WO2005117987A1 - Antibody conjugates targeting to ocular proteins - Google Patents

Abstract

A method of delivering a biologically compatible agent to an ocular structure is provided. According to the method, a biologically compatible agent coupled to an antibody is administered to the eye of a subject, and the antibody is bound to a structure of the eye to deliver the molecule to a desired location. Embodiments of the invention are useful in changing refractive index, treating collagen-based diseases, and treating presbyopia and/or cataractogenesis.

Description

ANTIBODY CONJUGATES TARGETING TO OCULAR PROTEINS

RELATED APPLICATIONS [0001] This application claims the benefit of priority of U.S. provisional application 60/575,746 filed in the U.S. Patent & Trademark Office on June 1, 2004 and U.S. provisional application 60/620,681 filed in the U.S. Patent & Trademark Office on October 22, 2004, the disclosures of which are incorporated herein by reference. FIELD OF THE INVENTION [0002] The invention relates to systems and methods of administering biocompatible agents to an eye, in particular a human eye, and more specifically of targeting the biocompatible agents conjugated to antibodies to specific locations in the eye. BACKGROUND OF THE INVENTION [0003] The eye, and in particular the human eye, is a complex organ that operates to convert incoming light into sensory signals that can be deciphered and interpreted by the brain. Light enters the eye through a tear film and an optically clear anterior cornea. The tear film and cornea are responsible for the initial convergence of incoming light. From there, the light passes through a variable size aperture (known as the pupil) of the iris, which has the appearance of a colored annulus. In a healthy or emmetropic eye, a physiological crystalline lens situated within a capsular bag is positioned posterior to the iris. Incoming light, already partially refracted by the tear film and cornea, is further refracted as the light passes through physiological crystalline lens. A properly operating crystalline lens causes the light to converge further so that the light passes through the vitreous fluid and arrives at the macula of the retina at a point known as the fovea. The amount of bending or refraction to which the light is subjected to as it passes through the eye and through the crystalline lens is referred to as the refractive power of the lens. The refractive power needed to focus on an object depends upon how far away the object is from the principle plane of the eye. More refractive power is required of the lens for converging light rays to view close objects clearly than is required for converging light rays to view distant objects clearly. [0004] A young and healthy physiological crystalline lens of the human eye has sufficient natural elasticity to have its shape altered through a process known as accommodation, to change its refractive power. The term accommodation refers to the ability of the eye to adjust focus between the distant point of focus, called the Punctum Remotum or pr (i.e., a far point beyond 20 feet or 6 meters away), and the near point of focus called the Punctum Proximum or pp (i.e., a near point within 20 feet or 6 meters away from the eye). Focus adjustment is performed in a young elastic lens using the accommodative-convergence mechanism. Ciliary muscle functions to shape the curvature of the physiological crystalline lens to an appropriate optical configuration for converging light rays entering the eye on the fovea of the retina. It is widely believed that near vision accommodation is accomplished via contracting the ciliary muscle, whereas far vision accommodation is accomplished by relaxing the ciliary muscle. [0005] With age, the eye undergoes various changes that can adversely affect eyesight and health. Perhaps the most common age-related change experienced by the eye is presbyopia. Presbyopia involves the deterioration of near vision accommodation ability. The onset of symptomatic age-induced presbyopia usually occurs around the age of 40. Those afflicted with presbyopia require near vision correction, typically in the form of reading glasses or the like, in order to perform near-vision tasks such as reading. According to one widely accepted theory, presbyopia is attributable to gradual hardening of the crystalline lens of the eye. As the lens hardens and loses its elasticity, ciliary muscles in the eye become less efficient at contracting the crystalline lens for near accommodation purposes. [0006] Another problem that arises with advanced age is the formation of cataracts, or cataractogenesis. Generally speaking, a cataract involves the development of clouding in the crystalline lens of the eye. If left untreated, cataracts can eventually lead to blindness. Aging is the primary risk factor of cataracts, although other factors such as smoking, steroid use, and eye injuries can increase the risk of cataracts. Treatment of advanced cataractogenesis typically involves surgical replacement of the damaged crystalline lens with a plastic intraocular lens. [0007] Another age-related affliction, which is the major cause of blindness in the Western world, is known as age-related macular degeneration (ARMD or AMD). Macular degeneration is a retinal degenerative condition (RDC) that leaves the afflicted individual with a "blind spot" or scotoma usually at or near the center of the individual's visual field. The blind spot or scotoma may appear as a black, gray, or distorted image. Objects in the central field of vision falling within the scotoma are not visible, thus limiting the afflicted individual's view of peripheral images around the blind spot. The visual field afforded by such peripheral images is often insufficient to allow the individual to perform routine activities such as reading, driving a vehicle, or even daily chores and errands. For example, when an individual having a RDC attempts to recognize another person at a distance, the individual may be able to discern the eccentric body portions of the viewed person peripherally, but the scotoma may "wipe out" the facial details of the viewed person, rendering the person unrecognizable. If left untreated, ARMD can lead to blindness. [0008] Corneal diseases represent an additional problem related to aging of the human eye. Examples of diseases of the cornea are epithelial basement membrane dystrophy and keratoconus. Corneal dystrophy is the gradual deterioration of one or more layers of the cornea. Epithelial basement membrane dystrophy causes the basement layer of the cornea to thicken and become irregular. If left untreated, cells buckle and break apart from the membrane. The breakdown of the membrane can lead to redness and mild to severe discomfort. Epithelial basement membrane dystrophy typically occurs after the age of 40, and more typically even later in life. The corneal disease keratoconus is a non-inflammatory eye condition in which the cornea progressively thins, eventually causing a vision-impairing, cone-like bulge on the cornea. [0009] The above afflictions, if left untreated, can have profound and life- altering effects on the afflicted individual. The effects range, for example, from the inconvenience of wearing corrective lenses to severe disabilities such as partial or complete blindness. Accordingly, it is highly desirable to provide methods and systems to treat these afflictions. [0010] Current known and theoretical techniques of administering drugs for the treatment of eyesight deficiencies and health problems include oral medications, topical ophthalmic eyedrops, injections in and around the eye, and chips implanted within the tissues of the eye. A common deficiency of many of these techniques is their ineffectiveness in reaching the targeted internal structures of the eye. Another deficiency of many of these techniques is their inability to target a specific component of the eye to the exclusion of non-targeted components. [0011] For example, because the crystalline lens is avascular and bathed on each side by avascular fluids, oral drug delivery, which is dependent on the bloodstream, is ineffective in delivering enzymatic molecules to the lens. Topical ophthalmic techniques can lack effectiveness if the administered drugs do not possess both hydrophobic domain and hydrophilic domain (i.e., amphipathic) to traverse the lipid-based corneal epithelium and high-water content corneal stroma, respectively. Also, topical ophthalmic drugs targeting structures located in the posterior chamber of the eye, e.g., the crystalline lens, may lose efficacy as the drops travel through the eye. [0012] Although injection techniques can circumvent some of these drawbacks, injections are often discouraged because of the risk that resulting trauma may induce cataract formation. "Chip"-like technologies are mostly theoretical, and are believed not to have been adopted because the surface of the lens is metabolically active and any attachment of a chip to the lens is likely to disturb the natural function of the eye and lead to a type of traumatic cataract. SUMMARY OF THE INVENTION [0013] It is an object of the invention to provide methods and systems of administering therapeutic biocompatible agents to the eye for changing the refractive index of a part or parts of the eye. [0014] It is another object of the invention to provide methods and systems of administering therapeutic biocompatible agents to the eye for treatment of age- related eyesight degeneration, such as experienced with persons afflicted with presbyopia and cataracts. [0015] It is still another object of the invention to provide methods and systems of administering therapeutic biocompatible agents to the eye for the treatment of diseases and age-related inflictions of the eyes, such as epithelial basement membrane dystrophy, keratoconus, macular degeneration and/or diabetic macular edema. [0016] In accordance with the purposes of the invention as embodied and broadly described herein, and to address above-discussed objectives, methods and systems are provided for delivering a biologically compatible molecule to the eye. The methods and systems generally involve administering a biologically compatible agent coupled (or ligated) to an antibody to an eye of a subject, and delivering the antibody with coupled agent to a targeted structure of the eye. [0017] The methods and systems of certain aspects and embodiments of the invention provided herein preferably deliver biocompatible agents to the eye via antibodies (or antibody fragments) designed for attachment to targeted optical structures the eye. By conjugating (or ligating) agents (e.g., molecules) to the antibodies, the antibodies act as a delivery system engineered to bind to specific substrates located in a selected one or plurality of optical structures. The eye has several proteins, collagens, and/or other molecules, tissues, and membranes that are unique to the eye and, in some cases, unique in their location in the eye with respect to other proteins, specifically collagens in the eye and in the subject's body. The specificity of the antigen-antibody reaction, coupled with inherent biological variation, makes an antibody a highly specific reagent capable of targeting and binding to specific structures of the eye, preferably without becoming bound to non- targeted structures. As a consequence, the agent attached to the antibody is delivered to the desired ocular structure. [0018] Specifying the antibodies to bind to unique proteins, collagens, lipids, and/or other substrates of various structures (e.g., membranes, fluid, and tissues) may result in any or all of the following advantages. First, this technique offers the opportunity to provide amphipathic agents for delivery of the biocompatible agents, as well as affording a wide selection of administration techniques from which to choose. Second, as the antibodies are customized to bind to substrates/tissues found specifically in the eye, the antibodies preferably do not attach to non-targeted tissues and, therefore, do not cause undesirable changes or side effects to the patient. Third, because certain eye structures treated in embodiments of the invention are avascular, e.g., the crystalline lens, introduction of antibodies is less likely to provoke an immune response in the eye. This may decrease the risk of inflammation, trauma and therapeutic delivery difficulties. Fourth, the therapy optionally can be initiated over a long period of time as a preventive measure and the therapy may be applied easily with a non-intrusive technique, such as an eye drop or the like. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS AND PREFERRED METHODS OF THE INVENTION [0019] Reference will now be made in detail to the presently preferred embodiments and methods of the invention. It should be noted, however, that the invention in its broader aspects is not limited to the specific details, representative systems and methods, and examples shown and described in this section in connection with the preferred embodiments and methods. The invention according to its various aspects is particularly pointed out and distinctly claimed in the attached claims read in view of this specification, and appropriate equivalents. [0020] The term "conjugated" and variations thereof are to be interpreted to include binding, attaching, joining, linking, ligating, or coupling, etc. [0021] The term "biocompatible" comprises an agent or agents generally understood as biologically compatible with the subject's eye. In particular, the agents are preferably substantially non-toxic, substantially non-hemolytic, and substantially non-irritant to the eye. Biocompatible agents may include, for example, molecules, polymers, surfactants, enzymes, etc. [0022] The terms "subject" and "patient" are used generally interchangeably herein, and may refer to a human or animal unless the context clearly dictates otherwise, e.g., a "human subject". [0023] In the below described aspects and embodiments, the therapeutic biocompatible agents (or molecules) may be administered to the eye using any suitable method, unless clearly stated otherwise. The administration method may be topical, e.g., an eye drop, in certain embodiments. To penetrate the cornea effectively, topical administrations preferably possess both hydrophobic domain and hydrophilic domain (i.e., amphipathic) to traverse the lipid-based corneal epithelium and high- water content corneal stroma, respectively. Injection is also possible in various embodiments. It is known in the art to include surfactants, toxins, and alcohols for increasing penetration. Other administering methods that may be employed include adenoviral vector, lipid vector such as lipid microspheres, electro- ionic transport, contact lens as therapeutic delivery system, slow drug release technology, and other techniques. The techniques are preferably performed in vivo, although in vitro procedures are also contemplated. [0024] The antibodies selected for the aspects and embodiments described herein can be polyclonal or monoclonal, although monoclonal antibodies are generally preferred. [0025] Cornea [0026] A first aspect of the invention provides methods and systems for delivering biocompatible molecules targeted to the cornea of the eye. The biocompatible agents are conjugated to antibodies that are delivered and bind to the cornea, preferably a substrate of the cornea selected from ocular collagens, proteins and/or other molecules (e.g., glycosaminoglycans (GAGs), proteoglycans) found on or within layers of the cornea. [0027] According to an embodiment of this first aspect of the invention, biocompatible agents are targeted to the cornea of the eye for the purpose of changing the refractive index of the cornea to alter the patient's refractive error. According to this embodiment, the biocompatible agents are selected to either increase or decrease the refractive index of the cornea, e.g., for making the recipient more nearsighted or farsighted. Biocompatible agents for changing refractive index include, for example and not necessarily limitation, plastics used in ophthalmology, such as polymethylmethacrylate, hydroxyethylmethacrylate, silicon hydrogels, silicon elastomers, and other biocompatible molecules (e.g., silicon, titanium), especially those used in ophthalmic surgery, such as in intraocular lens implantation, implantable lens procedures, polymeric sutures, etc. [0028] According to another embodiment of this first aspect of the invention, the biocompatible agents are targeted to the cornea of the eye for the purpose of treating diseases of the cornea, such as epithelial basement membrane dystrophy, keratoconus and other collagen-based diseases. In a particularly preferred implementation of this embodiment, the biocompatible agents partly or fully replenish tissues or cells of the cornea that have been damaged by the disease. For example, treatment of epithelial basement membrane dystrophy may involve administration of collagens (conjugated to antibodies) of the same type (e.g., Type 7) as the collagens damaged by the dystrophy. [0029] Potential targets in the cornea include ocular collagens, proteins and/or other molecules (e.g., glycosaminoglycans (GAGs), proteoglycans). The human cornea is a multi-layer membrane. Each membrane layer is composed of a discrete structure comprised of collagen, with the collagen types varying from layer to layer. The comeal layers are arranged, from anterior to posterior in direction, in the following order: epithelium; epithelium basement membrane; Bowman's membrane; corneal stroma; Descemet's membrane; and endothelium. Targeting of a particular layer or membrane is facilitated by selection of, for example, collagens of the targeted layer/membrane . [0030] The epithelial basement membrane (EPBM), also known as the basal lamina, is composed of two layers, the lamina lucida and the lamina densa. The EPBM is composed of primarily Type 7 collagen but also includes Type 6 collagen. Other components of the EPBM include laminin, heparan sulfate proteoglycan, fibronectin and fibrin. [0031] The Bowmans membrane, located immediately behind the EPBM, has a thickness of approximately 8-12 microns and is made of randomly arranged collagen fibrils. The Bowmans membrane is made of Types 1, 3, 5, and 6 collagen. [0032] The comeal stroma is a dense connective tissue of remarkable regularity. The co eal stroma makes up the vast majority of the cornea and predominantly comprises a 2 micron thick flattened collagenous lamellae (200-250 layers) orientated parallel to the comeal surface and continuous with the sclera at the li bus. Between the lamellae lie extremely flattened, modified fibroblasts known as keratocytes. The collagen fibers are predominantly of Type 1 (30 ran diameter, 64-70 mn banding), with some Types 3, 5 and 6 and 12 collagen. The transparency of the cornea is highly dependent upon the regular spacing of the collagen fibers (interfibrillary distance), which in turn is regulated by glycosaminoglycans (GAG) and proteoglycans forming bridges between the collagen fibrils. The GAGs in the human cornea are predominantly keratan sulphate and chondroitin (dermatan) sulphates. [0033] The Descemet's membrane is composed of Types 4, 5, 6, and 8 collagen, but is primarily Type 4 collagen, laminin and fibronectin. The Descement's membrane is a thin, homogenous, discrete, PAS-positive layer between the posterior stroma and the endothelium, from which it can become detached. Descemet's membrane is 8-12 microns in thickness and represents the modified basement membrane of the corneal endothelium. The anterior third of the Descemet's membrane is banded, whereas the posterior two thirds of the Descemet's membrane is homogeneous or non-banded. The Descemet's membrane is rich in basement membrane glycoproteins as well. The anterior banded region is reported to contain Type 8 collagen. Types 5 and 6 collagen may be involved in maintaining adherence at the interface of Descemet's membrane with the most posterior lamellae of the stroma. [0034] The corneal epithelial basement membrane, Bowman's membrane, the comeal stroma, descemet's membrane, and epithelium, either alone or in any combination, are preferred substrates for targeting. The particular membrane (or substrate) of the cornea subject to targeting may be predetermined by basing the selection of the antibody on its ability to bind to different membranes, i.e., to bind to collagen-type found in a certain membrane but not others. [0035] Crystalline Lens [0036] A second aspect of the invention provides methods and systems for administering biocompatible agents targeted to the crystalline lens of the eye. The biocompatible agents are conjugated to antibodies that are delivered and bind to the crystalline lens. As described in further detail below, potential target substrates of the crystalline lens include, for example, heat shock proteins (e.g., alpha, beta, and gamma crystallins), membrane proteins (e.g., extrinsic and intrinsic), cytoskeleton proteins (e.g., microfilaments, microtubules, intermediate filaments, and beaded-chain filaments), phospholipids, glycosphingolipids, sphingomyelins, cholesterols and other membrane lipids. [0037] Specifying the antibodies to bind to lens proteins and other molecules found in the crystalline lens of the eye has several potential advantages. First, it offers a direct avenue for drag delivery specifically to the crystalline lens. Second, because the antibodies are customized to bind to proteins and other substrates found only in the crystalline lens, the antibodies should not attach to other tissues lacking the targeted substrate and cause undesirable changes or side effects. Third, because the lens is avascular, introduction of antibodies is less likely to provoke an immune response in the generally biologically inactive lens. Fourth, a method of delivery that does not risk trauma to the human lens may be selected. Fifth, the therapy optionally can be initiated over a long period of time as a preventative measure for incipient presbyopes or cataractogenesis as the therapy may be applied easily with an eye drop or injection. [0038] According to one embodiment of the second aspect of the invention, the biocompatible agents may be targeted to the crystalline lens of the eye for the purpose of changing the refractive index of the crystalline lens to alter the patient's refractive index. According to this embodiment, the biocompatible agents are selected to either increase or decrease the refractive index of the crystalline lens, e.g., making the recipient more nearsighted or farsighted. Biocompatible agents for changing refractive index include, for example and not necessarily limitation, plastics used in ophthalmology, such as polymethylmethacrylate, hydroxyethylmethacrylate, silicon hydrogels, silicon elastomers, and other biocompatible molecules (e.g., silicon, titanium), especially used in ophthalmic surgery, such as in intraocular lens implantation, implantable lens procedures, polymeric sutures, etc. Targets for this embodiment may include, for example, collagens and proteins of the crystalline lens. [0039] Another embodiment of this aspect of the invention involves the targeting of the biocompatible agents to the crystalline lens of the eye for the purpose of disrupting, preventing, and/or reversing mechanisms believed responsible for presbyopia and cataract formation. These mechanisms will now be described in greater detail. [0040] The normal young lens is exquisitely transparent, appearing homogeneous and uniform in structure. The lens also is avascular, enclosed by a metabolically inert basement membrane composed of protein resembling collagen, as well as glycoproteins and perhaps other components. The human lens has a very high protein content of approximately 35 percent based on a wet weight basis in the periphery, which increases to approximately 40 weight percent in the central region of the tissue. Most of the proteins in the lens are represented by "crystallins," the so- called structural proteins. The crystallins make up fiber cells of the lens and their proximal periodicity (arcangement regularity) allows light to pass relatively unimpeded through their protein structure. [0041] Alpha crystallins are large macromolecular weight aggregates, about 800,000 d (Daltons). Alpha crystallin is a heteromeric complex containing 30-40 copies of two closely related subunits, alpha A and alpha B crystalline in roughly a 3:1 ratio. The alpha A subunit is found exclusively in lens, making it an especially useful target for isolated therapeutic treatment. The alpha B subunit is also found in skeletal muscle, skin, brain and other tissues, making it less preferred than the alpha A subunit but still a possible target. Both subunits are members of the small heat shock protein family. Alpha crystalline macromolecules in the normal lens are composed of polypeptide chains held together by non-covalent interaction. [0042] The beta-crystallin group is the most abundant crystalline group. There is believed to be four major size populations of beta-crystallin, with the larger species called beta High (H) and smaller components beta Low (L). These aggregates, like those of alpha-crystallin, are bound together noncovalently in a young healthy lens. Beta crystallins have properties intermediate between alpha and gamma crystallins. [0043] The gamma-crystallins are unique in being the only major group of lens proteins found entirely in the monomeric form. The beta and gamma crystallins are relatively rich in thiol groups. The gamma crystallins are found mainly at the central core region of the mammalian eye lens where the refractive index is a maximum and the water content a minimum. There is little or no protein turnover within the central core region. [0044] The hardening of a crystalline lens that accompanies presbyopia and opacification mechanisms associated with cataract formation are believed to be inextricably intertwined with one another. It is believed that presbyopia and cataract formation each generally involve the formation of disulfide bonds and/or the oxidation of thiols, such as methionine and cysteine, in the crystalline lens. More specifically, lens membrane proteins undergo changes as a result of aging of the human crystalline lens, hi particular, in oxidation of thiol groups in young normal lenses appears to be minimal and confined to proteins associated with the membrane, and protein aggregates generally are not covalently bonded in young healthy lenses. In older lenses, oxidation is found to a slight extent, but is confined to the membrane or membrane-associated components. This oxidation effect is believed to cause the formation of intramolecular disulfide bonds characteristic of cataractogenesis. In cataract, extensive oxidation is found in all factions and oxidized thiol groups lead to disulfide bonds. These disulfide bonds and other cross-links, higher oxidation states of sulfur, and unusual chromophores result in the formation of high molecular weight (HMW) aggregates. With increasing opacity, there appears to be increased accumulation of this type of giant macromolecule. Although the genesis of cataracts is not completely understood, it is believed that the native structure is perturbed, causing an unfolding of the macromolecule and exposure of oxidizable groups. [0045] Opacification of the human lens is accompanied by oxidation of cysteine sulfhydryl groups to half-cystine residues. In normal transparent lenses, cys- 131 and cys-142 from alpha A crystalline are present as a mixture of cysteine sulfhydryl and half-cystine disulfide groups, while identical analysis from cataractous lenses demonstrated undetectable levels of the cysteine sulfyhdryl group. The reactive sulfydryl groups in cysteines can interact with one another to form disulfide bridges, methionine can oxidize to polar sulfoxide, and photo-oxidation of aromatic residues can lead to a variety of products. Other covalent linkages include disulfide bridge formations, calcium salt bridges, and nonsulfide covalent cross links. Among the latter, transamination is the latest mechanism proposed for aggregate formation. [0046] Although any of the above heat shock proteins may be targeted, gamma crystallins present a potentially highly desirable target for the biocompatible molecule-antibody conjugate of this embodiment of the invention. Gamma crystallins are rich in the sulfur-containing residues cysteine and methionine, and aromatic residues, all of which are susceptible to oxidative processes and disulfide covalent bonding. [0047] It is also believed that presbyopia and cataract formation also are associated with changes in the amounts of certain certain membrane proteins (e.g., extrinsic and intrinsic), cytoskeleton proteins (e.g., microfilaments, microtubules, inteπnediate filaments, and beaded-chain filaments), phospholipids, glycosphingolipids, sphingomyelins, cholesterols and other membrane lipids. [0048] For example, the content of Vimentin (intermediate filament) by human membranes steadily decreases with aging. The MP43 protein increases in human fiber membranes in an age-dependent fashion. The human MP 10-12 protein is absent in the membranes from fetal lenses, appears first at birth and rises linearly in its relative weight fraction throughout the life span. The human lens fiber membrane main intrinsic protein consists of two polypeptides of 26,000 and 22,000 d molecular weight undergo a reciprocal gradual change in relative abundance from a predominance of MP26 at birth to equality or a predominance of MP22 after midlife.

Some of these membrane components are also components of the lens fiber cell cytoskeleton (spectrin, alpha actinin, the beaded-chain filament protein, vimentin and actin). The major age-dependent change in human fiber membranes consists of the gradual conversion of MP26 to MP22 throughout the life span. [0049] As mentioned above, the amount of cell membrane lipids of the crystalline lens also undergoes change with aging of the eye. Lipids make up to 55% of the lens fiber cell plasma membranes. Lens lipids include phospholipids, cholesterol and gylcosphingolipids. The relatively high cholesterol content of lens fiber plasma membranes is widely believed to contribute along with membrane proteins to creating a more rigid membrane. [0050] The major phospholipids are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin, and a minor fraction consisting of phosphatidyl-glycerol, phosphatidic acid, diphosphatidylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, and plasmogens. The only sterol in the lens is cholesterol. The most remarkable and potentially significant aging changes in the lipid composition of the membranes appear to be the loss of phospholipids and the conversion of the membranes into less fluid, more rigid structures through the retention of cholesterol, sphingomyelin and intrinsic proteins. 4,5- dihydrosphingomyelin (DH-SPH) species is 43% of all the phospholipids in lens membranes. Other sphingomyelin species, phosphatidylcholine species and phosphatidylethanolamine species together make up 37% of all phospholipids in lens membranes. This is unique as it is not found in any other naturally occurring membranes. PC, PE, SPH and overall chain saturation have all been shown to increase steadily with age. [0051] Glycosphingolipids are known to be found in the lens but have not been widely studied. Glycosphingolipid gangliosides detected in human include predominantly G_MI and G_M3. The long chain bases of the sphingolipid ceramide fraction of human lenses consist primarily of sphinganine and 4-sphinganine. The sphingomyelin concentration also increases in the transition from fetal to adult lens. Unusually high concentrations of sphingomyelin are attained in the human lens upon aging. 4,5-dihydrosphingomyelin (DH-SPH) is 43%) of all phospholipids in the lens. This is not found in any other naturally occurring membranes. [0052] In accordance with one implementation of this embodiment of the invention, the biocompatible molecules are selected from those that break disulfide cross-linking and/or disrupt oxidative processes that lead to cross linking and aggregation of proteins. Without necessarily wishing to be bound by any theory, it is believed that when the antibody attaches to the specified site, preferably the heat shock proteins (e.g., alpha, beta, and gamma crystallins), the conjugated molecule disrupts the oxidative process responsible for presbyopia and cataract formation. For example, the molecule may prevent oxidation of a site, may function as a crosslinking inhibitor, and/or may cleave disulfide bond crosslinks. [0053] Breaking of the disulfide bonds in a protein can either be carried out oxidatively or reductively. The following reagents may be selected for disulfide disruption and cleavage. The following list is not comprehensive, but is demonstrative only. Enzymatic reagents include thioredoxin and glutaredoxin. Chemical reagents include, for example, N-acetyl cysteine (NAC), tri-(2- carboxyethyl)phosphine (TCEP), glutathione (reduced from GSH), dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethylamine, homocysteine, and perfomiic acid (Devlin 74). [0054] Perfomiic acid is an example of a reagent for carrying out oxidative cleavage of disulfide bonds. A drawback to the use of performic acid to oxidize cystine groups is that perfonnic acid also reacts with methionine side chains to form the methionine sulfoxide. With regard to reductive cleavage, to prevent reoxidation of the cysteine side chains after reductive cleavage, the free mercaptains are generally alkylated to prevent reoxidation to disulfide bonds. The most commonly used alkylating agent is iodoacetate. [0055] According to another embodiment, the disulfide reducing capability localized to the monoclonal antibody recognition site is generated using an antibody- directed enzyme-prodrug therapy (ADEPT) approach, which permits conjugation of disulfide reducing enzymes to the antibody without destroying the structure of the antibody or its specific recognition capability. Generally, the approach retains the conjugated enzyme and antibody in oxidized form, and then delivers a prodrug comprising a reducing agent and an oxidized disulfide bond disrupter/breaker. Upon reacting with the reducing agent, the conjugated enzyme activates and remains in close proximity to the antibody recognition zone even if the antibody loses its recognition upon activation. The activated enzyme then reduces the disulfide bond disrupter/breaker at the site of the antibody recognition. An exemplary procedure is set forth below: [0056] 1) The reducing power is supplied in the form of a prodrug comprising two inactive components, such as reduced nicotinamide adenine dinucleotide (NADPH) and either oxidized glutathione (GSSG) or oxidized thioredoxin enzyme. These components are inactive towards disulfide bonds. [0057] 2) The monoclonal antibody and its conjugated enzyme (e.g., glutathione reductase or thioredoxm reductase) are fused (or ligated) together using molecular cloning technique (described below) and are delivered in the eye to recognize specific tissues. [0058] 3) Upon application of a mixture of NADPH and either GSSG or oxidized thioredoxin, NADPH turns on the conjugated enzyme and the "activated" enzyme reduces either GSSG or oxidized thioredoxin to generate reduced glutathione (GSH) or reduced thioredoxm at the site of antibody recognition only. Subsequently GSH or reduced thioredoxin can reduce disulfide bonds localized to the antibody recognition area. [0059] Cloning of the monoclonal antibody-enzyme conjugate: The enzyme capable of reducing either glutathione or thioredoxin will be fused to the N-terminus of variable region of either heavy (VH) or light chain (LH) of the monoclonal antibody. Since the conjugated enzymes (thioredoxm reductase or glutathione reductase) are functional only in dimeric form held by non-covalent forces, a monomeric functional conjugate enzyme has to be engineered. To achieve this goal, two copies of the enzyme will be fused in tandem separated by a suitable linker. The length of this linker will be optimized by testing activity as a function of linker length (5-30 A). Initially the conjugated enzyme (Er) will be purified either from bacteria or other hosts (yeast, insect cells) to optimize the linker, length between the two monomers. This procedure should generate a functional monomer retaining the activity of the naturally occurring dimeric form. The protein coding region of this optimized enzyme Er will be excised from a suitable plasmid and will be ligated to the variable region of the antibody in such a way that they express a fusion protein. A medium to long range linker composed of 25-50 amino acids will be placed between the Er and the antibody variable region by molecular cloning so that Er does not interfere with the antigen recognition and also can access large distance (30-50 A) from the antibody to reduce target disulfide bonds. Finally for protein purification purpose, 6-8 histidine residues in tandem will be introduced either at the amino or carboxy terminal of the whole construct. [0060] Expression and purification of the fusion constructs: The recombinant antibody-enzyme conjugate fusion construct may be transformed in suitable mammalian cells. Chinese Hamster Ovary (CHO) cells are the most popular choice for antibody production. However other types of cells such as murine lymphoid cells (NS0, SP2/0) can be tested. The desired agent can be engineered to be secreted using techniques known in the art. Alternatively, the agent may be isolated via non-secretion techniques as known in the art. Antibodies can be easily purified by the histidine-affinity column (Ni-NTA matrix). [0061] The above procedures may be modified as appropriate and employed for other aspects and embodiments of the invention. [0062] According to another implementation of this embodiment of the invention for treating presbyopia or cataract formation, the biocompatible agents may be administered to the crystalline lens of the eye for the purpose of offsetting the effects of aging, e.g., increasing or decreasing the amount of membrane proteins (e.g., extrinsic and intrinsic), cytoskeleton proteins (e.g., microfilaments, microtubules, intermediate filaments, beaded-chain filaments), phospholipids, glycosphingolipids, sphingomyelins, cholesterols and other membrane lipids, and combinations thereof. Table 1 sets forth a partial listing of membrane protein and other targets for this embodiment of the invention. Table 1 - Polypeptide composition of Vertebrate Lens fiber Plasma Membranes

[0063] In a particular implementation of this embodiment, the biocompatible agents partly or fully replenish proteins, lipids, etc. of the crystalline lens that have lessened in abundance with age. The description above identifies examples of proteins and lipids that undergo a reduction in quantity with aging. In another implementation of this embodiment, biocompatible agents are delivered to the crystalline lens to break down proteins, lipids, etc. of the crystalline lens that have increased in abundance with age. For example, appropriate agents, such as surfactants, may be delivered using antibodies to break down phospholipids. The surfactant may be selected from anionic surfactants, cationic surfactants, non-ionic surfactants, ampholytic surfactants, and combinations thereof. Examples of surfactants include sodium cholate, sodium deoxycholate (DOC), N-lauroylsarcosine sodium salt, cetyltrimethyl-ammoniumbromide (CTAB), and bis(2- ethylhexyl)sulfosuccinate sodium salt. [0064] Vitreous Cavity [0065] According to another aspect of the invention, a method and system are provided for delivering biocompatible or therapeutic agents targeted to the vitreous cavity of the eye. The agents are conjugated to antibodies that are delivered and bind to a vitreous cavity substrate, wherein the substrate is preferably selected from collagens, proteins (e.g., hyaluronic acids), and/or other molecules found within the vitreous cavity. [0066] The vitreous cavity is the largest cavity of the eye, representing approximately two thirds the volume of the eye. The vitreous cavity is bound anteriorly by the lens, posterior lens zonules and ciliary body, and posteriorly by the retinal cup. Contained within the vitreous cavity is a fluid known as the vitreous humour or vitreous. The vitreous is a transparent, viscoelastic gel that is 98% water with a refractive index of about 1.33. Its viscosity is 2-4 times that of water and is dependent on the concentration of Na-hyaluronate. The vitreous contains fine diameter Type II collagen fibers (8-12 nm diameter), which entrap large coiled hyaluronic acid molecules. The vitreous cavity is shaped like a sphere with an anterior depression, the hyaloid fossa (also known as patellar or lenticular fossa). The vitreous is traditionally regarded as consisting of two portions: (1) a cortical zone, characterized by more densely arranged collagen fibrils, and (2) a more liquid central vitreous. [0067] In a preferred embodiment of this aspect, the biocompatible agents increase or decrease the refractive index of the vitreous in the vitreous cavity, e.g., for countering native or age-related farsightedness or nearsightedness. Biocompatible agents for changing refractive index include, for example and not necessarily limitation, plastics used in ophthalmology, such as polymethylmethacrylate, hydroxyethylmethacrylate, silicon hydrogels, silicon elastomers, and other biologically compatible agents (e.g., silicon, titanium) used in ophthalmic surgery, such as in intraocular lens implantation, implantable lens procedures, polymeric sutures, etc. [0068] In another preferred implementation of this embodiment, the biocompatible agents conjugated to antibodies are delivered to the vitreous cavity to prevent, disrupt, and/or reverse vitreous degeneration, syneresis and/or floaters. The human vitreous begins to degenerate at adolescence leading to the appearance of liquid filled cavities and fibrillar strands (such as the retrolental, preretinal etc). The gel state of the vitreous humor is maintained by a dilute network of long, thin collagen fibrils containing Types 2, 5, 9 and 11 collagen. The distance between any two vitreal fibers is spaced such that the vitreous transmits light to the retina. As there is a progressive lateral fusion of the individual fibers with aging, the result of which causes an impediment to light traveling through the eye and can result in a thicker fiber that casts a shadow on the retina appearing to the subject as having an object floating in their eye. This form of vitreal degeneration is known as vitreous floaters. On occasion, an area where the vitreous is firmly attached to the retina can detach, leaving a thick, membranous opacity floating around the cavity which casts a shadow on the retina. This form of vitreal deneration is known as a posterior vitreous detachment. To counteract these aging mechanisms, the delivered conjugate may comprise a reducing agent for inhibiting or cleaving the disulfide crosslinks, such as a collagen cross-linking inhibitor, specifically molecules that cleave disulfide bonds specific to Type 2, Type 5, Type 9, Type 11 collagen and hyaluronic acid cross-links. The following list of disulfide disruption and cleaving reagents is not comprehensive, but is demonstrative only. Enzymatic reagents include thioredoxin and glutaredoxin. Chemical reagents include, for example, N-acetyl cysteine (NAC), tri-(2- carboxyethyl)phosphine (TCEP), glutathione (reduced from GSH), dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethylamine, homocysteine, and performic acid (Devlin 74). A [0069] Additionally, the antibodies may deliver anti-inflammatory molecules to inhibit or cure vitreous inflammations such as posterior uveitis. These therapies may be delivered individually or in conjunction with one another. [0070] Administration techniques described above may be used for this aspect of the invention, although injection is preferred. However, if the targets are specific to the vitreous and not found elsewhere in the eye, topical applications (e.g., eye drops) may be administered. Further, the antibody-directed enzyme prodrug therapy (ADEPT) described above may be used for this embodiment. [0071] Bruch's Membrane [0072] According to another aspect of the invention, a method and system are provided for delivering a biocompatible agent conjugated to an antibody targeted to Bruch's membrane of the eye for treatment of age-related macular degeneration (AMD) and/or diabetic macular edema. [0073] Age-related macular degeneration is the maj or cause of blindness in the western world. There are two forms of AMD. The first is the "dry" or nonexudative form, which involves atrophic and hypertrophic changes to the retinal pigment epithelium (RPE). The second is a "wet" or exudative form involving development of abnormal blood vessels called choroidal neovascular membranes (CNVs) under the retina, causing the leakage of fluid and blood. [0074] The Bruch's membrane is the basal lamina of the RPE. Changes in Bruch's membrane have been suggested to contribute to vascular permeation into the overlying retinal layers and formation of deposits (drusen), leakages of blood and other proteins, lipids and calcifications which can lead to retinal hypoxia, death of photoreceptors and neovascularization, which contribute to AMD. Material that accumulates within inner collagenous layer of Bruch's membrane is termed basal linear deposit. A basal linear deposit is primarily composed of granular and vesicular material with foci of wide-spaced collagen. The accumulation of basal linear deposit can be focal or diffuse. A cleavage plane between the RPE and Bruch's membrane tends to develop at the level of basal linear deposit. This accounts for the tendency of CNVs (choroidal neovascular membranes) to grow between the inner collagenous layer of Bruch's membrane and the RPE plasma membrane in AMD for the tendency of pigment epithelial detachments to occur, and for the surgical plane of CNV dissection to be between the RPE basement membrane and the inner collagenous layer of Bruch's membrane in AMD. Macrophages and foreign body giant cells near Bruch's membrane become more common when basal linear deposit is present. Macrophages might digest Bruch's membrane and can release cytokines that stimulate CNV growth. [0075] Basal laminar deposit develops first over-thickened or basophilic segments of Brach's membrane, widened intercapillary pillars, or small drusen, suggesting that its accumulation is a response to altered filtration through Brach's membrane at these sites. [0076] Brach's membrane is composed of Types 1-4, 6-8, and 18 collagen, integrins, vitronectin, laminin, fibronectin, and endostatin. The collagen is predominantly Types 1, 3, and 4, with very little Type 2 collagen and substantially no Type 5 collagen. Coated and uncoated vesicles, glycoproteins, and glycosaminoglycans are also found within the basal lamina. [0077] The accumulation of material in the inner and outer collagenous layers of Brach's membrane induces a change in Brach's membrane composition, as the vesicular material has high lipid content. [0078] It is believed that collagen Type XVIII may play a protective role in the accumulation of lipids and degeneration of Brach's membrane. Additionally, cross linking of the collagen in Brachs membrane also has been implicated in aging changes related to ARMD. The solubility of Brach's membrane collagen declines with age due to increased crosslinking both in the macular region and in the periphery. In contrast, the amount of non-collagen protein increases in Brach's membrane under the macula but not in the periphery. Protein deposition and crosslinking in Brach's membrane might prevent debris (e.g., vesicles) from passing through Brach's membrane to the choriocapillaries and result in accumulation of extracellular material in the inner aspect of Brach's membrane. [0079] In accordance with this embodiment of the invention, a method and system are provided for delivering therapeutic agents to the basement membrane of the RPE, Brach's membrane. The agents are specifically designed via antibody technology to deliver additional collagens of Type 1-8, in particular Types 1, 3, and 4 collagen and in addition collagen 18 which may play a protective role against AMD. The agents preferably inhibit and/or lyse the formation of lipids that are found in drusen. These may be delivered in conjunction with collage Type 18, both breaking down the lipids and providing a protective role at the same time. The agents may comprise enzymes to break collagen cross-linking in Brach's collagens. This might prevent debris from passing through Brach's membrane and help to maintain its integrity. [0080] In the event that injection is selected as the administration technique for this embodiment, injection may involve, for example, retrobulbar injection, injection into the vitreous, or intravenous injection. However, if the targeted proteins are specific to the Bruch's membrane and not found elsewhere in the eye, topical applications (e.g., eye drops) preferably are administered. [0081] Molecules that may be delivered to the retina (or Brach's membrane) for the above purposes include one or more of the following, alone or in combination: lutien; xeaxanthine; other antioxidants; carotenoids; micronutrients including zinc, selenium; interferon alpha; thalidomide and antineovascularization agents; angiostatic steroids; anti VEGF(vascular endothelial growth factors) agents; triamcinolone acetonide; and RhuFAB (Genentech compound).

[0082] The embodiments described herein may be administered individually or in conjunction with one another. The antibodies of the various embodiments may be polyclonal or monoclonal antibodies, and may also comprise molecules that are fragments and derivatives of such antibodies, including, for example, F(ab')₂, Fab' and Fab fragments. Such antibodies may be monospecific, or may comprise bispecific antibodies, such as chimeric antibodies, hybrid antibodies, etc. having at least two antigen or epitope binding sites. Monoclonal antibodies are preferred. [0083] Methods for isolating or obtaining such immunoglobulins are well- known in the art (Kohler, G. & Milstein, C, Nature 256:495-497 (1975); Taggart & Samloff, Science 219: 1228-1230 (1983); Kozbor et al., Immunology Today 4:72-79 (1983); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Takeda et al., Nature 314:452-454 (1985)); Biocca, S. et al., EMBO J. 9:101-108 (1990); Bird, R. E. et al., Science 242:423-426 (1988); Boss, M. A. et al., Nucl. Acids Res. 12:3791-3806 (1984); Boulianne, G. L. et al., Nature 312:643-446 (1984); Bukovsky, J. & Kennett, R. H., Hybridoma 6:219-228 (1987); Diano, M. et al., Anal. Biochem. 166:223-229 (1987); Huston J. S. et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Jones, P. T. et al., Nature 321:522-525 (1986); Langone, J. J. & Vunakis, H. V. (Editor), Methods Enzymol. 121, Academic Press, London (1987); Morrison, S. et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Oi, V. T. & Morrison, S. L., BioTechiiiques 4:214-221 (1986); Riecbmami, L. et al., Nature 332:323-327 (1988); Tramontano, A. et al, Proc. Natl. Acad. Sci. USA 83:6736-6740 (1986); Wood, C. R. et al. Nature 314:446-449(1985); and Ladner, U.S. Pat. No. 4,946,778, issued Aug. 7, 1990. [0084] Polyclonal and monoclonal antibodies are widely commercially available, as are a variety of products for conjugating agents to the antibodies. Use of such commercial products is within the scope of this invention. Alternatively, the antibodies and conjugation techniques may be developed using procedures that are known in the art and/or specified herein. [0085] Polyclonal antibodies may be produced through any of a variety of well known methods. For example, various animals may be immunized for this purpose in known manner by injecting them with an antigen (for example a lens protein, collagen, or crystallin, or another molecule sharing an epitope of such molecules). Such antigen molecules may be of natural origin or obtained by DNA recombination or synthetic methods, or may comprise fragments thereof. Desired polyclonal antibodies can be recovered from the resulting sera and purified by known methods. Alternatively, intact cells that array the antigen molecule may be used. Various adjuvants may also be used for increasing the immune response to the administration of antigen, depending on the animal selected for immunization. Examples of these adjuvants include Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactant substances such as polyanions, peptides, oil emulsions, haemocyanins, dinitrophenol or lysolecithin. [0086] There are several accepted methods of monoclonal antibody production, which typically include the step of isolating the antigenic target of the antibody. Methods of isolating antigen are standard practice and well understood in the art. [0087] The following materials, methods, and techniques are presented by way of example and illustration, and are not necessarily limiting upon the scope of the invention and its various embodiments. [0088] (1) Selection of a Cell Culture Medium [0089] Two common culture media used in laboratories are Dulbecco's minimum essential medium (DMEM) and RPMI 1640. According to a known technique, the powder form is sterilized once dissolved. The quality of water is important. Water purification units for different purposes produce different quality water; the unit used should be intended for tissue culture. Medium is conveniently used, for example, in 500 ml aliquots and stored, for example, at 4°C before use. Medium should be warmed to 37°C prior to use. According to an example technique, to the medium add glutamine (e.g, 2mM), penicillin (e.g, 100 IU ml^"1), streptomycin (e.g, 100 mg ml^"1) and fetal bovine serum (FBS) to 10%. If the medium is stored (e.g, for more than 2 weeks at 4°C), the levels of the essential amino acid glutamine, and the antibiotics penicillin and streptomycin may be replenished. [0090] (l)(a) Fetal Bovine Serum (FBS) [0091] Serum may be used to provide additional nutrients to the medium to support cell growth. FBS is a commonly used serum additive for tissue culture media. Different batches of FBS support cell growth to different degrees. Batches of FBS can be screened for ability to support hybridoma growth, although prescreened batches are available commercially. Mixtures of sera have been used for hybridoma culture, and the addition of mouse serum has been reported to increase hybridoma yields. A-gamma (Ig depleted) calf serum has been shown to produce twice as much immunoglobulin as standard FBS, in both human and mouse hybridoma lines (Torres et al, 1992), and purification of monoclonal antibody subsequently is easier if there is no bovine IgG present. [0092] (1) (b) Serum-Free Media [0093] The risk of introduction of pathogens such as bovine viruses or prions (which cause diseases such as bovine spongiform encephalopathy and Creutzfeldt- Jacob syndrome) and the presence of unwanted proteins in downstream processing has generated the desire to use completely defined serum-free medium. [0094] (2) Selection of Fusion Medium [0095] Medium containing hypoxanthine, aminopterin and thymidme (HAT) may be used to selectively grow hybrids following fusion. Aminopterin blocks the main biosynthetic pathway for DNA synthesis, while thymidine and hypoxanthine feed the salvage pathways. For each fusion, a fresh bottle of HAT medium may be made, for example, by the addition of 100 x stock HAT to the culture medium. A useful amount is about 100 ml of HAT medium/10⁸ lymphocytes fused. Medium containing hypoxanthine and thymidine (HT) may be used to maintain hybridoma growth. Because hypoxanthine and thymidine are used up by cells in culture whilst aminopterin is not, cells will die unless HT medium is added, until the aminiopterin has been diluted out or removed. From 7 days following the fusion, the hybridomas are maintained in medium with HT. Some laboratories wean hybridomas off HT medium once the aminopterin is depleted, but the effort involved outweighs the savings in HT. [0096] (2)(a)Lysis Medium [0097] If spleen cells are used as the source of immune cells for fusion, the erythrocytes are commonly lysed prior to fusion, using isotonic ammonium chloride or Gey's hemolytic medium. However, this step is not essential and can compromise the quality of the lymphocytes . [0098] (2) (b)Poly ethylene glycol (PEG) [0099] PEG is the fusion-inducing agent. Batches of PEG vary in their toxicity and ability to induce fusions. PEG may be stored in the dark, to avoid degradation by photooxidation. Some groups add dimethylsulfoxide (DMSO, 15% (v/v)) to the PEG for fusion, but the value of DMSO in the fusion process is questionable. [0100] (3) Immunization [0101] (3) (a) Selection of Antigen [0102] As much as 1 mg of antigen may be used for the immunization and screening. The antigen should be as pure as possible because there will be an immune response against contaminants in the preparation. The purity of the antigen used in the detection assay is important; methods of screening for antibody against a component of a mixture, such as western immunoblotting or biological assays, are considerably more labor intensive than ELISA. The protein should be no smaller than 3kDa, and should differ in amino acid sequence from the corresponding endogenous protein, in order to induce an immune response. Smaller or endogenous molecules can be made immunogenic by conjugation to a carrier protein, such as diphtheria toxoid. [0103] Synthetic peptides that correspond to an amino acid sequence of the antigen can be prepared or purchased for use as antigen. The peptide should correspond to a sequence that is present on the exterior of the antigen molecule, and should be predicted to be antigenic, on the basis of the literature or antigenicity programs such as Mac Vector. If the synthetic peptide is small it should be conjugated to a carrier protein. [0104] (3) (b) Standard Immunization Protocol [0105] The response to soluble antigens is greatly potentiated by using an adjuvant. Complete Freund's adjuvant (CFA) is efficient. Other adjuvants include GMDP (GERBU Adjuvant 01, GERBU Biotechnik, Gailberg, Germany). [0106] A booster immunization is commonly given about 2-4 weeks following the primary immunization; this should not be given in CFA because the risk of anaphylactic shock, but may be given in incomplete Freund's adjuvant (which is the same as CFA except that the mycobacterial component is missing). Preferably, the same aqueous adjuvant as for the primary immunization is used. [0107] Antibody liters greater than 1 in 100 mouse serum is the minimum required to consider using the splenocytes in a fusion. If the mouse was immunized with an impure preparation of antigen and then screened with the same material it is necessary to confirm a specific immune response against the antigen, for example, by western blotting to identify the antigen by molecular weight, or by inhibition of the antigen's biological activity. [0108] (3) (c) Sample Procedure [0109] 6 week old female mice > immunize each mouse with 50 micrograms antigen> wait 2 weeks> immunize each mouse with 50 micrograms antigen> wait 2 weeks >collect serum from mice and determine polyclonal titer> is the serum titer sufficient for fusion? - yes, then you have fusion- no, go back to immunizing mice. [0110] (3) (d) Alternative Immunization Protocols [0111] Several techniques have been used to overcome the problems of very low levels of antigen, including in vitro immunization, intrasplenic immunization and lymph node deposition. Small amounts of antigen purified on nitrocellulose membranes have been used to immunize animals either by an intrasplenic route or in vitro. These methods generally result in monoclonal antibodies of the IgM isotype and often of low affinity. [0112] (3)(e)Example Protocol for Conjugation of Antigen to Carrier Protein [0113] Materials: carrier protein (e.g. diphtheria toxoid) (the mass ratio of carrier protein to antigen should be 4:1); glutaraldehyde solution (0.1 M); dialysis membrane with a molecular weight cut off that will allow unconjugated hapten to dialyse out but retain conjugate; TBS buffer (Tris Base, 0.1M; NaCl, .15 M, ρH8) [0114] 1. Dilute the antigen to a concentration of 2.5 mg ml^"1 in TBS. [0115] 2. Dilute carrier protein to a concentration of 2 mg ml^"1. [0116] 3. Mix carrier and protein in 4:1 mass ratio in a beaker with a magnetic stirring bar. [0117] 4. Add Glutaraldehyde solution 1 volume per 2.4 volumes of protein solution. Add the glutaraldehyde solution to the continuously stirred protein over a period of 20 min and continue stirring for 90 min at room temperature. [0118] 5. Dialyse the reaction mixture against 2000 volumes of TBS for 16h at 4°C. [0119] (3)(f)Example Protocol for Immunization with Soluble Protein (available in quantity) [0120] Materials: 5 six week old female Balb/c mice; approx. 250 micrograms of the antigen; sterile isotonic saline or phosphate buffered saline ρH7; 10 microgram vial of GMPD adjuvant (GERBU adjuvant 10, GERBU Biotechnik, GmbH, Gailberg, Germany); 1 ml syringe and a 27 gauge injection needle. [0121] Resuspend 250 micrograms of the antigen in 1 ml of the aqueous solution [0122] Transfer the resuspended antigen to a 10 microgram vial of GERBU adjuvant and agitate to dissolve the adjuvant. [0123] Take the antigen into a 1 ml syringe. Tap the syringe with the nozzle facing upwards in order to dislodge bubbles from the internal surface of the syringe. Attach the needle to the syringe and depress the piston to check that the solution flows through the needle. [0124] A single injection of 200 microliters of the solution is made into the intraperitoneal cavity. [0125] (4) Cell Fusion [0126] Various myeloma cell lines are available for fusion, including, for example, SP2/0Agl4 (Shulman et al, 1978); P3-X63-Ag8.653 (Kearney et al, 1979); FO (De St. Groth and Scheidegger, 1980); and P3-NSl/l-Ag4-l (secretes Kappa chain) (Kohler et al, 1976). Rat, mouse, chicken, hamster and other mammals may be used as fusion partner. [0127] The maintenance and health of the myeloma fusion partner is of importance in the eventual success of the fusion. Fresh myeloma cells are preferable to ones that have been growing a long time. [0128] (4) (a)Fusion Protocol [0129] Established fusion protocols may use PEG to induce membrane fusion. Electroporation and electroacoustic techniques are alternatives that are especially useful when low numbers of specific B cells are available for fusion. When combined with in vitro immunization methods, the purification of antigen- specific B cells followed by electroporation enables the production of monoclonal antibodies against low amounts of antigen. [0130] There are many variations of the standard fusion protocol that may be used. [0131] (4)(b)Post-Fusion Care [0132] According to an example procedure, seven days after plating out the cells in HAT medium, half the medium is replaced with fresh medium containing HT, instead of HAT. At this time, small colonies of hybridoma cells may be visible. As the colonies grow, medium is withdrawn and antibody activity is tested for in the screening assay. Medium in the wells is replaced with fresh HT medium, as the medium becomes acidic (yellow). [0133] (4) (c) Example Procedure [0134] 1 week> inspect wells, identify hybridomas and mark these wells>replace medium if 2 weeks have elapsed since last replacement>is the medium sufficiently conditioned for screening >NO- go back to beginning. YES - screen the conditioned medium on 2 separate occasions > aspirate conditioned medium and replace with fresh medium > do any of the hybridomas test positive to antigen? NO> have all hybridomas been screened twice? > NO - go back to 1 week >YES - dispose of negative hybridomas>. If hybridomas test positive for antigen, clone and expand positive hybridomas. [0135] (5) Screening Assay/Cloning [0136] One of the keys to successful development of a monoclonal antibody is the screening assay. The more specific and simple the screening test, the better the chance of obtaining a monoclonal antibody of interest. The nature of the antigen will often dictate the screening assay. For example, antibodies to surface antigens of cells in suspension can be examined quickly and easily by imrmmofiuorescence, whereas immunoenzyme techniques are suitable for tissue sections and enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) for soluble antigens. Antibodies that react against fixed tissue will not necessarily react with fresh tissue. It is important to have purified antigen for the assay, since antibodies against impurities in the immunizing material will react if the test material contains the same impurities. The assay is preferably specific, sensitive and capable of screening large numbers of samples quickly. Appropriate positive and negative controls are in every assay. [0137] Once the screening assay indicates that a well contains an antibody of interest, the contents of the well should be cloned as soon as possible. It is important to clone positive wells so as to prevent them being overgrown by negative clones, and to avoid working with mixed clones. While there are several cloning methods, the most common is that of limiting dilution. [0138] (6) Cryopreservation [0139] Preserving cells in liquid nitrogen ensures long term availability of hybridomas. Cells may be frozen down as soon as possible and detailed record kept of what the cells are and when they were stored. The remaining steps are well known in the literature and include: (a) specificity and isotyping; (b) mycoplasma contamination detection; (c) large scale antibody production via Ascitic fluid, cell factory, or perfusion cell culture; (d) antibody purification including precipitation, chromatography and/or use of Protein G; and (e) storage and quality control. [0140] (7) Example Schedule for Making Monoclonal Antibodies [0141] 1. Prepare antigen and develop screening assay. [0142] 2. Immunize animals, a minimum of two for each antigen. [0143] 3. One week before fusion, thaw myeloma cells and scale-up. About 10⁸ cells for every 10⁸ mouse cells (one spleen) to be fused. [0144] 4. Reimmunize the animals 3-4 days before fusion. [0145] 5. Split myeloma cells 1 : 1 with fresh medium on the day before fusion. [0146] 6. Fuse cells, plate out in HT medium (hypoxanthine, thiamine medium). [0147] 7. After 24 hours carefully replace the HT medium with HAT medium (hypoxanthine, aminopterin and thymidine medium). [0148] 8. Seven days after fusion feed cells with HT medium. [0149] 9. About 7 days later, refeed with HT medium. [0150] 10. Test supematants from wells with colonies, as the supernatant turns yellow and the cells are about 50-90% confluent. [0151] 11. Clone positive wells and refeed with HT medium. [0152] 12. Test clones. [0153] 13. Scale-up and cryopreserve positive clones. [0154] (8) Recombinant Antibody Fragments [0155] Several strategies have been used to combine the target regions of antibodies into small functional proteins. Fv fragments are non-covalently associated heterodimers of VH and VL. These synthetic antibody derivatives can be stabilized by a hydrophilic flexible peptide linker joining the two domains and resulting in single-chain Fv molecules (scFv's). The peptide linker pennits an orientation of the CDR toward targets similar to that of natural antibodies. The most commonly used linker is a flexible decapentapeptide containing a combination of glycine and serine residues (Gly4Ser)3. The smallest of the antibody fragments is the minimal recognition unit (MRU) that can be derived from the peptide sequence of a single CDR. Antibody fragments containing the CDR regions, such as F(ab )2, Fab, and scFv's (miniantibodies), may have particular advantages over intact antibodies. Extended serum half-life and antibody stability are advantageous for some therapeutic applications and whole antibodies are better than small antibody fragments in this respect. F(ab )2 is the largest proteolytic fragment that retains the bivalent binding sites of an antibody and remains in the blood much longer than smaller antibody fragments, such as Fab or scFv . Some attempts have been made for engineering the stability of these smaller antibody fragments and for increasing the circulating life in vivo of antibodies through chemical coupling to PEG (pegylation). However, scFv's or Fab's may also be preferred in situations where the effector functions of the Fc portion are dispensable and only the antigen-binding site is required. Antibody fragments are particularly important in processes such as tumor imaging, where better tissue penetration to the specific target and rapid clearance from the body is required. Fab and scFv fragments are usually screened and selected by phage display teclinology. Recently, a helper phage has been described to increase the number of scFv fragments displayed on phage particles by more than 2 orders of magnitude. Once a Fab or scFv fragment with high affinity and specificity for a target antigen has been obtained, it may prove useful to genetically reconstruct these fragments into an intact fully human antibody. [0156] (9) Fusion and Bispecific Antibodies [0157] Several approaches have been developed in order to increase the efficiency of antibodies or antibody fragments, particularly in regard to their effector functions. Conjugation of antibodies to effector compounds, such as bacterial or plant toxins or cytotoxic drags, by covalent coupling allows increased antibody efficiency but can inactivate antibody-binding sites or alter the effector agents as a result of the chemical manipulation during coupling. The advent of genetic engineering permitted the design of several antibody constructs, where the antibody or fragment may be endowed with new properties through fusion with a non-immunoglobulin molecule (ligand). The ligand should not block the regions of the antibody that are designed to perform their biological action. Therefore, Fab-fusion proteins have the antigen- binding site free and can be linked to ligands that improve effector functions such as toxins (commonly used to target cancer cells), cytokines, or enzymes (functioning as a drug or pro-drug converting system). Fc fusion proteins or immunoadhesins maintain the immune effector functions of the Fc isotype and, in addition, bind to the complementary receptor of the ligand inserted. Ligand and Fc portion function independently as a result of the flexibility conferred by the hinge region. Fusion proteins can also be used to add other properties to antibodies such as the introduction of a lipid anchor into an scFv fragment to produce a membrane bound fusion antibody. [0158] An alternative method to improve effector functions is the production of bispecific antibodies, Ab molecules that have two different specific antigen- binding sites: a target-binding arm and an effector-binding arm. These molecules are generated by somatic cell hybridization (hybrid hybridoma bsAb's, recently improved), chemical conjugation (heteroconjugates), or genetic engineering. Antibody monomers, scFv and Fab, can also associate to produce multispecific and multivalent molecules. For example, conjugation of Fab fragments can give rise to bispecific F(ab )2 molecules (chemically linked by the disulfide exchange reaction or non-covalently associated via interaction of leucine zippers) or trimeric Fab molecules (prepared by chemical conjugation with Celltech's reagent TFM). Biomolecular engineering allows the production of bispecific chimeric Ab's or bispecific scFv, offering new hypotheses in immunoassays, immunodiagnostics, and therapy. Recently, fusion proteins of scFv and Fab small fragments with peptides binding Ab effector functions (pepbodies) have been expressed in bacteria. These fusion proteins were able to initiate Ab effector functions and are an example of antibody-like proteins with effector functions. [0159] The foregoing detailed description of the preferred embodiments of the invention has been provided for the purposes of illustration and description, and is not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention for various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention cover various modifications and equivalents included within the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS: 1. A method of delivering a biologically compatible agent to an ocular structure, comprising: administering to an eye of a subject a biologically compatible agent coupled to an antibody; and binding the antibody to a structure of the eye.

2. A method according to claim 1, wherein said administering comprises administering to the eye of a human subject.

3. A method according to claim 1, further comprising selecting a monoclonal antibody as the antibody administered to the eye of the subject.

4. A method according to claim 1 , wherein said binding comprises binding the antibody to the cornea of the eye.

5. A method of delivering a biologically compatible agent to an ocular structure to alter refractive index, comprising: selecting a biologically compatible agent that changes refractive index of an ocular structure; administering to an eye of a subject the biologically compatible agent coupled to an antibody; and binding the antibody to the ocular structure.

6. A method according to claim 5, wherein the ocular stracture comprises a cornea.

7. A method according to claim 5, wherein the ocular structure comprises a crystalline lens.

8. A method according to claim 5, wherein the ocular stracture comprises a vitreous cavity.

9. A method according to claim 5, wherein the antibody comprises an antibody fragment.

10. A method of delivering a biologically compatible agent to an ocular stracture to treat a collagen-based disease, comprising selecting a biologically compatible agent for treating a collagen-based disease of an ocular structure; administering to an eye of a subject the biologically compatible agent coupled to an antibody; and binding the antibody to the ocular structure.

11. A method according to claim 10, wherein the ocular structure comprises a cornea.

12. A method according to claim 10, wherein the ocular stracture comprises a crystalline lens.

13. A method according to claim 10, wherein the ocular structure comprises a vitreous cavity.

14. A method according to claim 10, wherein the antibody comprises an antibody fragment.

15. A method of delivering a biologically compatible agent to an ocular stracture to treat presbyopia and/or cataract, comprising selecting a biologically compatible agent disraptive to crosslinking processes characteristic of at least one of presbyopia and cataract fonnation; administering to an eye of a subject the biologically compatible agent and an antibody; and binding the antibody to a crystalline lens of the subject.

16. A method according to claim 15, wherein the crosslinking process comprises a collagen crosslinking process, and wherein the biologically compatible agent comprises a member selected from a collagen cross-linking inhibitor and a collagen cross-linking cleaver.

17. A method according to claim 15, wherein the ocular stracture comprises a cornea.

18. A method according to claim 15, wherein the ocular structure comprises a crystalline lens.

19. A method according to claim 15, wherein the ocular stracture comprises a vitreous cavity.

20. A method according to claim 15, wherein the antibody comprises an antibody fragment.