WO1995017673A1 - Diagnostics et therapies oculaires - Google Patents

Diagnostics et therapies oculaires Download PDF

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Publication number
WO1995017673A1
WO1995017673A1 PCT/US1994/014691 US9414691W WO9517673A1 WO 1995017673 A1 WO1995017673 A1 WO 1995017673A1 US 9414691 W US9414691 W US 9414691W WO 9517673 A1 WO9517673 A1 WO 9517673A1
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Prior art keywords
vitronectin
drusen
accumulation
ocular
probe
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PCT/US1994/014691
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English (en)
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Gregory S. Hageman
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Ocutech, Inc.
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Priority to AU14413/95A priority Critical patent/AU1441395A/en
Publication of WO1995017673A1 publication Critical patent/WO1995017673A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • vitronectin is a component of ocular deposits known as drusen. Methods of diagnosis and therapy are provided.
  • AMD AMD is the leading cause of new blindness in adults over age 60 in the United States. Based upon current demographic projections, the segment of the US population over age 50 will increase by approximately 75% in the next decade to over 70 million. The segment over age 85 is now growing at a rate six times faster than the rest of the population. Thus, the number of individuals afflicted with AMD is predicted to approach 4 million by the year 2000 and 6.3 million by the year 2030. Cataract can be effectively treated by replacing the diseased lens with a plastic intraocular lens or IOL. Glaucoma is associated with an increase in the pressure inside of the eye, the so-called intraocular pressure, or IOP, that eventually results in the degeneration of a portion of the retina. Once detected, glaucoma usually can be arrested by treatment with pressure-lowering drugs. Delayed detection and treatment, however, lead to impaired vision and eventual blindness.
  • IOP intraocular pressure
  • AMD is characterized by a progressive loss of vision in the critical central portion of the visual field. This visual loss is caused by degeneration of the macula, a small circular region of the neural retina that is responsible for one's ability to appreciate detail and read fine print.
  • AMD is clinically divided into two subtypes.
  • the more severe form of AMD is typically diagnosed in the late stages of the disease and is associated with the ingrowth of blood vessels into the retina.
  • This "neovascular" form of AMD is responsible for approximately 90% of the severe visual loss (20/200 or worse) occurring in patients with AMD.
  • An earlier atrophic, stage of the disease is manifested by abnormal deposition of cellular and extracellular materials in association with Bruch's membrane and the nearby vascular bed (the choriocapillari ⁇ ) , especially in the macular region.
  • drusen based on the German word for nodule. Drusen are concentrated between the retinal pigmented epithelium (RPE) basal lamina and the choriocapillaris. Ethnicity and age influence the incidence of drusen and AMD. Other deposits, termed “basal linear deposits” and
  • basal laminar deposits are located between the RPE basal lamina and the basal surface of the RPE. All of these deposits are of undetermined origin. Additional fibrillar deposits, also of unknown origin and composition, are observed within the connective tissue of the choroid in human eyes with drusen. Furthermore, various additional accumulations of extracellular material are associated with choroidal blood vessels, especially the choriocapillaris, with the elastic lamina of Bruch's membrane, and in the trabecular meshwork in many eyes with drusen.
  • Drusen can assume various forms and are typically classified as “soft” or “hard” depending upon ophthalmoscopic appearance. Drusen vary in size, shape, and/or number and they tend to increase with advancing age. Because people with large and numerous deposits typically report characteristic problems with reading in dim light and in color perception, drusen and other deposits can be considered a predictor for the subsequent development of AMD. The presence of large or extensive drusen is clearly correlated with an increased risk of developing the most severe and blinding form of AMD, that involving neovascularization of the retina.
  • Photocoagulation treatments various surgical approaches, and interferon injections have been proposed as treatments for AMD.
  • more promising approaches would involve the prevention of the initial development of this disease, and/or halting or reversing its progression at an early stage.
  • the invention identifies vitronectin as a principal molecular constituent of drusen and other abnormal deposits associated with AMD.
  • the invention describes chemical classes and specific agents for: a) dissolving or removing drusen and other deposits in the eyes of human patients with AMD; and b) blocking the deposition of vitronectin and other molecular species that comprise drusen and these other deposits.
  • the invention also describes diagnostics for early AMD based on the presence of abnormal concentrations or depositions of vitronectin, abnormalities in the proportions of specific vitronectin isoforms, the presence of aberrant forms of vitronectin, and the presence of autoantibodies to vitronectin in the bloodstream.
  • Methods of ameliorating an extracellular accumulation which includes vitronectin comprise administering an effective amount of a vitronectin- ameliorative compound.
  • the administration can be in vitro, in vivo, or ex vivo .
  • the recipient of the compound is preferably human tissue or a human being, although it could be lower animal tissue or an animal.
  • In vivo methods of diagnosis of an accumulation of vitronectin are also provided. Such methods comprise administering an effective amount of a vitronectin probe to a mammal suspected of having the accumulation, and observing for an interaction between the probe and the accumulation of vitronectin, thereby diagnosing the accumulation. Also provided are in vitro methods of diagnosis of an ocular accumulation of vitronectin in a mammal. These methods comprise obtaining a sample from the mammal, selecting a vitronectin probe, and performing an assay for vitronectin by applying the probe to the sample, thereby diagnosing the ocular accumulation.
  • Fig 1A is a schematic cross-sectional view of a human eye.
  • Fig. IB is a closeup of the boxed area of Fig. 1A showing three major layers or sheaths of the eye.
  • Fig. 2 is an expanded detail of Fig. IB showing the choroid and retinal layers in more detail.
  • the invention resides in the discovery that a major molecular component of drusen and other abnormal ocular deposits is vitronectin, a serum glycoprotein synthesized in the liver.
  • vitronectin a major molecular component of drusen and other abnormal ocular deposits
  • Preissner, K. T. Annu . Rev. Cell . Biol . 7:275-310 (1991).
  • Preissner, K. T. a serum glycoprotein synthesized in the liver.
  • vitronectin The primary amino acid sequence vitronectin has been deduced from cDNA. See Suzuti, et al., EMBO J. 4:2519-24 (1985) . In human serum, vitronectin is present at a concentration of about 0.25 to 0.45 mg/ml. Methods of purification and production of vitronectin are known. See, for example, U.S. Pat. No. 5,077,393 to Hayashi.
  • vitronectin Conventional ex vivo and in vitro uses for vitronectin generally involve its ability to promote cell adherence to surfaces and, perhaps, to each other. For example, the addition of vitronectin to tissue cultures controls the spreading of the cultured cells.
  • the eye includes three major layers or sheaths.
  • One is the sclera 4 which is a fairly tough, fibrous outer layer in continuity with the cornea 6 anteriorly and the sheath or dura 24 of the optic nerve 26 posteriorly.
  • the lens 8 separates the anterior chamber 28 and the posterior chamber from the vitreous chamber 30.
  • the anterior and posterior chambers contain aqueous humor or fluid and the vitreous chamber contains vitreous humor or fluid.
  • the middle layer is the choroid 10 which is a thin pigmented vascular layer between the sclera 4 superficially and the retina 12 which is deep to the choroid.
  • the retina is the innermost major layer of the eye.
  • the retina 12 includes an outer pigmented layer, the RPE 14, and the more inner neuronal layer or neural retina 22.
  • the neuronal layer includes neurons, ganglia and photoreceptor cells. Rod and cone cells, so named for their shapes, are photoreceptor cells responsible primarily for black/white and color vision, respectively.
  • Bruch's membrane is a term referring collectively to the basal laminae of the choriocapillaris 16 and the RPE 14, and all of the region between the two.
  • the capillary bed directly adjacent to the RPE, the choriocapillaris has a high rate of blood flow and oxygen tension, and is the major source of serum proteins, including vitronectin, in this region.
  • Drusen 18 tend to form or deposit primarily within
  • the depth of drusen deposition varies from roughly the choroidal basement membrane 16 through the RPE layer 14 and is designated schematically by the arrow numbered 20.
  • the passage of serum proteins, including vitronectin, from the circulation or other cellular or extracellular sources into the extracellular space adjacent to the RPE followed by localized binding to components of Bruch's membrane could, over an extended period of time, result in or contribute to the formation of aggregates of proteins and associated molecules that are recognized clinically as drusen and other abnormal deposits. Formation of these deposits could be linked to levels and/or specific isoforms and/or aberrant forms of circulating vitronectin and/or other components of these deposits, including serum proteins.
  • vitronectin normal or abnormal
  • vitronectin could be synthesized differentially by photoreceptor, RPE and/or choroidal cells and subsequently become deposited into the extracellular matrix.
  • treatment refers to improvement in the recipient's status.
  • the improvement can be subjective or objective and related to features such as symptoms or signs of the disease or condition being treated. For example, if the patient notes improved visual acuity or improved color perception, then successful treatment has occurred. Similarly, if the clinician notes objective improvement on a visual acuity test, such as using an eye chart, then treatment has also been successful. Alternatively, the clinician may note a decrease in drusen or other abnormal deposits upon examination of the patient's eye. This would also represent an improvement or a successful treatment in that the risk of developing the exudative form of AMD would be reduced. Prevention of deterioration of the recipient's status is also included by the term.
  • ameliorate in referring to vitronectin, drusen, a deposit or an accumulation can mean a decrease of vitronectin and/or other molecular components associated with drusen or other abnormal deposits or accumulations. It can also refer to a decrease in the total number of, or area covered by, deposits and/or amounts of various components of deposits. Additionally, “ameliorate” includes a decrease in the impact of deposits such as drusen, or an accumulation containing vitronectin, as well as prevention of worsening or enlargement of the deposit or accumulation. The term includes any of the arrest, prevention, elimination, and disruption of the accumulation.
  • RNA-ameliorative compounds are those compounds which can ameliorate vitronectin, and examples are listed in Tables I and II.
  • “Recombinant” means that the subject product is the result of the manipulation of genes by human intervention into new or non-native combinations.
  • Complementary DNA refers to DNA that is derived from a messenger RNA sequence (mRNA) , for example, using reverse transcriptase. Reverse transcriptase is an enzyme that polymerizes DNA using an RNA template.
  • mRNA messenger RNA sequence
  • a “vector” is a sequence of DNA, typically in plasmid or viral form, which is capable of replicating in a host. A vector can be used to transport or manipulate DNA sequences.
  • An "expression vector” includes vectors which are capable of expressing DNA sequences contained therein, typically producing a protein product.
  • the coding sequences are linked to other sequences capable of effecting their expression, such as promoters and enhancers.
  • Expression vectors are capable of replicating in a host in episomal form; others can integrate into a host cell's chromosome.
  • the expression vectors have a selectable marker, for example, neomycin resistance, which permits the selection of cells containing the marker.
  • a “promoter” is a DNA sequence 5• of the protein coding sequences which affects transcriptional activity. RNA polymerase first binds to the promoter to initiate transcription of a gene.
  • An “enhancer” is a DNA sequence that can positively affect transcriptional efficiency.
  • oligonucleotide is a polymer molecule of two or more nucleotides including either deoxyribonucleotides or ribonucleotides.
  • “Host cells” refer to cells which are capable or have been transformed with a vector, typically an expression vector.
  • a host cell can be prokaryotic or eukaryotic, including bacteria, insect, yeast and mammalian cells.
  • Cellular material refers to animal tissue, cells or portions thereof which can include, for example, whole cells, parts of cells and extracellular material and lysates of cells, or parts of cells and extracellular material.
  • tissue embraces extracellular material and acellular material of animal origin.
  • the invention includes immunoglobulins, especially im unoglobulins directed against vitronectin.
  • Immunoglobulins or antibodies are proteins that bind to an antigen.
  • the term "immunoglobulin” or “antibody” refers to an entire immunoglobulin or antibody or any functional fragment of an immunoglobulin molecule. Examples include complete antibody molecules, antibody fragments, such as Fab, F(ab') 2 , CDRs, V L , V H , and any other portion of an antibody.
  • Immunoglobulins are typically composed of four covalently bound peptide chains. For example, an IgG antibody has two light chains and two heavy chains. Each light chain is covalently bound to a heavy chain.
  • each heavy chain is covalently linked to the other to form a "Y" configuration, also known as an immunoglobulin conformation.
  • Fragments of these molecules, or even heavy or light chains alone, may bind antigen.
  • vitronectin or fragments thereof can be an antigen.
  • Antibodies, fragments of antibodies, and individual chains are all referred to herein as immunoglobulins.
  • a normal antibody heavy or light chain has an N- terminal (NH 2 ) variable (V) region, and a C-terminal (-COOH) constant (C) region.
  • the heavy chain variable region is referred to as V H (including, for example, V ⁇ )
  • the light chain variable region is referred to as V L (including V ⁇ or V ⁇ ) .
  • the variable region is the part of the molecule that binds to the antibody's cognate antigen, while the Fc region (the second and third domains of the C region) determines the antibody's effector function (e.g., complement fixation, opsonization) .
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, also called complementarity-determining regions or CDRs.
  • the extent of the framework region and CDRs have been defined (see, "Sequences of Proteins of
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.
  • the two types of light chains, K and ⁇ are referred to as isotypes.
  • Isotypic determinants typically reside in the constant region of the light chain, also referred to as the C L in general, and C ⁇ or C ⁇ in particular.
  • the constant region of the heavy chain molecule also known as C H , determines the isotype of the antibody.
  • Antibodies are referred to as IgM, IgD, IgG, IgA, and IgE depending on the heavy chain isotype.
  • the isotypes are encoded in the mu ( ⁇ ) , delta ( ⁇ ) , gamma (7) , alpha ( ⁇ ) , and epsilon (e) segments of the heavy chain constant region, respectively.
  • the heavy chain isotypes determine different effector functions of the antibody.
  • the heavy chain isotype determines the secreted form of the antibody.
  • Secreted IgG, IgD, and IgE isotypes are typically found in single unit or monomeric form.
  • Secreted IgM isotype is found in pentameric form; secreted IgA can be found in both monomeric and dimeric form.
  • Immunoglobulins are frequently classified according to their valency.
  • IgG is a bivalent antibody and IgM is a polyvalent antibody.
  • the valency refers to the number of binding sites on the immunoglobulin.
  • Monovalent means that one antibody molecule binds to one receptor
  • bivalent means that the antibody binds to exactly two receptors
  • polyvalent or multivalent means that it binds to two or more receptors.
  • Polyclonal antibodies generally comprise a mixture of bivalent antibodies.
  • Methods of the invention relating to vitronectin binding molecules include the use of monovalent immunoglobulins. In preferred embodiments, a single monovalent monoclonal antibody or a mixture of monovalent antibodies, such as two or more monoclonal antibodies, is used.
  • a particular embodiment is a monospecific (as monoclonal antibodies are by definition), monovalent (i.e Fab fragment or single chain antibody) monoclonal probe.
  • Murine, human, chimeric or other immunoglobulins, especially those of mammalian origin, may be used with the invention. While specificity of an immunoglobulin is desirable, the degree of specificity required varies with the application of the immunoglobin. For example, absolute specificity is not required in ocular diagnostics.
  • ocular structures labeled by a fluorochrome-conjugated vitronectin probe and visualized by fundus photography can be conclusively identified as drusen based on their size, shape, distribution and intensity of labeling.
  • the immunohistochemical data herein indicates that there are relatively high concentrations of vitronectin associated with drusen and that "background" labeling of other ocular structures containing lower vitronectin concentrations is lesser in intensity. This also applies in vivo. See Scheiffarth et al., J-nvest. Ophthalmol . Vis . Sci . 31:272-276 (1990), for example.
  • multiple vitronectin probes with differing patterns of background binding, but identical drusen-binding patterns can be employed to confirm identification of labeled structures as drusen.
  • the immunoglobulins can be prepared in a variety of ways known in the art, depending upon whether monoclonal or polyclonal antibodies are desired.
  • a vertebrate typically a domestic animal
  • blood from the vertebrate is collected shortly after immunization and the gamma globulin is isolated.
  • Suitable methods for preparing polyclonal antibodies are described in the Handbook of Experimental Immunology, 3d ed. , Weir (ed.), Blackwell Scientific Publications (1978).
  • a small animal typically a rat or mouse
  • the spleen is removed and the lymphocytes are fused with myeloma cells in the presence of a suitable fusion promoter.
  • the resulting hybrid cells or hybridomas are screened to isolate individual clones, each of which secrete a single antibody species to the antigen.
  • the individual antibody species are each the product of a single B cell generated in response to a specific antigenic site recognized on the antigen or immunogenic substance.
  • the process for obtaining monoclonal antibodies is described by Kohler and Milstein, Nature, 256:495 (1975) . See also Harlow and Lane, Antibodies : A Laboratory Manual, Cold Spring Harbor Publications, N.Y.
  • the peptides or antigens used to generate the antibodies may be used directly in the immunization procedure as immunogenic components associated with living or fixed cells or they may be bound to a suitable carrier protein, such as keyhole limpet hemocyanin (KLH) , human or bovine serum albumin (HSA or BSA) , and the like.
  • KLH keyhole limpet hemocyanin
  • HSA human or bovine serum albumin
  • Use of the antigen with a carrier protein is preferred for immunization.
  • the invention relates not only to amino acid sequences, but also to DNA sequences.
  • the DNA sequences associated with this invention include, for example, DNA subsequences encoding amino acid sequences of the antibody heavy or light chains, or fragments thereof, which determine binding specificity for a vitronectin receptor protein. These sequences may be ligated, for example, into human constant region expression vectors, and inserted into a host cell. The host cell can then express a recombinant chimeric or hybrid antibody that is specific for binding to a vitronectin receptor protein or polypeptide.
  • An F(ab') 2 fragment lacks the C-terminal portion of the heavy chain constant region, and has a molecular weight of approximately 110 kiloDalton (kDa) . It retains the two antigen binding sites and the interchain disulfide bonds in the hinge region, but it does not have the effector functions of an intact IgG molecule.
  • An F(ab') 2 fragment may be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, supra.
  • An "Fab" fragment comprises a light chain and the N- terminus portion of the heavy chain to which it is linked by disulfide bonds.
  • Fab fragments may be obtained from F(ab') 2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents (see Harlow and Lane, supra) .
  • Chimeric antibodies may also be used in this invention.
  • Chimeric antibodies or “chimeric peptides” refer to those antibodies or antibody peptides wherein one portion of the peptide has an amino acid sequence that is derived from, or is homologous to, a corresponding sequence in an antibody or peptide derived from a first gene source, while the remaining segment of the chain(s) is homologous to corresponding sequences of another gene source.
  • a chimeric antibody peptide may comprise an antibody heavy chain with a murine variable region and a human constant region. The two gene sources will typically involve two species, but will occasionally involve one species.
  • a successful human/murine chimeric antibody is one for carcinoembryonic (CEA) antigen described by Beidler, et al., J. of Immunology, 141:4053 (1988).
  • CEA carcinoembryonic
  • Other methods for constructing chimeric antibodies and binding fragments are described in Brown et al., Cancer Research 47:3577-3583 (1987); Kameyama et al., FEB 2:301-306 (1989);
  • a chimeric antibody is any antibody in which either or both of the heavy or light chains are composed of combinations of sequences mimicking the sequences in antibodies of different sources, whether these sources are differing classes, differing antigen responses, or differing species of origin, and whether or not the fusion point is at the variable/constant boundary.
  • chimeric antibodies can include antibodies where the framework and complementarity-determining regions are from different sources.
  • non-human CDRs are integrated into human framework regions linked to a human constant region to make "humanized antibodies.” See, for example, PCT
  • a "human-like framework region” is a framework region for each antibody chain, and it usually comprises at least about 70 or more amino acid residues, typically 75 to 85 or more residues.
  • the amino acid residues of the human-like framework region are at least about 80%, preferably about 80 to 85%, and most preferably more than 85% homologous with those in a human immunoglobulin.
  • humanized or “human-like immunoglobulin” refers to an immunoglobulin comprising a human-like framework region and a constant region that is substantially homologous to a human immunoglobulin constant region, e .g. , having at least about 80% or more, preferably about 85 to 90% or more and most preferably about 95% or more homology. Hence, most parts of a human-like immunoglobulin, except possibly the CDRs, are substantially homologous to corresponding parts of one or more native human immunoglobulin sequences.
  • Hybrid antibody refers to an antibody wherein each chain is separately homologous with reference to a mammalian antibody chain, but the combination represents a novel assembly so that two different antigens are recognized by the antibody.
  • one heavy and light chain pair is homologous to that found in an antibody raised against one epitope, while the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope. This results in the property of multi- functional valency or multivalency, i.e., ability to bind at least two different epitopes simultaneously.
  • Such hybrids may, of course, also be formed using chimeric chains.
  • the present invention encompasses, inter alia, a chimeric antibody, including a hybrid antibody or a humanized or human-like antibody. It also encompasses a recombinant DNA sequence encoding segments of the antibody or any peptide specific for vitronectin or a fragment of vitronectin. Variants of these sequences are also included, such as substitution, addition, and/or deletion mutations, or any other sequence possessing substantially similar binding activity to the sequences from which they are derived or otherwise similar to.
  • an immunoglobulin, antibody or other peptide is specific for vitronectin or a fragment thereof if the immunoglobulin antibody or peptide binds or is capable of binding vitronectin or the fragment as measured or determined by standard antibody-antigen or ligand-receptor assays.
  • assays include competitive assays, saturation assays, and standard immunoassays such as ELISA or RIA.
  • assays that measure binding of a test compound in the absence of an inhibitor may also be used.
  • the ability of a molecule or other compound to bind vitronectin can be detected by labeling the molecule of interest directly or it may be unlabeled and detected indirectly using various sandwich assay formats.
  • Assays for measuring binding of a test compound to one component alone rather than using a competition assay are also available.
  • immunoglobulins can be used to identify the presence of vitronectin. Standard procedures for monoclonal antibody assays, such as ELISA, may be used (see , Harlow and Lane, supra) .
  • assay formats may involve the detection of the presence or absence of various physiological or chemical changes that result from the interaction, such as down modulation, internalization or an increase in phosphorylation as described in Receptor-Effector Coupling - A Practical Approach, ed. Hulme, IRL Press, Oxford (1990) .
  • Such techniques relate to, for example, the antibodies' ability to stain tissue or deposits via histoche ical means, to react with intact tissue on a Fluorescence-activated cell sorter (FACS) , or to react with purified vitronectin in an immunolabeling assay, an immunoprecipitation assay or a Western blot assay.
  • FACS Fluorescence-activated cell sorter
  • variable regions and CDRs may be derived from a hybridoma that produces a monoclonal antibody that is specific for vitronectin.
  • Nucleic acid sequences relating to the present invention which are capable of ultimately expressing the desired chimeric antibodies can be formed from a variety of different nucleotide sequences (genomic or cDNA, RNA, synthetic oligonucleotides, etc.) and components (e.g., V, J, D, and C regions) , as well as by a variety of different techniques. Joining appropriate genomic sequences is presently a common method of production, but cDNA sequences may also be utilized (see, European Patent Publication No. 0239400 and Reichmann, L. , et al., Nature, 332:323-327 (1988).
  • a similar approach can be taken to isolate and subclone the sequences encoding the constant regions of the heavy and light chains that originate from another mammalian species.
  • the enhancers to the heavy and light chain can be included in the isolated heavy chain fragments, or can alternatively be isolated and subcloned.
  • Human constant region DNA sequences are preferably isolated from immortalized B-cells, see e .g. , Heiter, et al., Cell 22:197-207 (1980), but can be isolated or synthesized from a variety of other sources.
  • the nucleotide sequence of a human immunoglobulin C ⁇ l gene is described in Ellison, et al., Nucl . Acid. Res . 10:4071 (1982); Beidler, et al. , J". Jim ⁇ u-nol. 141:4053 (1988); Liu, et al., PNAS USA 84:3439 (1987).
  • the CDRs for producing the immunoglobulins of the present invention preferably are derived from monoclonal antibodies capable of binding to the desired antigen, vitronectin receptor protein, and produced in any convenient mammalian source, including, mice, rats, rabbits, hamsters, or other vertebrate host cells capable of producing antibodies by well known methods.
  • Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("ATCC”) (Catalogue of Cell Lines and Hybridomas , Fifth edition (1985) Rockville, Maryland, U.S.A.
  • substantially homologous modified immunoglobulins can be readily designed and manufactured utilizing various recombinant DNA and synthetic techniques known to those skilled in the art. Modifications of the genes may be readily accomplished by a variety of well known techniques, such as site-directed mutagenesis. See, for instance, Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S., et al.. Nature 328:731-734 (1987). Alternatively, polypeptide fragments comprising only a portion of the primary antibody structure may be produced, which fragments possess binding and/or effector activities.
  • the cloned variable and constant regions can be isolated from plasmids and ligated together into a mammalian expression vector, such as pSV2-neo, and pRSV-gpt, to form a functional transcription unit. These expression vectors can then be transfected into host cells.
  • Mouse myeloma cells such as SP 2/0 or P3X cells, are a preferred host because they do not secrete endogenous immunoglobulin protein and contain all of the components used in immunoglobulin expression.
  • Myeloma cells can be transfected using appropriate techniques as described above. Other types of promoters and enhancers specific for other host cells are known in the art. See Kameyoma, supra.
  • the DNA sequence encoding the chimeric antibody amino acid sequence can be linked to yeast promoters and enhancers and transfected into yeast by methods well known in the art.
  • the CDRs can then be ligated to the framework regions and constant regions to form a chimeric antibody. See PCT No. GB88/00731 (1989) .
  • the CDRs could be cloned in an expression vector comprising, for example, human framework and constant regions.
  • Another example is a recombinant DNA sequence comprising the heavy and/or light chain CDR1, CDR2, and CDR3 of one species, such as mouse, and the framework regions of human heavy chain to encode an antibody specific for vitronectin.
  • Other possibilities include using CDRs specific for vitronectin; using part of the variable region encompassing CDR1 and CDR2 from one mammalian species, and then ligating this sequence to another encoding the framework portions of a second mammalian species to the CDR3 of the first; or transfecting a host cell line with a recombinant DNA sequence encoding a vitronectin specific heavy chain CDRs derived from a first mammalian species, interspersed within the framework of a second mammalian species with a light chain containing a variable region DNA sequence derived from the first species and the constant region derived from the second species.
  • Antibodies may be expressed in an appropriate folded form, including single chain antibodies, from bacteria such as E. coli . See Pluckthun, Biotechnology 9:545 (1991); Huse, et al.. Science 246:1275 (1989) and Ward, et al.. Nature 341:544 (1989) .
  • the immunoglobulins may either be labeled or unlabeled.
  • Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the first antibody, such as antibodies specific for human immunoglobulin constant regions.
  • second antibodies labeled antibodies
  • the antibodies can be directly labeled.
  • labels may be employed, such as radionuclides, fluors including fluorophores and fluorochromes, chromophores, enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors, ligands (particularly haptens) , etc. Numerous types of immunoassays are available and are well known to those skilled in the art.
  • the immunoglobulins can be coupled to another compound for a variety of reasons.
  • a coupling may enhance transport, absorption, bioavailability and distribution of the immunoglobulin.
  • the compound to which the immunoglobulin is coupled may make the immunoglobulin less susceptible to breakdown by normal enzymatic activity or it may make the immunoglobulin more transportable to certain physiologic compartments.
  • the immunoglobulin could be rendered more transportable across the blood brain barrier or perhaps more lipid soluble and thus more likely to be received into tissues with a high lipid content.
  • Organic compounds may be synthesized with similar biological activity by first determining the relevant contact residues and conformation involved in vitronectin binding by an antibody peptide of this invention.
  • Computer programs to create models of proteins such as antibodies are generally available and well known to those skilled in the art. See Kabat, et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, National Institutes of Health (1987) ; Loew, et al., J-nt. J. Quant . Chem . Quant . Biol . Symp.
  • the invention provides proteins such as anti- vitronectin antibodies. Protein purification is known in the art.
  • the proteins of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity and fraction column chromatography, gel electrophoresis and the like. See, generally, Scopes, R. , Protein Purification , Springer-Verlag, N.Y. (1982) and U.S. Pat. No. 4,512,922 disclosing general methods for purifying protein from recombinantly engineered bacteria. Proteins may be expressed and purified from eukaryotic cells and prokaryotes. Following the growth of the recombinant cells and concomitant secretion of protein into the culture media, this "conditioned media" is harvested.
  • the conditioned media is then clarified by centrifugation or filtration to remove cells and cell debris.
  • the proteins contained in the clarified media are concentrated by adsorption to any suitable resin such as, for example, an anion exchange resin, or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration.
  • suitable resin such as, for example, an anion exchange resin, or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration.
  • suitable resin such as, for example, an anion exchange resin, or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration.
  • Other routine means known in the art may be equally suitable.
  • the purification of proteins secreted by cultured cells may require the additional use of, for example, affinity chromatography, ion exchange chromatography, sizing chromatography or other protein purification techniques to obtain homogeneity.
  • the expressed protein may not be excreted. If the cell line has a cell wall, then initial extraction in a low salt buffer may allow the protein to pellet with the cell wall fraction. Then all the protein may be eluted from the cell wall with high salt and then dialyzed. If the cell line allows the protein to be glycosolated, the purified glycoprotein may be enhanced by using a Con A column. Anion exchange columns (MonoQ, Pharmacia) and gel filtration columns may be used to further purify the protein. A highly pure preparation may be achieved at the expense of activity by denaturing preparative polyacrylamide gel electrophoresis.
  • the peptides used in the invention can be synthesized in solid or liquid phase as is known in the art.
  • the peptide can be synthesized at different substitution levels and the synthesis may follow a stepwise format or a coupling approach.
  • the stepwise method includes condensing amino acids to the terminal amino group sequentially and individually.
  • the coupling, or segment condensation, approach involves coupling fragments divided into several groups to the terminal amino acid.
  • Synthetic methods include azide, chloride, acid anhydride, mixed anhydride, active ester, Woodward reagent K, and carbodiimidazole processes as well as oxidation-reduction and other processes. These processes apply to both solid and liquid phase synthesis.
  • vitronectin-ameliorative compounds can include anti-vitronectin antibodies, vitronectin receptor molecules (integrins) , thrombin, anti-thrombin-3, thrombospondin, thrombomodulin, heparin, heparan sulfate, heparin cofactor 2, plasminogen, tissue plasminogen activator (TPA) , plasminogen activator inhibitors, endorphins, amyloid, serum amyloid P component, coumadin, somatomedin B, C5b-9 complement complex, fibrin, keratin, elastin, perforin, factor X, transglutaminase, protein kinases, sulfotransferases,
  • Compounds are preferably initially tested using immunohistochemical assays because such assays are accepted in the medical field as accurately reflecting in vivo conditions. Such assays are used extensively, for example, in human tumor diagnosis. See Miettinen, M. , Annals of Medicine 25:221-233 (1993) . Similarly, the art accepts the correlation between structures labeled in vivo by fluorochrome-labeled antibodies and structures labeled in histologic sections examined by fluorescence microscopy. See Scheiffarth (1990) .
  • Preferred compounds for use with the invention are those which act by binding to existing deposits and, by stearic interference and/or other molecular mechanisms, block additional deposition. Preferred amongst such compounds are those with monovalent binding characteristics and without other functional characteristics that might cause undesirable side effects. For instance, monovalent anti-vitronectin antibody fragments (e.g. Fab fragments derived from proteolytic cleavage of IgG or antibody fragments obtained by recombinant DNA cloning and expression) and/or relatively inert vitronectin-binding polypeptides derived synthetically or by cleavage of known vitronectin-binding proteins are preferred examples.
  • monovalent anti-vitronectin antibody fragments e.g. Fab fragments derived from proteolytic cleavage of IgG or antibody fragments obtained by recombinant DNA cloning and expression
  • a preferred, route of administration for such compounds is by subretinal or choroidal injection at or near sites of drusen deposition.
  • a preferred dosage is about a tenfold excess of compound to the estimated weight of the deposits. For example, assuming that the protein content of a 3 cm 2 confluent drusen deposit weighs about 1-100 micrograms, a tenfold excess of vitronectin/drusen-binding compound (i.e. 10-1000 micrograms) constitutes a preferred single dose.
  • Alternative compounds are those which act by binding to potential components of deposits while those components exist in soluble form (e.g. in serum or interstitial fluid) and inhibiting their ability to participate in the molecular interactions necessary to form a relatively insoluble deposit.
  • Preferred amongst such compounds are those with monovalent binding characteristics and without other functional characteristics that might cause undesirable side effects.
  • monovalent antibody fragments directed against one or more vitronectin-binding molecules e.g. Fab fragments derived from proteolytic cleavage of IgG or antibody fragments obtained by recombinant DNA cloning and expression
  • relatively inert polypeptides derived synthetically or by proteolytic cleavage
  • Preferred routes of administration of such compounds are by injection intravenously, subretinally or choroidally, typically in conjunction with sustained release compounds.
  • a preferred dosage is determined based on quantification of circulating and/or local levels of the target vitronectin-binding molecule and adjustment of dosages so as to sustain excess levels of deposition-inhibiting therapeutic molecules.
  • Therapeutic compound(s) also include generally compounds that either remove drusen and/or other deposits, remove components of deposits and/or remove molecules that induce the synthesis of components of deposits or the secretion of components of deposits.
  • This molecular interaction can be either covalent or noncovalent. Examples of such compounds include proteolytic enzymes, glycosidic enzymes, lipases, reducing agents, ionic or non-ionic detergents, polyanionic or polyanionic molecules, high or low salt mixtures, and high or low pH solutions.
  • proteolytic enzymes and/or glycosidic enzymes of with highly selective substrate specificity and high specific activity are preferred amongst these. These characteristics allow effective treatment with a minimum of adverse side effects and at relatively low enzyme concentrations.
  • a preferred route of administration for such compounds is by subretinal or choroidal injection at or near sites of drusen deposition. For example, assuming that the protein content of a 3 cm 2 confluent drusen deposit to be 1-100 micrograms, as much as a one-hundredfold excess of enzymatic units (i.e. the number of units required to hydrolyze 1-100 micrograms) constitutes a preferred single dose.
  • a therapeutic compound can be a protease, a glycosidase, a lipase, a reducing agent, or an ion.
  • the protease can be any of a number of enzymes including trypsin, plasmin, and plasminogen activator.
  • the glycosidase can be an endoglycosidase or an exoglycosidase.
  • Preferred endoglycosidases are endoglycosidase-F and chondroitinase.
  • the reducing agent chosen can disrupt disulfide bonds involved in maintaining the tertiary structure of drusen. Such reducing agents include /3-mercaptoethanol and glutathione.
  • ions which may be suitable for therapeutic use include the ions of sodium, potassium, calcium, chloride, magnesium (and other divalent cations) hydrogen, and hydroxyl.
  • the tissue is preferably human tissue.
  • tissue includes blood and its derivatives. Most preferably, the tissue is human ocular tissue. Typically, the tissue is derived from or includes any of the retina, the choroid and the trabecular meshwork, including the canal of Schlemm.
  • the tissue can be in vitro, in vivo or ex vivo.
  • an in vitro diagnostic method can be performed on a retinal punch biopsy from a donor eye.
  • treatment is in vivo or ex vivo.
  • An example of an ex vivo treatment is removal of a tissue or fluid sample, such as blood or vitreous humor, followed by treatment of the tissue or fluid. After treatment, the tissue or fluid, or a fraction thereof, is returned to the patient. Dialysis is another example of an ex vivo method.
  • hybrid molecules containing regions of nucleotide or amino acid sequences with the capacity to bind one or more of the identified molecular components of drusen and/or other deposits.
  • hybrid molecules may be derived by the chemical coupling of all or parts of two or more molecular species or by the expression of genetically engineered recombinant proteins.
  • the vitronectin-binding domain(s) of the heavy and/or light chains of an anti- vitronectin antibody could be coupled to a proteolytic enzyme known to digest vitronectin.
  • the genetic sequences encoding these two molecular species may combined and expressed as a recombinant hybrid protein.
  • AMD is a primary target for diagnosis and treatment using the invention.
  • other ocular conditions such as glaucoma, which include an abnormal synthesis, circulation, deposition or accumulation of vitronectin and/or other extracellular or cell associated deposits, can be treated using the invention.
  • the abnormal extracellular deposits associated with AMD are located in the same ocular compartment as those associated with glaucoma such as primary open angle glaucoma. An interference with fluid flow dynamics due to these deposits can be a common feature of the two conditions.
  • Visual acuity is one feature which can be used to assist the practitioner in deciding to diagnose and treat a patient using the invention. Typically, a visual acuity of 20/100 is considered significantly impaired. Some practitioners prefer the more significant impairment of 20/200 as a guideline. Visual acuity is measured by any of a number of means accepted in the art such as, for example, the Bailey- Lovie chart.
  • IOP is used to assist with diagnosis and evaluation of therapy, as in, for example, glaucoma.
  • Measurement of IOP is well known to those of skill in the art, and involves the use of a tonometer or similar instrument.
  • Characteristic isotypes and/or abnormal molecular forms of serum vitronectin may correlate with drusen or AMD.
  • a diagnostic test can be based on biochemical/chemical characterization (e.g. electrophoretic separation, immunoblotting, protein sequencing) of vitronectin in a serum sample.
  • biochemical/chemical characterization e.g. electrophoretic separation, immunoblotting, protein sequencing
  • the invention's evidence for the presence of autoantibodies indicates a serologically detectable immune response to molecules that co-migrate at the same molecular weight as vitronectin in some AMD patients. This immune response also serves as the basis of a diagnostic technology.
  • Therapeutic benefit includes any of a number of subjective or objective factors indicating a response of the condition being treated as discussed herein.
  • compositions are typically administered to human patients parenterally.
  • the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts.
  • the compositions may also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to form pharmaceutical compositions for animal or human administration.
  • diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are water, the various saline solutions, Ringer's solution, dextrose solution, and Hank's solution.
  • composition or formulation may also include additives such as other carriers; adjuvants; or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • additives such as other carriers; adjuvants; or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Effective amounts of such diluent or additive are those amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubility, biological activity, etc.
  • composition of the invention includes a diagnostic or therapeutic compound which may be formulated with conventional, pharmaceutically acceptable, vehicles for parenteral administration by injection.
  • Formulations may also include small amounts of adjuvants such as buffers and preservatives to maintain isotonicity, physiological and pH stability.
  • compositions of the invention are presented for administration to humans and animals, preferably in unit dosage forms such as vials, ampules, tablets, caplets, pills. powders, granules, eyedrops, oral or ocular solutions or suspensions, ocular ointments, and oil-in-water emulsions.
  • Topical preparations typically include vehicles suitable for use on the skin (including the cornea1/conjunctival epithelium) , including emollients, emulsifiers, wax, fats, alcohols, and/or oils.
  • unit dosage form refers to physically discrete units suitable as unitary doses for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce a desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle.
  • a peptide of the invention may be administered in water, saline or buffered vehicles with or without various adjuvants, excipients, or diluting agents.
  • the proportion of peptide and adjuvant can be varied over a broad range so long as both are present in effective amounts.
  • aluminum hydroxide can be present in an amount of about 0.5% of the composition mixture (A1 2 0 3 basis) .
  • the peptide composition may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried.
  • Lyophilization permits long-term storage in a stabilized form.
  • the peptide compositions are typically formulated to contain a final concentration of peptide in the range of from 0.2 to 200 ⁇ g/ml.
  • Slow or extended-release delivery systems including any of a number of biopolymers (biological-based systems) , systems employing liposomes, and polymeric delivery systems, can be utilized with the compositions described herein to provide a continuous or longterm source of ophthalmic composition to the eye. Examples of these delivery systems are described by Mitra (ed.), Ophthalmic Drug Delivery Systems , Marcel Dekker, Inc., New York, NY (1993). Slow release delivery systems are preferred for choroidal injection, transscleral injection or placing a scleral patch (discussed more fully below) .
  • Preferred methods of administration include choroidal injection, transscleral injection or placing a scleral patch, and selective arterial catheterization.
  • Other preferred deliveries are intraocular, including transretinal, subconjunctival bulbar, scleral pocket and scleral cutdown injections.
  • the agent can be alternatively administered intravascularly, such as intravenously (IV) or intraarterially.
  • choroidal injection and scleral patching are similar.
  • the clinician uses a local approach to the eye after initiation of appropriate anesthesia, including painkillers and ophthalmoplegics.
  • a needle containing the therapeutic compound is directed into the patient's choroid or sclera and inserted under sterile conditions.
  • the needle When the needle is properly positioned the compound is injected into either or both of the choroid or sclera.
  • the clinician may choose a sustained release or longer acting formulation.
  • the procedure may need repetition only every several months or several years, depending on the patient's tolerance of the treatment and response.
  • Therapeutic agents of the invention can also be delivered or administered topically, by transdermal patches, intraperitoneally (IP) , intraarterially, subcutaneously (SQ) , in aerosol form, orally, and in drops among other methods.
  • IP intraperitoneally
  • SQ subcutaneously
  • the agent can be delivered as a bolus, a short term infusion or a continuous, longer term infusion.
  • the dosage of compounds used in accordance with the invention vary depending on the class of compound and the condition being treated.
  • the age, weight, and clinical condition of the recipient patient; and the experience and judgment of the clinician or practitioner administering the therapy are among the factors affecting the selected dosage.
  • the dosage of an immunoglobulin such as an antibody will range from about 1.0 microgram per kilogram per day to about 1 milligram per kg per day for polyclonal antibodies and about 5% to about 10% of that amount for monoclonal antibodies.
  • the immunoglobulin can be administered once daily as an intravenous infusion.
  • the dosage is repeated daily until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
  • the dosage of the therapeutic agent administered depends on the class of the agent, the patient, the patient's medical history, and the severity of the disease process (See Table II) .
  • the dose should be sufficient to ameliorate drusen or vitronectin deposition and attendant sequelae without producing significant toxicity to the cells of the retina, RPE, choroid, sclera, trabecular meshwork, other ocular and non-ocular tissues.
  • the dosage of the specific compound for treatment depends upon many factors that are well known to those skilled in the art.
  • An effective amount of the compound is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • the dosing range varies with the compound used, the route of administration and the potency of the particular compound. See Table II for preferred dosages and routes of administration.
  • the compound may be administered by any conventional method for the administration of parenteral agents.
  • an effective amount is that amount of compound which is sufficient for and appropriate to the diagnostic test employed.
  • ELISAs require very small amounts of sample and compound. Such amounts are known to those of skill in the art.
  • Vitronectin Probes The invention provides vitronectin probes or compounds which specifically bind to vitronectin. Usually the probe is a peptide such as an antibody.
  • fluorescein-labeled antibodies have been injected into the ear vein of a rabbit and visualized in the eye up to 24 hours later. The specific binding of the antibody probes to the targeted chorioretinal lesions was confirmed in subsequent histologic examination of the ocular tissue using fluorescence light microscopy. See Scheiffarth at page 275. Similar use of antibody probes in humans has been documented for tumor immunodetection and immunotherapy. See, for example, Miettinen (1993) .
  • Intravenously injected antibodies or fluorescein are capable of reaching the retina/choroid region of the eye. Because the endothelial cells of the choroidal vasculature are highly fenestrated, they allow the passage of relatively large proteins from the capillary lumen to the adjacent extravascular space. In the eye, this space is directly contiguous with the interface between Bruch's membrane and the retinal pigmented epithelium, the site at which drusen deposits are formed. Since the extravascular space presents no barrier to the diffusion of proteins such as antibodies or other drusen-binding molecules, the intravenously applied anti-drusen probes have free access to their target ligands.
  • labeled drusen-binding antibodies and other drusen-binding molecules can reach and bind to drusen deposits in the eye following intravenous injection or other routes of administration.
  • Vitronectin probes may be utilized in different ways, particularly for diagnostic purposes.
  • Utilities include, but are not limited to, in vitro, ex vivo, and in vivo diagnosis of an accumulation having vitronectin, especially an abnormal, extracellular, ocular accumulation.
  • Solid support refers to any surface that is transferable from solution to solution or forms a structure for conducting peptide-based assays, and includes beads, membranes, microtiter wells, strings, plastic strips, or any surface onto which peptide probes may be immobilized.
  • Examples of peptide probes include both capture probes and signal probes.
  • a capture probe specific for vitronectin is typically permitted to bind a target unknown sample.
  • the capture probe may be attached to a solid support. Additionally, the capture probe may be involved in a complex consisting of a target peptide and a signal probe. If the target contains vitronectin, it binds with the capture probe and the signal probe.
  • a signal probe is typically be attached to a labeling substance or a marker that is detectable using means well known in the art.
  • the capture probe and the signal probe need not comprise the entire length of the vitronectin protein.
  • These probes can be oligopeptides comprising sequences that are specific for vitronectin and the length of the capture and signal probes may vary considerably. These peptides are usually produced by synthetic or recombinant techniques.
  • Sandwich assays are a convenient way to apply the methods just described but other methods may be employed.
  • a sandwich assay is generally performed as follows. First, a capture peptide is hybridized to an unknown target peptide under suitable conditions known to those skilled in the art. The resulting complex of capture probe and target is permitted to bind to the signal probe. The final complex comprises the capture probe bound to the target which is in turn bound to the signal probe. This resulting complex can be attached to the solid support or tissue via the capture probe. Alternatively, the complex can be suspended in fluid such as serum or vitreous humor.
  • Unbound peptides, including signal probes and targets, and any debris are washed away while the complexes of interest remain attached to the solid support or tissue.
  • the complexes can be concentrated in fluid such as serum or vitreous humor by methods such as centrifugation or precipitation.
  • the signal is detected in the final complex and means of detection depend upon the marker employed. For example, if a fluorescent label is present, a fluorometer may be used as a detection device.
  • the vitronectin probe is not limited to antibodies, however. Any agent that binds to vitronectin could be used. For examples, see Table I. Molecules such as those listed in Table I could be used as vitronectin probes by methods involving, for example, labeling the molecule by means well known in the art.
  • the label is usually a detectable marker such as a fluorophore or chromophore or a radioactive marker such as I 125 .
  • the probe binds with any vitronectin in the sample and subsequently the sample is evaluated for detection of the label by appropriate means such as a fluorometer, a radiosensitive plate or counter, and a scintillation counter.
  • an in vitro method can include evaluating samples such as biopsy material or extracted fluid for vitronectin.
  • samples such as biopsy material or extracted fluid for vitronectin.
  • a biopsy can be taken from the retina, and a fluid sample can be taken from the vitreous humor, blood or serum.
  • the vitronectin detected can be in a deposit such as drusen or in suspension in fluid.
  • Diagnostic assays include, for example, ELISA, RIA, and Western blot assays.
  • Example 1 Immunohistochemical Characterization of Drusen A series of experiments were performed on fixed and unfixed sections of retina/RPE/choroid obtained from approximately 250 human donor eyes (age 1-96 years, with and without AMD) within three hours following death. For fixation, tissues were immersed in 4% paraformaldehyde in 100 mM sodium cacodylate buffer at pH 7.4 for two to three hours at 4°C. Fixed and unfixed tissues were subsequently infiltrated and embedded in acrylamide and/or OCT (Optimal Cutting Temperature Compound, available from Miles, Inc., Elkhart, Indiana) and sectioned to a thickness of 5-8 microns on a cryostat at -20°C. Additional fixed tissue from each eye was processed for correlative examination by electron microscopy. Individual sections were examined immunohistochemically using a variety of antibodies and lectins. Other sections from the same eye(s) served as the controls.
  • OCT Optimal Cutting Temperature Compound
  • control solutions were applied at the same weight to volume concentration as in the experimental condition; they contained one of the following reagents: pre- immune serum, non-immune serum, an irrelevant antibody or lectin, primary antibody plus an excess of antigen, or buffer solution with or without bovine serum albumin.
  • putative drusen-associated proteins are fractionated by gel electrophoresis and/or high performance liquid chromatography (HPLC) and are further characterized by amino acid sequencing.
  • Proteoglycan core proteins are analyzed similarly and their carbohydrate composition and sequence are characterized using chromatographic methods.
  • lipid-containing, drusen-associated molecules are fractionated by thin layer chromatography and/or HPLC, and are characterized similarly using immunoprobes and molecular analyses.
  • electrophoretic separations of proteins in RPE/choroidal tissue from donors with and without drusen revealed the presence of 28 kDa, 35 kDa and 36 kDa bands that were present only in drusen-containing samples.
  • Amino acid sequence analyses of the drusen- associated bands indicated that the 28 kDa band is an immunoglobulin kappa light chain. The other molecular species have blocked amino termini and are being sequenced by other means.
  • Example 3 Early Development of Drusen and Associated Abnormalities Biochemical and immunohistochemical studies of eyes from human donors, with and without AMD, identical to those described above, were done and focused on characterization of the earliest detectable vitronectin-associated abnormalities that occur at the choroid-RPE-retina interface. In addition to vitronectin, of special interest was the presence of other molecular components that correlated with the early immunohistochemical evidence of vitronectin deposition associated with drusen. Most striking were small "nuclei" (less than 1 ⁇ m diameter) that reacted with antibodies directed against vitronectin, Type IV collagen and wheat germ agglutinin.
  • Example 4A Identification of Agents that Ameliorate Drusen Data relating to the molecular organization and biochemical properties of drusen and other deposits was obtained by treating sections of human retina, RPE and choroid containing demonstrable drusen with agents representative of each of the major categories listed in Table II. Table II lists examples of agents for amelioration of vitronectin- containing deposits such as drusen.
  • Such agents are derived from a number of classes of biologically active agents such as proteases, glycosidases, lipases, reducing agents, inorganic ions and biological solvents/detergents (Table II) .
  • proteases e.g. trypsin
  • enzymes with narrower specificities e.g. plasmin, plasminogen activator
  • Proteases are applied at concentrations ranging from about .0001 to about 10.0 units per milliliter of appropriately buffered reaction solution (as suggested by the supplier) .
  • Proteases are applied to sections for periods ranging from about 1 minute to about 24 hours at temperatures ranging from about 2°C to about 45°C.
  • Glycosidases This group includes a range of endo- and exoglycosidases as well as enzymes with narrower specificities selected based on the basis of the oligosaccharide sequences of vitronectin-associated carbohydrates. See Table II for some examples.
  • Glycosidases are applied individually and in combinations at concentrations ranging from about .0001 to about 10.0 units per milliliter of appropriately buffered reaction solution (as suggested by the supplier) .
  • One unit of glycosidase activity is defined, using endoglycosidase F as an example, as that amount of enzyme that will hydrolyze 1.0 ⁇ mole of dansyl-Asn(GlcNAc) 2 (Man) 5 in 60 minutes at 37°C at pH 5.0.
  • Glycosidases are applied to sections for periods ranging from about 1 minute to about 24 hours at temperatures ranging from about 2°C to about 45°C.
  • Lipases This group includes agents of broad or narrow specificity selected for their ability to disrupt lipid components of vitronectin-containing drusen and other abnormal deposits. Some examples are listed in Table II. One unit of lipase is defined as that amount of enzyme that will hydrolyze 1.0 microequivalent of fatty acid from a triglyceride in one hour at 37°C.
  • This group includes a range of agents, with relatively strong (e.g. j8-mercaptoethanol) to relatively weak (glutathione) reducing activity, that act to disrupt disulfide bonds involved in maintaining the tertiary structure of vitronectin. Such agents are applied at concentrations ranging from about .001 millimolar (mM) to about 100 mM for periods ranging from about 1 minute to about 24 hours at temperatures ranging from about 2°C to about 45°C.
  • Inorganic Ions This group includes a variety of ionic species (e.g. Mg ++ , Mn ++ , Na + , K + , C ++ , Cl") in addition to H + and OH" (i.e.
  • pH that will act to disrupt noncovalent interactions responsible for vitronectin accumulation.
  • agents are applied at concentrations ranging from about .0001 M to about 10.0 M for periods ranging from about 1 minute to about 24 hours at temperatures ranging from about 2°C to about 45°C.
  • Biological Solvents/Detergents This group includes a variety of compounds which act by disrupting noncovalent interactions responsible for vitronectin accumulation. See Table II for examples. Such agents are applied at concentrations ranging from about .0001 M to about 1.0 M for periods ranging from about 1 minute to about 24 hours at temperatures ranging from about 2°C to about 45°C.
  • Tissue punches were incubated in solutions of the above-listed agents for 10 minutes, 30 minutes and 60 minutes. Following incubation, tissues were fixed in 4% paraformaldehyde and 100 mM sodium cacodylate buffer at pH 7.4 for 2-3 hours, at 4°C. Control tissues were incubated in balanced salt solution in parallel with enzyme-containing solutions. Following fixation, tissues were infiltrated and embedded in acrylamide and sectioned to a thickness of 5-8 ⁇ m on a cryostat at -20°C.
  • Sections were incubated with various drusen-reactive antibodies (including antibodies directed against vitronectin) and lectins (see Table III) , as well as with hematoxylin and eosin (HE) and periodic acid Schiff (PAS) stains to determine whether any of the agents resulted in dissolution or disruption of drusen.
  • Drusen in the test sections were disrupted by chondroitinase ABC and DMSO by 10 minutes and, by 60 minutes, were completely dissolved in many cases.
  • drusen and drusen-reactive molecules, such as vitronectin were unaffected in control sections.
  • elastase-treated tissue exhibited some disruption of drusen by 60 minutes at the dosage utilized.
  • Example 4B Agents that Ameliorate Drusen
  • chondroitinase ABC, DMSO, trypsin and urea/NP-40 disrupted or removed drusen.
  • treatment with the proteases chymotrypsin, collagenase, dispase, elastase, Factor Xa and trypsin reduced the binding of some drusen-binding lectins and antibodies.
  • treatment with the glycosidases amylase Ila, ⁇ - galactosidase, lysozyme, N-glycosidase and neuraminidase reduced the binding of some drusen-binding lectins.
  • Treatment with SDS//3-mercaptoethanol and exposure to alkaline pH (NaOH) reduced the binding of some drusen-binding lectins.
  • Chloroform-methanol-HCl (2:1:1) extraction reduced the binding of all lectins and Sudan Black staining of drusen.
  • RPE cell cultures from (1) eyes with drusen and (2) eyes without drusen were established from corresponding donor eyes with and without drusen, respectively.
  • the donors were of various ethnic backgrounds. Protocols utilized for the isolation and in vitro culture of human RPE cells are well known to those trained in the art. See, for example, Pfeffer, Prog. Retinal Res . 10 (1991).
  • these cultures have been shown to have drusen-associated differences in cellular growth rate and in the types of proteins secreted into the culture medium.
  • the invention provides a comparison of normal and abnormal RPE cell lines. This comparison is exploited to elucidate, among other things, gene expression of proteins including vitronectin.
  • a nonhuman species known to display clinical characteristics of AMD is the rhesus monkey, some of which have macular drusen similar, if not identical, to those in humans. See Monaco and Wormington, "The Rhesus Monkey As An Animal Model For Age-Related Maculopathy,” Optometry and Vision Science 67(7)532-537 (1990) and Hope, et al . , "A Primate Model For Age-Related Macular Drusen," British Journal Ophthal . 76:11-16 (1992). Aged monkeys are available from various Regional Primate Centers as well as other commercial sources. Sources include university primate colonies, such as at the University of Puerto Rico and drug companies such as Lederle.
  • Monkeys in the AMD colony are given periodic retinal examinations to determine the course of drusen development.
  • fundus examinations are conducted prior to drug administration and periodically thereafter.
  • Serum is collected and screened for abnormalities, including autoantibodies. This colony is a resource for analyses of the onset of AMD and the preliminary testing of potential pharmacologic agents.
  • Detection of vitronectin-related abnormalities can be a reliable, predictable indication of the early phases of AMD or other related diseases. Such detection is the groundwork for an early diagnostic test for AMD.
  • the diagnostic test can be an immunoassay, such as an enzyme-linked immunoassay (ELISA) , which detects in serum one or more of the following: 1) presence of vitronectin antibodies; 2) abnormal levels of vitronectin protein; 3) abnormal vitronectin isoform ratios; and 4) aberrant forms of vitronectin.
  • ELISA enzyme-linked immunoassay
  • patients with serum values of vitronectin above a certain normal level would be judged at increased risk for the development of extensive drusen, reduced visual acuity or, perhaps, the potentially-blinding neovascular form of AMD.
  • Example 8 An in situ Diagnostic Test A patient suspected of either suffering from the early stages of AMD or having a significant predisposition to AMD warranting prophylactic intervention is selected for a clinical diagnostic trial.
  • a drusen-binding molecule(s) such as vitronectin antibody, vitronectin protein, or vitronectin protein fragment, is conjugated to an appropriate fluorochrome, such as fluorescein, using methods well known in the art.
  • the binding molecule-fluorochrome conjugate is injected beneath the sclera and into the suprachoroidal space above the retina where the drusen are largest and most prevalent. Typically, this region includes the region overlying the macula.
  • the imaging agent can be injected intravenously.
  • vitronectin-fluorochrome conjugate diffuses into the region where the drusen are located and binds to them. Whereas detection and visualization of drusen larger than 60-100 ⁇ m in diameter can be accomplished using conventional fundus photography, visualization and detection of much smaller drusen deposits is improved significantly by fluorescence enhancement.
  • the amount and pattern of the vitronectin deposition detectable is normal, then no further intervention may be indicated. However, if the amount and pattern of vitronectin are abnormal or indicate early drusen development, early AMD, or a significant risk to development of AMD, then therapeutic intervention may be indicated. Enhanced visualization, in turn, is exploited therapeutically in conjunction with an appropriate ophthalmic laser that allows for the selective targeting and disruption of drusen while leaving surrounding tissues relatively intact.
  • a patient having the severe neovascular or exudative form of AMD is selected for treatment.
  • the patient has vision of 20/100 or worse as measured by the Bailey-Lovie chart.
  • a full ophthalmological examination including fluorescein angiography, fundus photography, scanning laser fundoscopy, visual acuity, contrast sensitivity, reading speed, color vision, electroretinography, and visual fields, is performed.
  • the patient is appropriately positioned on the operating table or chair.
  • a retrobulbar injection of 2% lidocaine or 0.5% marcaine is administered to induce local anesthesia; this is combined with a sedative, if necessary.
  • Cycloplegics and adrenergic agents such as cyclogel and neosynephrine, may also be employed.
  • a 360° peritomy is performed and a suture is placed on the lateral rectus and the muscle is subsequently disinserted.
  • the globe is rotated nasally (abducted) , a region near the inferior oblique muscle insertion site is identified, and a sclerotomy is performed.
  • a cannula is inserted into the suprachoroidal space; the cannula may be first attached or later attached to a syringe or other delivery system containing the therapeutic compound.
  • the therapeutic agent such as chondroitinase ABC
  • the therapeutic agent is injected into the choroid and/or the suprachoroidal space and the cannula is subsequently withdrawn.
  • the therapeutic agent is contained within a slow release vehicle and such vehicle is implanted into the suprachoroidal space or choroidal stroma.
  • the sclerotomy site is closed with a single, interrupted 9-0 or 10-0 nylon or vicryl suture, the lateral rectus is sutured to its insertion site and the peritomy is closed.
  • the patient is evaluated as immediately as possible for ophthalmologic complications. After the anesthetic- and cycloplegic-induced effects have worn off, the patient is reevaluated using the same examination outlined above. Visual acuity is assessed at weekly to monthly intervals for a one- year period. Thereafter, visual acuity is assessed at yearly intervals. The therapy may be repeated every 12 to 24 months as necessary.
  • patients with AMD or drusen who are receiving single, intravenous injections (or multiple injections over a defined period of time) of potential drusen-ameliorating drugs (such as streptokinase, tissue plasminogen activator, coumadin, warfarin, wydase, heparin, nitroglycerin) for non-ophthalmic (e.g. cardiovascular) indications, are given a full ophthalmological examination prior to initial drug administration.
  • This examination includes of fluorescein angiography, fundus photography, scanning laser fundoscopy, visual acuity, contrast sensitivity, reading speed, color vision, electroretinography and visual fields.
  • a map of drusen distribution is generated prior to initial drug administration. As soon as possible following drug administration, the full ophthalmological examination is repeated. The patient is reassessed at weekly to monthly intervals for the ensuing year. A significant redistribution and/or loss of drusen, combined with visual improvement, is a successful outcome, and suggests that the drug utilized is a potential drusen-ameliorating compound.
  • choroidal abnormalities were identified in human donor eyes based on conventional electron microscopical and immunocytochemical observations. In most eyes, the severity of these abnormalities increased with increasing drusen grade. In eyes possessing significant amounts of hard drusen, a unique fibrillar system was identified throughout the choroid. These fibrillar networks, which are composed of aggregates of distinct filamentous structures surrounded by an amorphous material, contain vitronectin and, perhaps, elastin.
  • connective tissue beams of the trabecular meshwork are similar in origin to, and the basal laminae of the meshwork endothelial cells are contiguous with, those of Bruch's membrane.
  • a drusen-ameliorative treatment is likely to have utility as glaucoma treatment.
  • the trabecular meshwork-containing tissues were fixed and prepared for immunocytochemical observations. Many of these specimens demonstrated a strong positive reaction with anti-vitronectin antibodies. Control specimens, collected from human eyes derived from donors without glaucoma did not exhibit the same reaction. These studies suggest that vitronectin-containing deposits are present within the extracellular matrix stroma associated with the trabecular beams of the trabecular meshwork in patients with glaucoma. In addition to vitronectin, thrombospondin was identified in associated with the trabecular meshwork in glaucomatous eyes.
  • Example 11 Retina as a Source of Ocular Vitronectin Northern blot analyses revealed vitronectin transcripts in monkey and human retinas, indicating that ocular vitronectin may not be derived solely from liver- synthesized plasma vitronectin.
  • TGF-B Growth Factors
  • Plasminogen Activator Inhibitor-1 (PAI-1)
  • TGF-B Transforming Growth Factor-B
  • Sialidase II II II (Neuramin- idase)
  • Lipoprotein o.lng-lOmg Various Subretinal, Sigma lipase Vehicles Intravenous, Chemical, (See text Choroidal St. Louis ⁇ pages 27-28)
  • WGA wheat germ agglutinin sWGA: succinylated wheat germ agglutinin
  • EBL elderberry bark lectin
  • PNA Phaseolus vulgaris (peanut) aggiutinin
  • LCA Lens culnaris agglutinin
  • PHA-L Phaseolus vulgraris agglutinin
  • Con A (weak) : Concanavalin A agglutinin
  • the lectins WGA, sWGA, LFA and PHA-L also bind basal laminar/linear deposits (BLD's)

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Abstract

Analyses de la composition de drusen et de dépôts sur la membrane vitrée qui sont impliqués dans la dégénérescence maculaire liée à l'âge (AMD). Fondée sur la découverte récente que la vitronectine est un constituant des drusen, la présente invention concerne des procédés de diagnostic et de thérapie de la dégénérescence maculaire liée à l'âge. La vitronectine est impliquée dans l'évolution de l'AMD. Elle est également identifiée comme étant un constituant du réseau trabéculaire chez les patients atteints de glaucome. Des procédés in vitro, in vivo et ex vivo sont également décrits.
PCT/US1994/014691 1993-12-21 1994-12-20 Diagnostics et therapies oculaires WO1995017673A1 (fr)

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WO2001084149A2 (fr) * 2000-04-29 2001-11-08 University Of Iowa Research Foundation Diagnostic et therapeutique pour des troubles lies a une degenerescence maculaire
JP2003504088A (ja) * 1999-02-19 2003-02-04 ザ ユニヴァーシティー オブ アイオワ リサーチ ファンデーション 黄斑変性に関する診断および治療
US7011952B2 (en) 2000-02-22 2006-03-14 University Of Iowa Research Foundation Diagnostics and therapeutics for macular degeneration-related disorders
US7108982B1 (en) 1999-02-19 2006-09-19 University Of Iowa Research Foundation Diagnostics and the therapeutics for macular degeneration
EP1804064A1 (fr) * 1999-02-19 2007-07-04 University Of Iowa Research Foundation Diagnostics et thérapeutiques pour la dégénérescence maculaire
EP2267027A3 (fr) * 2002-11-08 2011-07-20 Ablynx N.V. Procédé d'administration de polypeptides thérapeutiques et polypeptides correspondants
WO2012075023A3 (fr) * 2010-11-29 2012-11-29 Novelmed Therapeutics, Inc. Néoanticorps pour le diagnostic d'une lésion tissulaire
WO2014036483A1 (fr) * 2012-08-31 2014-03-06 University Of Maryland, Baltimore Méthodes et compositions pour détecter des druses et prédire la dégénérescence maculaire liée à l'âge
US9243065B2 (en) 2002-11-08 2016-01-26 Ablynx N.V. Polypeptide constructs including VHH directed against EGFR for intracellular delivery
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US9371381B2 (en) 2002-11-08 2016-06-21 Ablynx, N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US10010447B2 (en) 2013-12-18 2018-07-03 Novartis Ag Systems and methods for subretinal delivery of therapeutic agents
US11253614B2 (en) 2016-08-23 2022-02-22 University Of Maryland, Baltimore Methods for detecting and/or predicting age-related macular degeneration and/or Alzheimer's disease
WO2023003949A1 (fr) * 2021-07-21 2023-01-26 Sanford Burnham Prebys Medical Discovery Institute Compositions et méthodes d'inhibition de la protéine vitronectine de sang humain

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JP2003504088A (ja) * 1999-02-19 2003-02-04 ザ ユニヴァーシティー オブ アイオワ リサーチ ファンデーション 黄斑変性に関する診断および治療
US7108982B1 (en) 1999-02-19 2006-09-19 University Of Iowa Research Foundation Diagnostics and the therapeutics for macular degeneration
EP1804064A1 (fr) * 1999-02-19 2007-07-04 University Of Iowa Research Foundation Diagnostics et thérapeutiques pour la dégénérescence maculaire
US20110182908A1 (en) * 2000-02-22 2011-07-28 University Of Iowa Research Foundation Diagnostics and Therapeutics for Macular Degeneration-Related Disorders
US7011952B2 (en) 2000-02-22 2006-03-14 University Of Iowa Research Foundation Diagnostics and therapeutics for macular degeneration-related disorders
US7344846B2 (en) * 2000-02-22 2008-03-18 University Of Iowa Research Foundation Diagnostics and therapeutics for macular degeneration-related disorders
US7351524B2 (en) 2000-02-22 2008-04-01 University Of Iowa Research Foundation Diagnostics and therapeutics for macular degeneration-related disorders
US7682804B2 (en) * 2000-02-22 2010-03-23 University Of Iowa Research Foundation Diagnostics and therapeutics for macular degeneration-related disorders
WO2001084149A3 (fr) * 2000-04-29 2002-07-18 Univ Iowa Res Found Diagnostic et therapeutique pour des troubles lies a une degenerescence maculaire
AU2001261103B2 (en) * 2000-04-29 2007-08-23 University Of Iowa Research Foundation Diagnostics and therapeutics for macular degeneration-related disorders
WO2001084149A2 (fr) * 2000-04-29 2001-11-08 University Of Iowa Research Foundation Diagnostic et therapeutique pour des troubles lies a une degenerescence maculaire
US9725522B2 (en) 2002-11-08 2017-08-08 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US9243065B2 (en) 2002-11-08 2016-01-26 Ablynx N.V. Polypeptide constructs including VHH directed against EGFR for intracellular delivery
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US9371381B2 (en) 2002-11-08 2016-06-21 Ablynx, N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
EP2267027A3 (fr) * 2002-11-08 2011-07-20 Ablynx N.V. Procédé d'administration de polypeptides thérapeutiques et polypeptides correspondants
WO2012075023A3 (fr) * 2010-11-29 2012-11-29 Novelmed Therapeutics, Inc. Néoanticorps pour le diagnostic d'une lésion tissulaire
US8940490B2 (en) 2010-11-29 2015-01-27 Novelmed Therapeutics, Inc. Neoantibodies for diagnosing tissue injury
US9816999B2 (en) 2010-11-29 2017-11-14 Novelmed Therapeutics, Inc. Neo antibodies for diagnostic imaging of tissue injury
WO2014036483A1 (fr) * 2012-08-31 2014-03-06 University Of Maryland, Baltimore Méthodes et compositions pour détecter des druses et prédire la dégénérescence maculaire liée à l'âge
US9801955B2 (en) 2012-08-31 2017-10-31 University Of Maryland, Baltimore Methods and compositions for detecting drusen and predicting age-related macular degeneration
US10010447B2 (en) 2013-12-18 2018-07-03 Novartis Ag Systems and methods for subretinal delivery of therapeutic agents
US11253614B2 (en) 2016-08-23 2022-02-22 University Of Maryland, Baltimore Methods for detecting and/or predicting age-related macular degeneration and/or Alzheimer's disease
WO2023003949A1 (fr) * 2021-07-21 2023-01-26 Sanford Burnham Prebys Medical Discovery Institute Compositions et méthodes d'inhibition de la protéine vitronectine de sang humain

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