sequence (HHHHHHHH; SEQ ID NO:8) were present at the carboxy terminus of the VHH anti-VEGF antibody depicted in FIG. 19. In some cases, the VHH anti-VEGF antibody does not comprise an enterokinase cleavage site or a poly (His) sequence.

Joint

As noted above, in some cases, suitable polypeptide-polymer conjugates comprise a linker group that links the polypeptide to the polymer. Suitable linkers include peptide linkers and non-peptide linkers.

The linker peptide may have any of a variety of amino acid sequences. Exemplary peptide linkers are between about 6 amino acids and about 40 amino acids in length, or between about 6 amino acids and about 25 amino acids in length. Exemplary linkers include poly (glycine) linkers (e.g., (Gly)_nWherein n is an integer from 2 to about 10); a linker comprising Gly and Ser; and similar joints.

Conjugation

Various conjugation methods and chemistries can be used to conjugate the polypeptide to the polymer. Various zero-length, homobifunctional and heterobifunctional crosslinking reagents can be used. Zero-length crosslinking reagents include direct conjugation of two intrinsic chemical groups without the introduction of an extrinsic material. Agents that catalyze disulfide bond formation belong to this class. Another example is a reagent which induces condensation of a carboxyl group and a primary amino group to form an amide bond, such as carbodiimide, ethyl chloroformate, Wydwold reagent K (2-ethyl-5-phenylisoxazole-3' -sulfonate) and carbonyldiimidazole. Homo-and heterobifunctional reagents typically contain two identical or two different sites, respectively, that can react with amino, sulfhydryl, guanidino, indole, or nonspecific groups.

In some embodiments, the polymer comprises an amino-reactive group for reacting with a primary amine group on the polypeptide or on the linker. Suitable amino-reactive groups include, but are not limited to, N-hydroxysuccinimide (NHS) esters, imidoesters, isocyanates, acyl halides, aryl azides, p-nitrophenyl esters, aldehydes, and sulfonyl chlorides.

In some embodiments, the polymer comprises a thiol-reactive group, e.g., for reacting with a cysteine residue in the polypeptide. Suitable thiol-reactive groups include, but are not limited to, maleimides, alkyl halides, pyridyl disulfides, and thiophthalimides.

In other embodiments, carbodiimides that are soluble in both water and organic solvents are used as the carboxyl-reactive reagent. These compounds react with free carboxyl groups to form pseudoureas, which can then be coupled to available amines to produce amide linkages.

As noted above, in some embodiments, the polypeptide is conjugated to the polymer using a homobifunctional crosslinker.

In some embodiments, the difunctional crosslinking agent is reacted with a primary amine. Homobifunctional crosslinkers that react with primary amines include NHS esters, imidoesters, isothiocyanates, isocyanates, acid halides, aryl azides, p-nitrophenyl esters, aldehydes, and sulfonyl chlorides.

Non-limiting examples of homobifunctional NHS esters include disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfo-DST), bis-2- (succinimidyloxycarbonyloxy) ethyl sulfone (BSOCOES), bis-2- (sulfosuccinimidyloxycarbonyloxy) ethyl sulfone (sulfo-BSOCOES), ethylene glycol bis (succinimidylsuccinate) (EGS), ethylene glycol bis (sulfosuccinimidylsuccinate) (sulfo-EGS), dithiobis (succinimidylpropionate) (DSP), and dithiobis (sulfosuccinimidylpropionate) (sulfo-DSP). Non-limiting examples of homobifunctional imidoesters include dimethyl malonimidoester (DMM), dimethyl succinimidoester (DMSC), dimethyl adipimidate (DMA), dimethyl pimelimide dimethyl ester (DMP), dimethyl octadiimide dimethyl ester (DMS), dimethyl-3, 3' -oxopropanimidate (DODP), dimethyl-3, 3' - (methylenedioxy) propanimidate (DMDP), dimethyl-3, 3' - (dimethylenedioxy) propanimidate (DDDP), dimethyl-3, 3' - (tetramethylenedioxy) propanimidate (DTDP), and dimethyl-3, 3' -Dithioiminopropionate (DTBP).

Non-limiting examples of homobifunctional isothiocyanates include: terephthalocyanurate (DITC) and 4,4 '-diisothiocyanato-2, 2' -disulfonic acid stilbene (DIDS). Non-limiting examples of homobifunctional isocyanates include xylene-diisocyanate, toluene-2, 4-diisocyanate, toluene-2-isocyanate-4-isothiocyanate, 3-methoxydiphenylmethane-4, 4' -diisocyanate, 2' -dicarboxy-4, 4' -azophenyl diisocyanate, and hexamethylene diisocyanate. Non-limiting examples of homobifunctional aryl halides include 1, 5-difluoro-2, 4-dinitrobenzene (DFDNB) and 4,4 '-difluoro-3, 3' -dinitrophenyl-sulfone. Non-limiting examples of homobifunctional aliphatic aldehyde reagents include glyoxal, malondialdehyde, and glutaraldehyde. Non-limiting examples of homobifunctional acylating agents include the nitrophenyl esters of dicarboxylic acids. Non-limiting examples of homobifunctional aromatic sulfonyl chlorides include phenol-2, 4-disulfonyl chloride and alpha-naphthol-2, 4-disulfonyl chloride. Additional non-limiting examples of amino-reactive homobifunctional reagents include erythritol dicarbonate, which reacts with amines to produce biscarbamates.

In some embodiments, the homobifunctional crosslinking agent reacts with free thiol groups. Homobifunctional crosslinkers that react with free mercapto groups include, for example, maleimides, pyridyl disulfides, and alkyl halides.

Non-limiting examples of homobifunctional maleimides include bismaleimide hexane (BMH), N '- (1, 3-phenylene) bismaleimide, N' - (1, 2-phenylene) bismaleimide, azophenylbismaleimide, and bis (N-maleimidomethyl) ether. Non-limiting examples of homobifunctional pyridyl disulfides include 1, 4-bis-3 '- (2' -pyridyldithio) propionamidobutane (DPDPDPB). Non-limiting examples of homobifunctional alkyl halides include 2,2 '-dicarboxy-4, 4' -diiodoacetamidoazobenzene, α '-diiodo-p-xylenesulfonic acid, α' -dibromo-p-xylenesulfonic acid, N '-bis (b-bromoethyl) benzylamine, N' -bis (bromoacetyl) phenylhydrazine, and 1, 2-bis (bromoacetyl) amino-3-phenylpropane.

As noted above, in some embodiments, the polypeptide is conjugated to the polymer using a heterobifunctional reagent. Suitable heterobifunctional reagents include amino reactive reagents comprising a pyridyl disulfide moiety; an amino reactive reagent comprising a maleimide moiety; an amino reactive agent comprising an alkyl halide moiety; and an amino-reactive reagent comprising an alkyl dihalide moiety.

Non-limiting examples of heterobifunctional reagents having a pyridyl disulfide moiety and an amino-reactive NHS ester include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl 6-3- (2-pyridyldithio) propionamidohexanoate (LC-SPDP), sulfosuccinimidyl 6-3- (2-pyridyldithio) propionamidohexanoate (sulfo-LCST-PDP), 4-succinimidyloxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene (SMPT), Divinylsulfone (DVS), and sulfosuccinimidyl 6- α -methyl- α - (2-pyridyldithio) benzoic acid amidohexanoate (sulfo-LC-SMPT).

Non-limiting examples of heterobifunctional reagents comprising a maleimide moiety and an amino-reactive NHS ester include maleimidoacetate succinimidyl ester (AMAS), 3-maleimidopropionate succinimidyl ester (BMPS), N-, γ -maleimidobutyryloxysuccinimidyl ester (GMBS), N-, γ -maleimidobutyryloxysulfosuccinimidyl ester (sulfo-GMBS), 6-maleimidohexanoate succinimidyl Ester (EMCS), 3-maleimidobenzoate succinimidyl ester (SMB), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (sulfo-MBS), Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), and sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate (sulfo-SMPB).

Non-limiting examples of heterobifunctional reagents comprising an alkyl halide moiety and an amino-reactive NHS ester include N-succinimidyl- (4-iodoacetyl) aminobenzoate (SIAB), sulfosuccinimidyl- (4-iodoacetyl) aminobenzoate (sulfo-SIAB), succinimidyl-6- (iodoacetyl) aminocaproate (SIAX), succinimidyl-6- (6- ((iodoacetyl) -amino) hexanoylamino) hexanoate (SIAXX), succinimidyl-6- (((4- (iodoacetyl) -amino) methyl) -cyclohexane-1-carbonyl) aminocaproate (SIACX) and succinimidyl-4 ((iodoacetyl) -amino) methylcyclohexane-1-carboxylate (SIAC).

A non-limiting example of a heterobifunctional reagent comprising an amino-reactive NHS ester and an alkyl dihalide moiety is N-hydroxysuccinimidyl 2, 3-dibromopropionate (SDBP). Non-limiting examples of heterobifunctional reagents comprising an alkyl halide moiety and an amino-reactive p-nitrophenyl ester moiety include p-Nitrophenyliodoacetate (NPIA).

Compositions, formulations, dosages and routes of administration

In some cases, the methods of the present disclosure comprise administering a polypeptide-polymer conjugate to an individual in need thereof, wherein the polypeptide-polymer conjugate is homogeneous, e.g., all polypeptides in the polypeptide-polymer conjugate comprise the same amino acid sequence. For example, in some embodiments, a composition to be administered to an individual comprises a plurality (e.g., multiple copies) of polypeptide-polymer conjugates, wherein each polypeptide-polymer conjugate molecule comprises polypeptides all having the same amino acid sequence.

In some cases, the methods of the present disclosure comprise administering to an individual in need thereof a composition comprising a polypeptide-polymer conjugate, wherein the composition comprises two or more polypeptide-polymer conjugates, e.g., a composition comprising a first polypeptide-polymer conjugate, wherein the first polypeptide-polymer conjugate comprises a polypeptide having a first amino acid sequence; and at least a second polypeptide-polymer conjugate, wherein the second polypeptide-polymer conjugate comprises a polypeptide having a second amino acid sequence that is different from the first amino acid sequence. In some cases, the composition comprises a third or another polypeptide-polymer conjugate. As one non-limiting example, the first polypeptide-polymer conjugate comprises a first polypeptide that is an anti-angiogenic polypeptide; and the second polypeptide-polymer conjugate comprises a second polypeptide that inhibits a cell signaling pathway. Various other combinations of the first, second, etc. polypeptides may be used. The ratio of the first polypeptide-polymer conjugate to the second polypeptide-polymer conjugate in the composition can be, for example, between about 0:001 and 10³To about 10³To within 0.001. Similarly, where the subject compositions comprise first, second, and third polypeptide-polymer conjugates, the ratio of the first, second, and third polypeptide-polymer conjugates can vary.

In addition to polypeptide-polymer conjugates, compositions suitable for use in the methods of the present disclosure may comprise one or more of: salts, e.g. NaCl, MgCl₂、KCl、MgSO₄Etc.; buffers, for example Tris buffer, N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), 2- (N-morpholino) ethanesulfonic acid (MES), 2- (N-morpholino) ethanesulfonic acid sodium salt (MES), 3- (N-morpholino) propanesulfonic acid (MOPS), N-Tris [ hydroxymethyl ] methane]Methyl-3-aminopropanesulfonic acid (TAPS); a solubilizer; detergent compositionFor example, nonionic detergents such as tween-20 and the like; a protease inhibitor; and similar agents.

Compositions suitable for use in the methods of the present disclosure can comprise a polypeptide-polymer conjugate (as described above) and a pharmaceutically acceptable excipient. Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or similar vehicles and combinations thereof. In addition, the vehicle may, if desired, contain minor amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffering agents. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17 th edition, 1985. The composition or formulation to be administered will in any case contain an amount of the agent sufficient to achieve the desired state in the subject being treated. Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are readily available to the public. Furthermore, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizing agents, wetting agents and the like are readily available to the public.

As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" are used interchangeably and include any material that does not substantially affect the biological activity of the conjugate, does not induce an immune response in the host, and does not have any substantial adverse physiological effect on the host when combined with the polypeptide-polymer conjugate. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions (such as oil/water emulsions), and any of a variety of types of wetting agents. Other carriers may also include sterile solutions, tablets including coated tablets and capsules. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clays, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also contain flavor and color additives or other ingredients. Compositions comprising such carriers may be formulated by well-known conventional methods.

The pharmaceutical composition can be formulated for a selected mode of administration, including, for example, intraocular administration, such as intravitreal administration.

Compositions comprising the conjugates can comprise an aqueous carrier, such as water, buffered water, saline, phosphate buffered saline, and the like. The composition may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.

The compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, the lyophilized formulation being combined with a sterile aqueous carrier prior to administration. The resulting aqueous solution can be packaged in a glass syringe. The pH of the formulation may range from 3 and 11, for example from about pH 5 to about pH 9, or from about pH 7 to about pH 8.

Suitable dosages of the conjugates for use in the methods of the present disclosure include about 1 μ g to about 10mg, e.g., about 1 μ g to about 5 μ g, about 5 μ g to about 10 μ g, about 10 μ g to about 20 μ g, about 20 μ g to about 25 μ g, about 25 μ g to about 50 μ g, about 50 μ g to about 100 μ g, about 100 μ g to about 150 μ g, about 150 μ g to about 250 μ g, about 250 μ g to about 500 μ g, about 500 μ g to about 750 μ g, about 750 μ g to about 1mg, about 1mg to about 5mg, or about 5mg to about 10mg per dose. In some cases, suitable doses of the conjugate for use in the methods of the present disclosure include 10mg to 100mg, e.g., 10mg to 20mg, 20mg to 25mg, 25mg to 50mg, 50mg to 75mg, or 75mg to 100mg per dose.

In some embodiments, multiple doses of the conjugate are administered. The frequency of administration of the conjugate can vary depending on any of a variety of factors, such as the severity of the symptoms, and the like. For example, in some embodiments, the conjugate is administered monthly, twice monthly, three times monthly, every other week (qow), weekly (qw), twice weekly (biw), three times weekly (tiw), four times weekly, five times weekly, six times weekly, every other day (qod), every day (qd), twice daily (qid), or three times daily (tid). In some embodiments, the conjugate is administered once every two months, once every three months, once every 6 months, or once a year.

In some cases, the composition comprising the conjugate is administered by intravitreal, transscleral, periocular, conjunctival, sub-tenon, intracameral, subretinal, subconjunctival, retrobulbar, or intratubular administration. In some cases, the composition comprising the conjugate is administered intravitreally. In some cases, the composition is delivered intravitreally or in close proximity to the posterior segment of the eye. In some cases, the composition is administered by intravitreal injection. In some cases, the composition comprising the conjugate is administered by intraocular injection.

Disorders of the disease

Ocular disorders that can be treated using the methods of the present disclosure include, but are not limited to, macular degeneration, choroidal neovascularization, macular edema, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, and ocular inflammation.

Ocular diseases that may be treated using the methods of the present disclosure include, but are not limited to, acute macular optic neurophathy; behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular region or location; ocular tumors; retinal disorders such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy and diabetic macular edema), Proliferative Vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease; sympathetic ophthalmia; vogat small willow-indigenous (Vogt Koyanagi-Harada, VKH) syndrome; grape membrane diffusion; a post-ocular condition caused by or affected by ocular laser therapy; a post-ocular condition caused by or affected by photodynamic therapy; photocoagulation, radiation retinopathy; a condition of the epiretinal membrane; retinal branch vein occlusion; ischemic anterior optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinal cleavage; retinitis pigmentosa; glaucoma, and glaucoma; elsholtzia syndrome, cone-rod degeneration; stargardt disease (yellow-spotted fundus); hereditary macular degeneration; choroidal retinal degeneration; leber congenital black; congenital stationary night blindness; choroids without veins; bard-boddler syndrome; macular telangiectasia; leber hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders including achromatopsia, achromatopsia and third color blindness.

In some cases, the ocular disease is glaucoma, retinitis pigmentosa, macular degeneration, retinal detachment, leber congenital black, diabetic retinopathy, achromatopsia, or achromatopsia.

Subject suitable for treatment

Subjects suitable for treatment with the methods of the present disclosure include individuals who have been diagnosed as having an ocular disease or disorder (e.g., any of the ocular diseases or disorders listed above). Subjects suitable for treatment with the methods of the present disclosure include individuals who have been treated for an ocular disease or disorder and who have not responded to treatment.

Individuals suitable for treatment with the methods of the present disclosure include individuals with decreased visual acuity due to ocular diseases or disorders. Individuals suitable for treatment with the methods of the present disclosure include individuals with abnormally high intraocular pressure due to an ocular disease or disorder. Individuals suitable for treatment with the methods of the present disclosure include individuals with pathological angiogenesis in the eye due to ocular diseases or disorders.

Visual acuity may be measured using, for example, a snellen chart, Bailey-Lovie chart, decimal progress chart, fleiburg visual acuity test, Minimum Angle of Resolution (MAR) measurement, or the like. Ametropia (visual distortion) can be measured using an Amsler chart. Contrast sensitivity can be measured using a Pelli-Robson chart. Diagnostic studies include, but are not limited to, standard ophthalmic examinations of the fundus, stereobiomicroscopy of the macula, intravenous fundus fluorescein angiography, fundus photography, indocyanine green video angiography, and optical coherence tomography. Subjects exhibiting abnormalities in one or more of these diagnostic studies (e.g., subjects falling outside the range considered normal for healthy eyes) can be treated according to the present disclosure. For example, a subject may be classified as having early, intermediate or late stage ARMD according to the classification scheme used in the study of age-related eye disease. Subjects falling within any of the categories described therein may be treated according to the methods of the present disclosure.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, e.g., bp, base pair; kb, kilobases; pl, picoliter; s or sec, seconds; min, min; h or hr, hours; aa, an amino acid; kb, kilobases; bp, base pair; nt, nucleotide; i.m., intramuscular (intramyogenic); i.p., intraperitoneal (intraperitoneally); s.c., subcutaneous (s.c.), etc.

Example 1:sFlt multivalent conjugates inhibit angiogenesis and improve half-life in vivo

To improve the intravitreal residence time of anti-VEGF drugs, multivalent bioconjugates of anti-VEGF proteins were synthesized. The conjugates comprised soluble fms-like tyrosine kinase-1 (sFlt) covalently grafted to a chain of hyaluronic acid (HyA). The conjugate is referred to as mvsFlt. Covalent conjugation to HyA chains was shown not to reduce the bioactivity of sFlt using a mouse corneal angiogenesis assay, and mvsFlt was comparable to sFlt in inhibiting corneal angiogenesis. In the rat vitreous model, a significant increase in intravitreal residence time of mvsFlt compared to unconjugated sFlt was observed after 2 days. The calculated intravitreal half-lives of sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, mvsFlt was shown to be more effective in inhibiting retinal neovascularization than the unconjugated form in an oxygen-induced retinopathy model, this effect likely being due to the longer half-life of mvsFlt in the vitreous. In summary, the results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF, and that this conjugation significantly improves drug half-life. These in vivo results indicate that multivalent conjugation can substantially improve drug half-life and, therefore, improve the efficacy of currently available drugs for diseases such as diabetic retinopathy, thereby improving the quality of life of patients.

Materials and methods

Expression of soluble Flt-1 receptor

The sFlt sequence [13] of the first 3 Ig-like extracellular domains of sFlt-1 was cloned into pFastBac1 plasmid (Life Technologies) and then transformed into DH10Bac Enterobacter coli, which was plated on triple antibiotic plates containing kanamycin (50. mu.g/mL Sigma Aldrich), gentamycin (7. mu.g/mL, Sigma Aldrich), tetracycline (10. mu.g/mL, Sigma Aldrich), IPTG (40. mu.g/mL, Sigma Aldrich) and Blu-gal (100. mu.g/mL, Thermo Fisher Scientific). Bacmids containing the sFlt gene were isolated from DH10Bac e.coli (Life Technologies) and transfected into SF9 insect cells for virus production (supplied by Tissue Culture Facility, UC Berkeley). The High Five insect cells (supplied by Tissue Culture Facility, UC Berkeley) were then infected with the virus to induce sFlt protein expression. After 3 days, the protein was purified from the supernatant using Ni-NTA agarose beads (Qiagen Laboratories). Recombinant sFlt was eluted from Ni-NTA beads using an imidazole (Sigma Aldrich) gradient and then concentrated and buffer exchanged with 10% glycerol/PBS using Amicon Ultra-15mL centrifuge (EMD Millipore). The protein solution was sterile filtered and the concentration was determined by BCA assay (Thermo Fisher Scientific).

Synthesis of mvsFlt conjugates

Conjugation of sFlt to HyA was performed according to the scheme in fig. 1 and as previously described ([12,14-16 ]). To prepare the thiol-reactive HyA intermediate, 3' -N- (epsilon-maleimidocaproic acid) hydrazide (EMCH, Pierce,1.2mg/mL), 1-hydroxybenzotriazole hydrate (HOBt, Sigma,0.3mg/mL) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Pierce,10mg/mL) were added to a 3mg/mL solution of 650kDa HyA (Lifecore Biotechnology) in 0.1M 2- (N-morpholino) ethanesulfonic acid (MES) (Sigma) buffer (pH 6.5) and allowed to react at 4 ℃ for 4 h. The solution was then dialyzed into pH 7.0 Phosphate Buffered Saline (PBS) containing 10% glycerol. Recombinant sFlt was treated with 10 molar excess of 2-iminothiolane to generate a thiol group for conjugation to a maleimide group on EMCH. Activated HyA-EMCH was then added to sFlt at a molar ratio of 1:10 (HyA to sFlt) and allowed to react overnight at 4 ℃ to synthesize the final mvsFlt conjugate. The mvsFlt conjugate was thoroughly dialyzed against 100kDa molecular weight cut-off (MWCO) Float-A-Lyzer G2(Spectrum Labs) in PBS pH 7.0 to remove unreacted sFlt. The BCA assay was used to measure the concentration of mvsFlt.

FIG. 1: schematic synthesis of mvsFlt. mvsFlt bioconjugates were synthesized using a 3-step reaction in which HyA was reacted with EDC and EMCH to produce thiol-reactive HyA-EMCH intermediates. The sFlt was then treated with 2-iminothiolane and reacted with a HyA-EMCH intermediate to synthesize the final mvsFlt bioconjugate.

Corneal angiogenesis assay

All experiments were performed with wild type 7 to 12 week old male and female littermate FVB/n mice. Mice were maintained under pathogen-free conditions in a UCSF barrier facility and were performed according to procedures approved by the UCSF Institutional Animal Care and Use Committee (IACUC). All experiments were approved by UCSF IACUC prior to work. Mice were anesthetized by inhalation of isoflurane (Abbott Laboratories, Abbott Park, IL), 10mg/kg carprofen (Sigma, st. louis, MO) and by topical application of 0.5% proparacaine (Bausch & Lomb, Rochester, NY) placed on the cornea. An alkaline burn was created by applying a 2.5mm diameter filter paper soaked in 0.1N NaOH (Sigma Aldrich) to the central cornea for 30 seconds followed by rinsing with 250 μ L of PBS. After chemical burn treatment, topical 0.5% proparacaine was added to the cornea for anesthesia. Mice were given subconjunctival injections of 5. mu.L of sFlt (150. mu.g/ml), mvsFlt (150. mu.g/ml) or PBS on days 1 and 3 after burn. 10 days after treatment, eyes were removed and the cornea dissected and fixed in 4% paraformaldehyde overnight at 4 ℃. The cornea was blocked with 3% BSA and stained with DAPI, rabbit anti-mouse CD31 primary antibody (Santa Cruz Biotechnology) and goat anti-rabbit Alexa Fluor 488 secondary antibody (Life Technologies) for visualization and quantification of blood vessels. The cornea was cut into quadrants and mounted flat onto a slide using fluorocount mounting medium (Sigma Aldrich). Imaging was performed using an automated slide scanner Zeiss Axioscan Z1(Zeiss Instruments). Corneal vascular coverage was quantified using NIH ImageJ software by comparing total corneal area to corneal vascularized area.

Determination of the residence time in the mvsFlt vitreous body

All residence time experiments were performed on 8-week-old Brown Norway rats obtained from Charles River Laboratories and treated according to protocols approved by the committee for animal care and use at the bureau of berkeley division, california university. All experiments were approved by the university of california, beckeley university, IACUC, before work was performed. Rats were anesthetized with a mixture of ketamine and xylazine (50 mg and 10mg/kg body weight, respectively) for surgical procedures. Mu.l PBS, sFlt or mvsFlt was intravitreally injected 1mm posterior to the limbus of the eye using a Hanmidton 30 syringe at 1mg/mL and monitored daily for signs of inflammation. This concentration was chosen to maximize fluorescence in the vitreous and to remain within the detection limits of the fluorometer after 48 hours. Rats in each group were CO administered at 0, 4, 12, 24 and 48 hours post injection₂The eyes were sacrificed by asphyxiation and immediately removed and placed on dry ice. The frozen vitreous was then removed from the eye and immersed in 100 μ L RIPA buffer. After shaking on ice for 2 hours, each vitreous sample was homogenized with a tissue disruptor (Bio Spectrum Products Co.) and fluorescence was measured using a fluorimeter (Molecular Devices). Quantification was performed by normalizing the fluorescence of the vitreous samples to the 0 hour vitreous fluorescence reading within their respective groups. Half-lives of sFlt and mvsFlt were calculated according to equation 1:

C_t＝C₀e^-kt (1)

wherein C is_tIs the concentration at time t, C₀Is the initial concentration and k is given by equation 2Elimination constant:

k＝log(2)/t_1/2 (2)

wherein t is_1/2Is the drug half-life. Value t for calculation_1/2Based on data from 48 hour time points.

OIR rat angiogenesis model

Pregnant Brown Norway rats were obtained from Charles River Laboratories. All animal experiments were performed in compliance with the declaration of ARVO for use in ophthalmic and vision research animals and approved by the animal care and use committee of the university of oklahoma institution. Newborn pups were assigned to PBS, sFlt or mvsFlt treated groups. The light was cycled using a 12 hour on, 12 hour off protocol and room temperature was maintained at approximately 21C. Rat pups were exposed to hyperoxia (75% O) on postnatal day 7 (P7) to P12₂). Oxygen-treated rats were placed in an incubator connected to an Oxycler Model A4(Redfield, NY) with oxygen and nitrogen, allowing the oxygen concentration to be adjusted to 75% + -2%. Rats were placed in an oxygen chamber with enough food and water to maintain them for 5 days. At P12, the animals were returned to room air and 2. mu.L of PBS, sFlt or mvsFlt was administered by intravitreal injection at 150. mu.g/mL to each eye. Rats of P17 were anesthetized and treated with high molecular weight FITC-dextran (2X 10)⁶(ii) a Sigma-Aldrich, St. Louis MO) perfusion, e.g., Smith et al [17]The method is as follows. The retina was dissected and flat-sealed and the vasculature was imaged using a fluorescence microscope (CKX 41; Olympus). Vascular coverage of P17 was quantified using NIH ImageJ by comparing total retinal area to vascularized area.

Statistical analysis

Values are expressed as mean ± Standard Deviation (SD). Statistical analysis was performed with a two-tailed t-test to compare the mean. In quantitative measurements, treatment groups were also compared using one-way (using Tukey post analysis) and two-way ANOVA (using Bonferroni post test) where appropriate (Prism, GraphPad Software). P values less than 0.05 were considered statistically significant.

Results

sFlt and mvsFlt equally inhibit corneal angiogenesis

Chemical injury-based models of corneal angiogenesis were used to determine whether sFlt conjugation to HyA reduced the biological activity of mvsFlt compared to sFlt in vivo. All mice treated with sFlt and mvsFlt showed similar inhibition curves of corneal angiogenesis 10 days after corneal injury (fig. 2). The cornea treated with PBS had 28.8 + -11.5% vascular coverage, compared to 12.8 + -3.8% and 15.8 + -7.1% vascular coverage of the cornea treated with sFlt and mvsFlt, respectively.

FIGS. 2A-2C: sFlt and mvsFlt equally inhibited corneal angiogenesis. A) A schematic diagram depicts a method for implementing a corneal burn model. Mice were treated twice with 5 μ l PBS, sFlt or mvsFlt on days 1 and 3 after chemical burn. B) Representative images of eyes treated with PBS, sFlt, and mvsFlt. CD31 positive (green) staining of corneal vessels. C) Quantification of corneal angiogenesis on day 10 post-treatment. One-way ANOVA to obtain p-value^**<0.01(n.s. -not significant;^*p<0.05；^**p<0.01). The scale bar corresponds to 20 μm.

Significantly longer residence times of mvsFlt in the vitreous

It was confirmed that vitreous bodies of Brown Norway rats can be used to determine the intravitreal residence time of molecules of different sizes using fluorescently labeled dextran of varying size (fig. 6). The difference in residence time between sFlt and mvsFlt was immediately apparent starting at 4 hours, at which time only 18.2. + -. 7.3% of the sFlt remained, compared to 105.8. + -. 9.8% of mvsFlt (FIG. 3). By 12 hours, only 2.6. + -. 1.9% of sFlt remained detectable, compared to 62.9. + -. 14.1% for mvsFlt. 2 days after injection, sFlt was barely detectable (1.2. + -. 0.5%), whereas 66.2. + -. 28.6% of mvsFlt remained in the vitreous. The half-life of sFlt in the vitreous was calculated to be 3.3 hours using equation 1 and equation 2, compared to 35 hours for mvsFlt.

FIGS. 3A-3B: mvsFlt has a longer residence time in rat vitreous. A) The schematic diagram depicts a method for determining the half-life of fluorescently labeled sFlt and mvsFlt in rat vitreous. The vitreous was injected with 5. mu.l Alexa Fluor 488-labeled sFlt or mvsFlt. At 0, 4, 12, 24 andafter 48 hours, rats were sacrificed and their eyes were removed and frozen for analysis. The vitreous is then removed, immersed in RIPA buffer and homogenized for subsequent fluorescence measurements. B) Conjugation with HyA significantly improved the residence time of sFlt in the vitreous after 48 hours compared to sFlt. Results are expressed as mean. + -. SD: (^*p<0.05， ^**p<0.01，^***p<0.001)。^*Indicating the difference between mvsFlt and sFlt at a given time point. Two-way ANOVA to obtain p-value^***<0.001。

FIG. 6: higher molecular weight dextrans show longer in vivo residence times. Experiments confirmed the effect of size on retention of fluorescently labeled dextran. 2MDa dextran (solid line) has significantly improved residence time over 40kDa (dashed line) within 48 hours. The half-lives of the 40kDa and 2MDa glucans were 3.2 hours and 5 hours, respectively.^*The difference between 40kDa and 2MDa dextran at a given time point is indicated. Two-way ANOVA to obtain p-value^*<0.05 (^**Corresponding to a P value less than 0.01).

mvsFlt is a more potent inhibitor of retinal neovascularization

OIR model of retinal angiogenesis assay was used to examine the effect of HyA conjugation of sFlt on inhibition of retinal angiogenesis. This short-term model allows indirect examination of the effect of mvsFlt half-life on prolonged angiogenesis inhibition. Neovascular coverage was calculated by comparing the area of vascular coverage to the total retinal area. After 5 days of treatment, the retinal vessel coverage of PBS injected eyes was 84.3 + -3.8%, and that of retinas treated with intravitreal sFlt injection was 85.4 + -6.1%. In contrast, retinas from rats treated with intravitreal injection of mvsFlt were significantly lower and had retinal vascular coverage of 72.9 ± 3.4% (fig. 4).

FIGS. 4A-4C: mvsFlt inhibits retinal angiogenesis. A) The schematic diagram shows a method for implementing the OIR model. Newborn rat pups were placed in normoxic conditions (21% oxygen, room air) from postnatal day (P)0-7 to allow normal retinal vasculature to develop and then transferred from P7-P12 to hyperoxic stripsThis induces vessel pruning. At P13, pups were transferred back to normoxic conditions and treated with 2 μ l PBS, sFlt or mvsFlt and sacrificed at P17. B) Representative images of retinas treated with PBS, sFlt, and mvsFlt. Green staining indicated CD31+ cells. The scale bar corresponds to 250 μm. The dashed box magnifies this portion of the tissue (scale bar corresponds to 100 μm). C) Quantitative retinal vessel formation was observed 5 days after treatment. The percent retinal vascularization is calculated by comparing the vascularization area in the image to the total retinal area. One-way ANOVA to obtain p-value^***<0.001(n.s. -not significant;^**p<0.01)。

both sFlt and mvsFlt had similar concentrations in the vitreous at the time of injection (fig. 5A). Over time, sFlt was low enough to clear from the vitreous, leaving a much lower concentration of drug (fig. 5B, top). This allows for an increase in intravitreal VEGF concentration, which induces angiogenesis. In contrast, mvsFlt has a much longer intravitreal residence time and is therefore able to act as an absorber of VEGF over time (fig. 5B, bottom), thereby inhibiting angiogenesis and maintaining basal levels of retinal vascularization.

FIGS. 5A-5B: the schematic diagram demonstrates the proposed mechanism of action of mvsFlt. A) sFlt (red, unconjugated) and mvsFlt (red conjugated blue strand of HyA) were injected into diabetic retinas in the presence of high concentrations of VEGF (green circles). B) After a given time t, most of the sFlt has been cleared from the vitreous and VEGF is therefore able to induce vascular growth. mvsFlt has a longer residence time in the vitreous and is able to bind and inhibit VEGF for a much longer period of time, resulting in delayed inhibition of retinal angiogenesis.

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43.Fishburn SC.The pharmacology of PEGylation:Balancing PD with PK to generate novel therapeutics.J Pharm Sci.2010；97(10):4167-83。

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Example 2: multivalent hyaluronic acid bioconjugates improve sFlt in vitro activity

Materials and methods

Expression of soluble Flt-1 receptor

The sFlt sequence [15] of the first 3 Ig-like extracellular domains of sFlt-1 was cloned into pFastBac1 plasmid (Life Technologies) and then transformed into DH10Bac Enterobacter coli, which was plated on triple antibiotic plates containing kanamycin (50. mu.g/mL), gentamicin (7. mu.g/mL, Sigma Aldrich), tetracycline (10. mu.g/mL, Sigma Aldrich), IPTG (40. mu.g/mL, Sigma Aldrich) and Bluo-gal (100. mu.g/mL, Thermo Fisher Scientific). Bacmids containing the sFlt gene were isolated from DH10Bac e.coli (Life Technologies) and transfected into SF9 insect cells for virus production (supplied by Tissue Culture Facility, UC Berkeley). The High Five insect cells (supplied by Tissue Culture Facility, UC Berkeley) were then infected with the virus to induce sFlt protein expression. After 3 days, the protein was purified from the supernatant using Ni-NTA agarose beads (Qiagen Laboratories). Recombinant sFlt was eluted from Ni-NTA beads using an imidazole gradient and then concentrated and buffer exchanged with 10% glycerol/PBS using an Amicon Ultra-15mL centrifuge device (EMD Millipore). The protein solution was sterile filtered and the concentration was determined using BCA assay (Thermo Fisher Scientific).

Synthesis of mvsFlt conjugates

Conjugation of sFlt to HyA was performed according to the scheme in FIG. 1, as previously described [16-18 ]. To prepare thiol-reactive HyA intermediates, 3' -N- (epsilon-maleimidocaproic acid) hydrazide (EMCH, Thermo Fisher Scientific,1.2mg/mL), 1-hydroxybenzotriazole hydrate (HOBt, Sigma Aldrich,0.3mg/mL) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Fisher Scientific,10mg/mL) were added to 3mg/mL solutions of various molecular weights of HyA (lifecore biotechnology) in 0.1M 2- (N-morpholino) ethanesulfonic acid (MES) (Sigma Aldrich) buffer (pH 6.5) and allowed to react at 4 ℃ for 4 hours. The solution was then dialyzed into pH 7.0PBS containing 10% glycerol. Recombinant sFlt was treated with 10 molar excess of 2-iminothiolane to generate a thiol group for conjugation to a maleimide group on EMCH. sFlt was then added to activated HyA-EMCH at molar ratios of sFlt to HyA of 10:1 and 30:1 to produce mvsFlt bioconjugates and allowed to react overnight at 4 ℃. The mvsFlt reaction was thoroughly dialyzed using a100 kDa molecular weight cut-off (MWCO) Float-A-Lyzer G2(Spectrum Labs) dialysis tube in PBS pH 7.0 to remove unreacted sFlt. Protein concentration of mvsFlt bioconjugates was measured using BCA assay. The conjugate was defined as 'low conjugation ratio' (LCR) mvsFlt at 10:1sFlt to HyA molar feed ratio and 'high conjugation ratio' (HCR) mvsFlt at 30:1 molar feed ratio.

SEC-MALS characterization of mvsFlt

Protein conjugation was characterized using size exclusion chromatography with multi-angle light scattering (SEC-MALS), as previously described [19 ]. Briefly, the SEC-MALS setup consisted of an Agilent HPLC 1100 with a DAWN-HELEOS II multi-angle laser light scattering detector and an Optilab relative refraction interferometer (Wyatt Technology, Santa Barbara, Calif.). The refractive index change was measured differentially using a 690nm laser and the UV absorbance was measured at 280nm using a diode array detector. Shodex OH pak SB-804 column was used for the separation (Phenomenex Corp.). Prior to analysis, mvsFlt conjugate was sterile filtered through a 0.45 μm filter and injected at 200 μ L with HyA concentrations between 0.2-0.5 mg/mL. The dn/dc values for HyA-EMCH and sFlt were determined to be 0.1447 and 0.185, respectively, using SEC-MALS. The UV extinction coefficients for HyA-EMCH and sFlt were also determined according to SEC-MALS and were 0.022 and 0.894, respectively. Data analysis was performed using Astra software (Wyatt Technologies).

SDS-PAGE analysis of mvsFlt

Samples were prepared using 5X SDS dye loading buffer, 2-mercaptoethanol, and boiled at 95 ℃ for 5 minutes. A pre-made Mini-Protean TGX 4-20% gradient gel (Bio-Rad Laboratories) was run at 110 volts for 90 minutes. The gel was then stained with Bio-Safe Coomassie stain (Bio-Rad Laboratories) for 2 hours, and then imaged using a BioRad molecular imager ChemiDoc XRS +. The protein intensity in the stack and gradient gels was analyzed using ImageJ to determine the amount of conjugated protein relative to free unconjugated protein that did not dialyze out of solution after the conjugation reaction.

DLS size characterization of mvsFlt

Hydrodynamic diameters of mvsFlt bioconjugates were determined using a Brookhaven goniometer and laser scattering system (BI-200SM, Brookhaven Instruments Inc.). Each sample was filtered at 0.45 μm and loaded into a 150 μ L cuvette (BI-SVC, Brookhaven). Data acquisition was performed using a 637nm laser at 90 degrees for 2 minutes. Data analysis was performed using a BI-9000AT signal processor using BIC dynamic light Scattering software (Brookhaven). The intensity average particle size is obtained using a non-negative least squares (NNLS) analysis method.

Binding competition ELISA

Use of VEGF₁₆₅Quantikine sandwich ELISA (R)&D System) analysis of mvsFlt conjugates to check that HyA conjugation inhibits VEGF for sFlt₁₆₅The influence of (c). The measurements were performed according to the manufacturer's instructions. Briefly, VEGF was prepared₁₆₅Added to PBS with different concentrations of sFlt or mvsFlt. Detection of free VEGF bound to the Capture antibody on the plate surface Using Horseradish peroxidase-conjugated detection antibody₁₆₅And quantified using a spectrophotometer at 450 nm.

HUVEC endothelial cell survival assay

Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from ATCC and cultured at 37 ℃ and 5% CO₂Next, in a wet incubator in EBM-2 medium(Lonza). To examine HyA conjugation for sFlt binding to VEGF₁₆₅And inhibiting VEGF₁₆₅Effect of the ability to Activity in vitro, use in VEGF₁₆₅And HUVECs grown in the presence of mvsFlt conjugates were subjected to survival assays. HUVECs were added at 10,000 cells/well to 96-well plates coated with 0.2% gelatin in M199 medium. Cells were plated in 2% FBS and 20ng/mL VEGF in the presence of sFlt or mvsFlt₁₆₅(R&D Systems). 72 hours after plating, the medium was aspirated and the cells were washed with PBS and then frozen for analysis using CyQuant (Life technologies). The total number of cells per well was determined by reading the fluorescence using a fluorimeter (Molecular Devices) at 480nm excitation and 520nm emission.

HUVEC tube formation assay

HUVEC tube formation assays were performed in 96-well plates coated with 80 μ Ι _ matrigel (Corning, NY) and incubated at 37 ℃ for 1 hour to allow gelation to occur. HUVEC were trypsinized and resuspended in VEGF with 2% FBS and 20ng/mL₁₆₅And treated with mvsFlt LCR conjugate. Wells were imaged 18 hours after plating and tube formation was quantified using ImageJ software.

HUVEC migration assay

The wells of a 12-well plate were coated with 0.2% gelatin. HUVECs were added to EBM-2 at 150,000 cells/well and allowed to attach and expand overnight. The cross was scored into the confluent layer of HUVEC using a 1mL pipette tip. The wells were then washed with excess PBS to remove cell debris and culture medium, and with VEGF-containing medium₁₆₅And medium replacement of mvsFlt. The scratch was imaged at 0 and 24 hours post scratch and the cell-free area was quantified using ImageJ and T-scratch software (CSE Laboratory software, ETH Zurich). The percent open wound area was calculated by comparing the open scratch area at 24 hours to the open scratch area at 0 hours.

Retention of mvsFlt in Cross-linked HyA gels

To model the chemistry and network structure of the vitreous, acrylated HyA (AcHyA) hydrogels were synthesized, as previously described[20，21]. Briefly, adipic acid dihydrazide (ADH, Sigma Aldrich) was added to HyA in deionized water (DI) in 30 molar excess. EDC (3mmol) and HOBt (3mmol) were dissolved in DMSO/water and added to HyA solution. The solution was allowed to react for 24 hours and then dialyzed thoroughly against DI water. HyA-ADH was precipitated in 100% ethanol and reacted with acryloxysuccinimide to generate an acrylate group on HyA. The resulting AcHyA was dialyzed thoroughly and lyophilized for storage. Using H¹NMR confirms the presence of grafted acrylate groups on the HyA chain.

To prepare a 1% AcHyA hydrogel, 8mg of AcHyA was dissolved in 800. mu.L of triethanolamine buffer (TEOA; 0.3M, pH 8). Prior to crosslinking, 5. mu.g of Alexafluor 4885-SDP ester (Life Technologies) labeled sFlt, 650kDa LCR mvsFlt or Bovine Serum Albumin (BSA) was added to AcHyA solution in a volume of 50. mu.L. Thiolated 5kDa-PEG cross-linker (Laysan Bio Inc.) was dissolved in 100. mu.L TEOA buffer and added to the dissolved AcHyA. Cell culture inserts (Millipore, Billerica, MN) with 4 μm large pores were added to the wells of a 24-well plate, and 70 μ L of a gel containing sFlt, mvsFlt, or BSA was added to each insert. The gel was crosslinked at 37 ℃ for 1 hour, then 150 μ Ι _ of PBS was added to the wells and the hydrogel was submerged. To determine release kinetics, well supernatants were collected and replaced completely on days 0, 1,2, 3, 7, 10, and 14. Samples were read with a fluorimeter to detect fluorescently labeled sFlt, mvsFlt and BSA in the supernatant.

Fluorescence bleach recovery (FRAP) diffusivity measurement

FRAP measurements were performed on 1% AcHyA hydrogels containing FITC labeled sFlt and 650kDa LCR mvsFlt. The total fluorescence intensity of the hydrogels was obtained using a Zeiss LSM710 laser scanning microscope (Carl Zeiss, Jena, germany) with a 20X magnification objective and an argon laser set at 488nm with 50% power. Photobleaching is performed by exposing 100x100- μm spots in the field of view to a high intensity laser. The area was monitored by 15 pre-bleaching scan images at low laser intensity (2%) and then bleached to 75% of the initial fluorescence intensity at 100% laser power. A total of about 300 image scans of less than 1 second per sample were acquired. Fraction of movement of fluorescent sFlt and mvsFlt molecules within hydrogels by comparison of fluorescence in bleached regions after complete recovery (F)_∞) Before bleaching (F)_Initial) And immediately after bleaching (F)₀) Is determined. The movement fraction R is defined according to equation (1):

enzymatic degradation with MMP-7

Matrix metalloproteinase-7 (MMP-7) has previously been shown to specifically degrade sFlt [22 ]. To determine whether HyA conjugation prevented mvsFlt degradation, sFlt and mvsFlt were treated with varying amounts of MMP-7(EMD Millipore). sFlt and mvsFlt were incubated with matrix metalloproteinase-7 (MMP-7) at 37C for 12 hours with high (1:1 molar ratio MMP-7: sFlt), medium (1:2), and low (1:4) molar ratios MMP-7 and sFlt, while shaking. The enzyme treated sFlt and mvsFlt were then loaded into a preformed Mini-Protean TGX 4-20% gradient gel (Bio-Rad Laboratories) and run at 110 volts for 90 minutes. The gel was then stained using a silver staining kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The extent of enzymatic degradation was assessed by quantifying the total amount of protein remaining in the gel after treatment with MMP-7. Each well was normalized to its corresponding MMP-7 free well and background intensity was subtracted from the blank well between groups on the gel.

Statistical analysis

All quantification experiments were performed in triplicate. Values are expressed as mean ± Standard Deviation (SD). One-way ANOVA with Tukey post hoc analysis was used for comparison treatment groups in quantitative measurements, where appropriate, and p <0.05 was used to assess statistical significance.

Results

The overall goal of this study was to synthesize protein-polymer bioconjugates to increase the residence time of anti-VEGF drugs in the vitreous for the treatment of patients with DR. Development of a drug for VEGF compared to drugs with short half-lives currently used for the treatment of DR₁₆₅Has an undisturbed affinity andlarge multivalent protein bioconjugates of good enzyme stability that show delayed diffusion and movement in an in vitro model of the vitreous.

FIGS. 7A-7D. multivalent sFlt synthesis and schematic. A) mvsFlt bioconjugates were synthesized using a 3-step reaction in which HyA was reacted with EDC and EMCH to produce cysteine-reactive HyA-EMCH intermediates. The sFlt was then treated with 2-iminothiolane and then reacted with a HyA-EMCH intermediate for the synthesis of the final product. B) Conjugation of proteins to HyA and subsequent conjugation to VEGF₁₆₅Schematic representation of the binding. The ratio a: b represents the valency of the sFlt molecule (a) covalently bound to the HyA single chain (b). C) Schematic representation of Low Conjugation Ratio (LCR) mvsFlt conjugates synthesized by reacting 10 sFlt molecules with 1 HyA chain. This reaction had a 61% conjugation efficiency as determined by SEC-MALS (see table 1). D) Schematic representation of High Conjugation Ratio (HCR) mvsFlt conjugates synthesized by reacting 30 sFlt molecules with 1 HyA chain (same molecular weight as HyA in (C)). This reaction had a 52% conjugation efficiency as determined by SEC-MALS (see table 1).

Synthesis of mvsFlt conjugates was performed according to the schematic shown in FIGS. 7A-7D. mvsFlt was produced at low (LCR, fig. 7C) and high (HCR, fig. 7D) conjugation ratios to determine whether certain valencies provided an enhancement of VEGF binding. sFlt was successfully conjugated to HyA at several different molecular weights and valencies, which was significantly greater than sFlt in its unconjugated form (fig. 8A-8D). As shown in table 1 and fig. 8A, SEC-MALS was used to characterize the molecular weights of the protein and polymer components of the conjugates. The conjugation efficiency of conjugates with sFlt to HyA feed ratio of 10:1 (referred to as low conjugation ratio, LCR) averaged 61.2 ± 12.5%, while conjugates with sFlt to HyA feed ratio of 30:1 (referred to as high conjugation ratio, HCR) averaged 51.8 ± 4.1%. SDS-PAGE of unbound sFlt showed protein bands at the predicted 50 kDa. In contrast, mvsFlt bioconjugates migrated only into the stacking portion of the gel, indicating inhibition of movement due to covalent attachment to much larger multivalent conjugates (fig. 8B). Gel analysis using ImageJ indicated that on average 76.4 ± 6.7% of the sFlt in the mvsFlt bioconjugates was covalently bound, while the remaining detected sFlt could interact non-specifically with the clear mass acid chains in solution and thus could not be removed by dialysis (fig. 8C).

Table 1: SEC-MALS analysis of all mvsFlt conjugates.

^aThe number average molecular weight is given in g/mol.

^bWeight average molecular weight given in g/mol

^cPolydispersity index given as Mw/Mn

^dFinal stoichiometric ratio of sFlt to HyA by total attached protein Mw divided by

sFlt MW (50kDa) to calculate

^*LCR is a low conjugation ratio conjugate (10:1sFlt/HyA strand feeding ratio)

⁺HCR is high conjugation ratio conjugate/(30: 1sFlt/HyA strand feed ratio)

All mvsFlt conjugates were also characterized by DLS to determine the hydrodynamic diameter of the conjugate in solution, since drug size is a critical factor in determining its movement through biological hydrogels (such as the vitreous). Unconjugated sFlt had diameters of 22.6 nm. + -. 3.1nm, whereas mvsFlt conjugates prepared with HyA molecular weights of 300kDa, 650kDa and 1MDa had diameters of 123.9. + -. 23.1nm, 236.3. + -. 38.7nm and 223. + -. 13.9nm, respectively (FIG. 8D). The size of mvsFlt conjugates depends on the HyA molecular weight of the 300 and 650kDa conjugates; however, there was no significant difference between the 650kDa and 1MDa conjugates (fig. 8D). Interestingly, HCR conjugates with molecular weights of 300 and 650kDa had lower diameters than their corresponding LCR conjugates, probably due to the increase in positive charge from sFlt on the negatively charged HyA backbone with increasing sFlt attachment, resulting in tighter folding of the conjugate around them.

FIGS. 8A-8D. A) A SEC-MALS chromatogram of cumulative weight fraction versus molar mass of 650kDa LCR and HCR bioconjugates is depicted. The dotted, dashed and solid lines represent the total molar mass, HyA molar mass and total bioconjugate molar mass, respectively, of all covalently linked sFlt proteins (all given as g/mol). B) 4-20% SDS-page gradient gels of sFlt and mvsFlt. Protein bands in the stacked gels indicate successful conjugation of the protein to HyA. The protein band within the gel indicates the proportion of protein that is not conjugated, but remains in solution after dialysis. C) Quantitative protein band intensities of SDS-PAGE gels. Percent sFlt bound was determined by dividing the protein strength in the stacked gels by the total protein strength in the corresponding wells. Free sFlt was determined by dividing the protein strength in the isolate gel by the total protein strength in the corresponding well. D) Dynamic light scattering analysis of the conjugates. sFlt is significantly less than all mvsFlt bioconjugates ((s))^***p<0.001). In the case of 300 and 650kDa mvsFlt bioconjugates, LCR mvsFlt was significantly larger than its corresponding HCR conjugate (s^**p<0.01). Values are given as ± SD.

FIGS. 9A-9B all mvsFlt bioconjugates in VEGF₁₆₅ELISA and VEGF₁₆₅Maintenance of VEGF inhibition in dependent HUVEC survival assays₁₆₅Ability to rely on activity. A) Binding of VEGF to Capture antibody by mvsFlt bioconjugate₁₆₅Dose-dependent inhibition of (a). Inhibition was independent of whether sFlt was bound to HyA or free in solution. There were no significant differences between any of the groups (table 2). B) In VEGF₁₆₅Dose-dependent inhibition of HUVEC survival with mvsFlt of different molecular weights and protein valencies in the presence. Inhibition was independent of whether sFlt was bound to HyA or free in solution (table 2). Values are given as mean ± SD.

Table 2: inhibition of VEGF from examination of mvsFlt₁₆₅ELISA and HUVEC survival assay of IC₅₀Value of

	ELISA(ng/mL)	HUVEC survival (ng/mL)
			Unconjugated sFlt	3.8±2.4	39.3±4.4
300kDa LCR	4.5±1.5	46.9±9.9
			300kDa HCR	4.7±1.7	44.4±2.6
650kDa LCR	3.9±2.6	41.7±6.7
			650kDa HCR	2.0±0.1	43.2±13.6
1MDa LCR	2.2±0.1	44.9±10.7
			1MDa HCR	3.3±1.6	45.4±2.2

Several different assays were used to determine whether conjugation of sFlt to HyA affects sFlt-affinity VEGF₁₆₅And alter VEGF₁₆₅Dependent cell function. Use of VEGF₁₆₅Specific ELISA, observing dose-dependent response; the responses indicated that conjugation of sFlt to HyA did not alter mvsFlt binding to VEGF₁₆₅Ability (fig. 9A). ELISA results indicate IC for sFlt₅₀Values of 3.8. + -. 2.4ng/mL, and IC of various mvsFlt conjugates₅₀The values averaged 3.4. + -. 1.1 ng/mL (Table 2).

10A-10E. mvsFlt inhibited HUVEC tube formation. A) Representative images of inhibited HUVEC tube formation on matrigel (BD Biosciences) when treated with 1 μ g/mL of all LCR mvsFlt bioconjugates. Cells were seeded at 20,000 cells/well on 100 μ L matrigel in 96-well plates and imaged at 18 hours. Scale bar 500 μm. B-E) quantification of the number of manifolds per well (B), the average tube length (C), the total number of nodes (branching points, D) and the manifold length per well (E) ((B-E)^***p<0.001)。

mvsFlt showed a dose-dependent decrease in survival in the survival assay using HUVEC, and similar to ELISA results, the effect was not associated with conjugation of HyA (fig. 9B, table 2). These results indicate that covalent conjugation of sFlt to HyA does not reduce the ability of sFlt to bind VEGF. This is very promising due to the fact that: other conjugation techniques (such as pegylation) conjugation has previously been reported to significantly reduce protein bioactivity [23,24 ].

For subsequent studies, only LCR conjugates of different molecular weights were studied, since VEGF was examined in vitro when assayed in ELISA and survival assays₁₆₅All conjugates performed equally well when inhibiting activity. In vitro tube formation and migration assays the in vitro mvsFlt conjugates can be examined for their effect on two additional processes involved in angiogenesis in vivo (tube formation by organization and cell migration). Similar to survival data, sFlt and mvsFlt have similar VEGs in both assays in which addition of sFlt and mvsFlt equally inhibited histocompatibility tubes (fig. 10A-10E) and wound closure (fig. 11A-11B)F₁₆₅Inhibition curves. In summary, ELISA and in vitro angiogenesis assays indicated that conjugation of sFlt to HyA did not affect sFlt binding to VEGF₁₆₅And all mvsFlt conjugates maintain their inhibition by VEGF₁₆₅The ability of signal transduction to mediate endothelial cell function.

FIGS. 11A-11B. mvsFlt inhibits VEGF₁₆₅Driven migration of HUVEC. Representative images of inhibition of HUVEC migration with LCR mvsFlt bioconjugates of varying molecular weight. HUVECs were allowed to grow to confluence in 12-well plates before scratching and were treated with 20ng/mL VEGF in the presence of 200 ng/mL mvsFlt₁₆₅And (6) processing. Cells were stained with CellTracker Green (Life Technologies) prior to inoculation. Scale bar 20 μm. B) Quantitative HUVEC migration following treatment with LCR mvsFlt, showing percent open wound area calculated by comparing open wound area at 24 hours to open wound area at time 0: (^***p<0.001)。

Cross-linked HyA gels [20,21] were used to examine how diffusion conjugation of sFlt with HyA affects diffusion. The hydrogel was selected as a model system for the study of in vitro vitreous based on compositional similarity with the high HyA content associated vitreous. The hardness of HyA hydrogels (fig. 14A) was higher than published reports examining bovine and porcine vitreous hardness [25], suggesting that the prediction of clearance of drugs from the vitreous using this model may not be sufficient to estimate the actual in vivo rate. However, given the compositional similarity of the model to the vitreous, it is expected that diffusion of sFlt and mvsFlt through these gels will help predict the benefit of conjugation to HyA. Several different models were used to characterize the mesh size of HyA hydrogels (table 3). Preliminary experiments to analyze size-dependent diffusion using 40kDa and 2MDa fluorescently labeled dextran (Life Technologies) confirmed that this hydrogel system was suitable for examining molecular weight and size-dependent diffusion (fig. 14B). Using this system, the kinetics of release of sFlt and all LCR mvsFlt bioconjugates from HyA gels were analyzed. The major effect of mvsFlt bioconjugates is expected to be a size-dependent reduction in motility. Only conjugates of varying sizes were analyzed while keeping valencies constant. After 14 days, only 30.8. + -. 1.9% of sFlt remained in the gel compared to 38.3%. + -. 2.2%, 63.8. + -. 0.5% and 62.8%. + -. 0.4% for the 300kDa, 650kDa and 1MDa LCR conjugates, respectively (FIG. 12A). In contrast, after 24 hours BSA was 100% released, this difference was more likely due to a significant difference in isoelectric points (BSA was 5.4[26] and sFlt was 9.5[27]), and the protein affinity for HyA was very low rather than due to protein size, since BSA and sFlt had similar molecular weights.

Table 3: based on dilatometric and rheological dataAnd grid size calculation.

Molecular weight between crosslinks

⁺ξ -according to [31 ]]Calculated grid size of 1% HyA gel

From mass expansion data

Calculation from rheological data

Sflt conjugation to HyA reduced migration and diffusion of mvsFlt in HyA gels. A) After day 1, Alexafluor 488-labeled LCR mvsFlt bioconjugates 650kDa and 1MDa encapsulated in 1% HyA hydrogels diffused out significantly slower than unconjugated sFlt and 300kDa mvsFlt ((^*p < 0.05) and last period until day 14Point (A)^***p < 0.001). B) Representative confocal images of FRAP experiments corresponding to FITC-labeled 650kDa LCR mvsFlt. F_InitialmvsFlt in gels before bleaching is depicted. F₀Is a fluorescence measurement taken immediately after 75% photobleaching; f_∞Corresponding to the maximum recovery of fluorescence at the end of the experiment. C) Normalized fluorescence recovery of FITC-labeled sFlt and 650kDa LCR mvsFlt after photobleaching [ f (t)]。

Fig. 14A-14b. (A) Rheological properties of 1% HyA hydrogel. (B) After 7 days, fluorescently labeled 40kDa dextran encapsulated in HyA hydrogel diffused out significantly faster than 2MDa dextran ((r))^***p＜0.001)。

To assess whether diffusion through the gel is Fickian diffusion, the curve in fig. 12A is fitted using equation (2), as described by Ritger et al [32 ]:

the diffusion index n indicates whether the diffusion is a Fickian diffusion, and if n equals 0.5, the transmission is Fickian. The n-values for BSA, sFlt and mvsFlt release were determined to be 0.3-0.4 (FIGS. 15A-15E), indicating that diffusion through the gel was not Fickian diffusion. Since the size of the protein is very small and the affinity to HyA hydrogel is very low due to charge repulsion, BSA is depleted from the gel by a rapid burst release, resulting in non-Fickian diffusion. In contrast, sFlt and mvsFlt conjugates released slower due in part to ionic affinity and size with HyA hydrogels. Although sFlt and BSA are similar in size (50kDa and 66kDa, respectively), sFlt has a much stronger ionic interaction with the matrix, which slows its diffusion from the gel, also causing non-Fickian diffusion due to this strong affinity. The size of the 300kDa mvsFlt conjugate was small enough (< 150nm, see FIG. 8D) relative to the estimated hydrogel ξ (Table 3) to be released as fast as sFlt. The diameter of the two largest conjugates is close to the lattice size (> 225nm) and due to the size significantly hinders their release, leading to gel release following a peristaltic diffusion mechanism [33 ].

Based on data from the sFlt release study, only 650kDa LCR bioconjugates were studied using FRAP, as this conjugate showed the greatest difference in gel retention compared to the unconjugated sFlt. The migration score of sFlt in the gel was 73.8 ± 4.4%, while the migration score of mvsFlt within the gel was 48.3 ± 3.0%, indicating that a significant portion of mvsFlt bioconjugates were large enough to become immobile within the gel due to the diameter similarity between the mesh sizes of mvsFlt and HyA gels. The experimental data in FIG. 12C was fitted to Soumpassis [34] to obtain a characteristic diffusion time. Interestingly, the characteristic diffusion time of mvsFlt (94.8. + -. 19.5s) was significantly faster than that of sFlt (176. + -. 18.1 s). This difference is likely due to the shielding of the positively charged sFlt by the negatively charged hyaluronic acid ions within the multivalent conjugate, which reduces the overall affinity of the conjugate for the gel, allowing faster diffusion. In contrast, unconjugated sFlt remains highly positively charged, which results in stronger ionic affinity within the HyA gel that slows its characteristic diffusion time. mvsFlt in the hydrogel also restored fluorescence to a significantly lower degree of 85.3 ± 0.8%, compared to sFlt restoring fluorescence to 91.6% ± 2.4%. Although mvsFlt conjugates showed faster diffusion, a much smaller percentage of mvsFlt bioconjugates were actually mobile and size significantly limited total fluorescence recovery, results also supported by the gel release data in fig. 12A. It is evident that although mvsFlt can diffuse faster, as shown by FRAP in fig. 12B, 12C, a much smaller portion of this sample is able to move due to size, and thus it releases much less over time, as evidenced by the gel release data in fig. 12A. In summary, the effect of size is expected to be the strongest determinant of in vivo residence time of mvsFlt.

Fig. 13A-13b conjugation to HyA reduces the susceptibility of protease degradation by MMP-7. A) A4-20% SDS-page gradient gel of sFlt and mvsFlt (650kDa LCR) after 12 hours of treatment with MMP-7 at high, medium and low molar ratios of MMP-7 to sFlt, which correspond to 1:1, 1:2 and 1:4 molar ratios of MMP-7 to sFlt. The band intensities were normalized to the absence of treatment with MMP-7 within each group,and blank wells were used to subtract background from each sample. B) Quantification of 4-20% gradient gels of sFlt and mvsFlt treated with MMP-7 at high, medium and low molar ratios of sFlt to MMP-7 for 12 hours ((^*p<0.05； ^**p<0.01)。

Fig. 15A-15e data from gel release data were fitted to check Fickian diffusion. (A-E) fitting made with Ritger et al [13] equation (2) to determine a plot of the diffusion index n and the characteristic constant k of the macromolecular network system and drug.

The effect of conjugation of sFlt to HyA on protease degradation of sFlt protein was studied using MMP-7, a protease that has been shown to specifically degrade sFlt [22 ]. It was found that conjugation of sFlt to HyA prevented sFlt degradation at all molar ratios of MMP-7 to sFlt (FIG. 13A). At high concentrations of protease (1:1 molar ratio of MMP-7 to sFlt), only 6.8. + -. 6.6% of sFlt remained detectable, compared to 34.8. + -. 1.8% of mvsFlt (FIG. 13B). Decreasing the ratio of MMP-7 to sFlt from 1:1 to 1:4 still caused significant degradation of sFlt and shielding of degradation of the conjugated form, with an increase in detectable sFlt to 34 ± 2.7%, compared to 74.8 ± 4.9% detectable mvsFlt (fig. 3B). It is believed that the shielding effect on HyA provides for maintenance of mvsFlt in vivo stability and bioavailability, which contributes to the prolonged anti-angiogenic effect of mvsFlt bioconjugates.

Reference to the literature

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Example 3:generation of multivalent conjugates with anti-VEGF VHH or with single chain variable fragment (scFv) anti-VEGF (anti-VEGF scFv)

Two multivalent conjugates prepared from two different anti-VEGF antibody formats were generated: single chain variable fragment (scFv) anti-VEGF antibodies and single domain camelid (VHH) anti-VEGF antibodies.

FIGS. 20A-20C: each antibody conjugate binds to human 500pg VEGF-A using an ELISA assay₁₆₅Is compared to the corresponding unconjugated antibody. Both multivalent conjugates were prepared with 860kDa hyaluronic acid (HyA). The valencies of the scFv anti-VEGF antibody and the VHH anti-VEGF antibody are 31 and 28, respectively. The data were fitted using a four parameter logistic curve and used to calculate IC50 for each treatment. Figure 20A shows the percentage of unconjugated scFv anti-VEGF antibody and conjugated multivalent scFv anti-VEGF antibody that did not bind VEGF. Figure 20B shows the percentage of unconjugated VHH anti-VEGF antibody and conjugated multivalent VHH anti-VEGF antibody that did not bind VEGF. Figure 20C shows IC50 values for the conjugated antibodies of figures 20A-20B compared to the unconjugated antibodies of figures 20A-20B. As summarized in fig. 20C, multivalent conjugation had no substantial effect on the IC50 values of the conjugates compared to the unconjugated antibody.

Example 4:anti-VEGF VHH multivalent (mv anti-VEGF) conjugates prepared with HyA show increased in vitro and in vivoHalf life

The diffusion rates of unconjugated anti-VEGF antibody VHH and multivalent anti-VEGF antibody VHH (28VHH/860kDa HyA) were compared by fluorescently labeling proteins and trapping them within a 1% HyA-PEG hydrogel prepared from commercially available components (BioTime). The expansion ratio and average molecular weight between crosslinks were estimated. The average mesh diameter of the hydrogel was estimated to be about 80 nm.

FIG. 21: the concentration of unconjugated protein and protein conjugate released from the hydrogel was measured every 2-3 days, and the data was fitted to an exponential decay curve to estimate its diffusion half-life. The half-life of the VHH anti-VEGF antibody conjugate is about 4 times greater than the half-life of the unconjugated VHH anti-VEGF antibody. The results shown in figure 21 are the average of three independent replicates.

After injection into rat eyes, the in vivo residence time of the unconjugated VHH anti-VEGF antibody and the multivalent VHH anti-VEGF antibody conjugate were compared. In this experiment, all VHH anti-VEGF antibodies were fluorescently labeled as reporters for concentration measurements, and 4 independent batches of mv anti-VEGF (860kDa MW HyA and valency in the 24-45 range) were used. In each eye, 5 μ L275 μ g/mL of VHH anti-VEGF antibody or multivalent VHH anti-VEGF antibody conjugate solution was injected. Each eye received a total of 1.375 μ g of VHH anti-VEGF antibody conjugated not or at any valency. At 0.5, 4, 22 and 45 hours post-injection, rats were euthanized and their eyes were removed as follows: n-8 eyes (0.5 hr), n-8 eyes (4 hr), n-4 eyes (22 hr), and n-2 eyes (45 hr).

Figure 22A shows the percentage of protein recovered for unconjugated VHH and multivalent VHH anti-VEGF antibody conjugates. Figure 22B shows half-lives of unconjugated VHH anti-VEGF antibody and conjugated multivalent VHH anti-VEGF antibody. The results show that the conjugated multivalent VHH anti-VEGF antibody has an in vivo half-life of 21.9 hours, compared to the unconjugated VHH anti-VEGF antibody which has an in vivo half-life of 1.9 hours.

Example 5: generation of VHH anti-VEGF antibody multivalent conjugates made with carboxymethyl cellulose

FIGS. 23A-23B show the generation of multivalent conjugates prepared from-17 repeats of a single domain camelid (VHH) antibody and carboxymethylcellulose (CMC, -700 kDa). The ability of this anti-VEGF antibody (anti-VEGF VHH-CMC) conjugate to bind human 500pg VEGF-a165 was compared to the corresponding unconjugated VHH anti-VEGF antibody using an ELISA assay as shown in figure 23A. As shown in fig. 23B, the data were fitted using a four-parameter logarithmic curve and used to calculate IC50 for each treatment.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

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Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val

225 230 235 240

Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr

245 250 255

Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys

260 265 270

Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His

275 280 285

Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys

290 295 300

Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys

305 310 315 320

Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val

325 330 335

Lys His Cys Asp Asp Asp Asp Lys His His His His His His

340 345 350

<210> 5

<211> 280

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 5

Met Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Glu Ile Ile His Ser

20 25 30

Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Tyr Leu Ala Ser

85 90 95

Thr Asn Gly Ala Asn Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

130 135 140

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser

145 150 155 160

Leu Thr Asp Tyr Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys

165 170 175

Gly Leu Glu Trp Val Gly Phe Ile Asp Pro Asp Asp Asp Pro Tyr Tyr

180 185 190

Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

195 200 205

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Gly Gly Asp His Asn Ser Gly Trp Gly Leu Asp

225 230 235 240

Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Pro Ser Thr

245 250 255

Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Gly Gly Cys Asp Asp Asp

260 265 270

Asp Lys His His His His His His

275 280

<210> 6

<211> 156

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 6

Ser Asn Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

1 5 10 15

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe

20 25 30

Ser Ser Tyr Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg

35 40 45

Glu Phe Val Val Ala Ile Ser Lys Gly Gly Tyr Lys Tyr Asp Ala Val

50 55 60

Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

65 70 75 80

Val Tyr Leu Gln Ile Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Ser Ser Arg Ala Tyr Gly Ser Ser Arg Leu Arg Leu Ala

100 105 110

Asp Thr Tyr Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Gly Gly

130 135 140

Cys Asp Asp Asp Asp Lys His His His His His His

145 150 155

<210> 7

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 7

Asp Asp Asp Asp Lys

1 5

<210> 8

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 8

His His His His His His

1 5