WO2009149205A2 - Lignées cellulaires qui secrètent des récepteurs de vegf solubles et leurs utilisations - Google Patents

Lignées cellulaires qui secrètent des récepteurs de vegf solubles et leurs utilisations Download PDF

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WO2009149205A2
WO2009149205A2 PCT/US2009/046163 US2009046163W WO2009149205A2 WO 2009149205 A2 WO2009149205 A2 WO 2009149205A2 US 2009046163 W US2009046163 W US 2009046163W WO 2009149205 A2 WO2009149205 A2 WO 2009149205A2
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seq
cells
vegf receptor
arpe
eye
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WO2009149205A3 (fr
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Weng Tao
Konrad Kauper
Paul Stabila
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Neurotech USA Inc
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Neurotech USA Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the field of encapsulated cell therapy.
  • transplantation patients must be immunosuppressed in order to avert immunological rejection of the transplant, which results in loss of transplant function and eventual necrosis of the transplanted tissue or cells.
  • transplant must remain functional for a long period of time, even for the remainder of the patient's lifetime. It is both undesirable and expensive to maintain a patient in an immunosuppressed state for a substantial period of time.
  • One major problem in treatment of such diseases is the inability to deliver therapeutic agents into the eye, due to the presence of the blood-retinal barrier, and to maintain them there at therapeutically effective concentrations.
  • BDNF and CNTF have been shown to slow degeneration of retinal ganglion cells and decrease degeneration of photoreceptors in various animal models. See, e.g., Genetic Technology News, vol. 13, no. 1 (Jan. 1993).
  • nerve growth factor has been shown to enhance retinal ganglion cell survival after optic nerve section and has also been shown to promote recovery of retinal neurons after ischemia. See, e.g., Siliprandi, et al., Invest. Ophthalmol. & Vis. ScL, 34, pp. 3232-3245 (1993).
  • a desirable alternative to transplantation procedures is the implantation of cells or tissues within a physical barrier which will allow diffusion of nutrients, metabolites, and secreted products, but will block the cellular and molecular effectors of immunological rejection.
  • a variety of devices which protect tissues or cells producing a selected product from the immune system have been explored. See, e.g. , US Patent No. 5,158,881;
  • WO92/03327 WO91/00119; and WO93/00128, each of which is incorporated herein by reference in its entirety.
  • These devices include, for example, extravascular diffusion chambers, intravascular diffusion chambers, intravascular ultrafiltration chambers, and implantation of microencapsulated cells. See Scharp, D. W., et al., World J. Surg., 8, pp. 221- 9 (1984). See, e.g., Lim et al., Science 210: 908-910 (1980); Sun, A. M., Methods in
  • the use of such devices would alleviate the need to maintain the patient in an immunosuppressed state.
  • none of these approaches have been satisfactory for providing long-term transplant function.
  • methods of delivering appropriate quantities of needed substances such as, for example, neurotrophic factors, anti-angiogenic factors, anti-inflammatory factors, enzymes, hormones, or other factors, or of providing other needed metabolic functions, to the eye for an extended period of time are needed.
  • nucleic acids encoding a Vascular Endothelial Growth Factor (VEGF) receptor wherein the nucleic acids comprise or consist of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13.
  • the invention also provides nucleic acid molecules encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.
  • nucleic acid molecules encoding a polypeptide having a sequence that is at least 95% identical to any SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14.
  • the nucleic acid molecules may be the complement of such a nucleic acid molecule.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the nucleic acid molecules of the invention can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, or a complement of any of these nucleotide sequences
  • SEQ ID NO: 1 e.g., a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, encoding a polypeptide having a sequence
  • soluble VEGF receptor molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • nucleic acids of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to soluble VEGFR nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 or a complement thereof. Oligonucleotides may be chemically synthesized and may be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13.
  • a nucleic acid molecule that is complementary to these nucleotide sequences is one that is sufficiently complementary to the nucleotide sequence that it can hydrogen bond with little or no mismatches, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Van der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13, e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of any of the soluble VEGF receptors of the invention.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.
  • Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
  • Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution.
  • Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 30%, 50%, 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 due to degeneracy of the genetic code and thus encode the same soluble VEGFR proteins as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13.
  • an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500, 1000, 1500, 2000, or more nucleotides in length.
  • an isolated nucleic acid molecule of the invention hybridizes to the coding region, for example SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60 0 C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50 0 C.
  • a non- limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40 0 C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50 0 C.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • polypeptide encoded by any of the nucleic acid molecules described herein can be soluble VEGF receptors.
  • the soluble VEGF receptors described herein bind (e.g., preferentially) to VEGF. The binding of these soluble VEGF receptors to VEGF inhibits endothelial cell proliferation and vascular permeability.
  • the invention also involves an isolated polypeptide that is at least 80% identical to a polypeptide having an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 14.
  • the isolated polypeptide is at least 80% homologous to a fragment (i.e., at least 6 contiguous amino acids) of a polypeptide having an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 14.
  • the invention also includes isolated polypeptides that are at least 80% homologous to a derivative, analog, or homolog of a polypeptide having an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 14.
  • the invention also provides an isolated polypeptide that is at least 80% identical to a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 14.
  • polypeptides should be encoded by a nucleic acid molecule capable of hybridizing to a nucleic acid molecule of SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13 under stringent conditions.
  • novel polypeptides of the invention include the soluble VEGF receptor polypeptides whose sequence is provided in SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14.
  • the invention also includes mutant or variant polypeptides any of whose residues may be changed from the corresponding residue shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14, while still encoding a polypeptide that maintains its soluble VEGF receptor activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to 20% or more of the residues may be so changed.
  • a soluble VEGF receptor variant that preserves soluble VEGFR-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution.
  • the invention also pertains to isolated soluble VEGFR polypeptides, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof.
  • Soluble VEGFR constructs described herein can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • the soluble VEGFR polypeptides of the invention are produced by recombinant DNA techniques.
  • a soluble VEGFR protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or biologically active portion thereof is substantially free of cellular material or other contaminating proteins or polypeptides from the cell or tissue source from which the soluble VEGFR polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of soluble VEGFR polypeptides in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of VEGFR polypeptide having less than about 30% (by dry weight) of non-VEGFR protein (also referred to herein as a "contaminating protein”), more preferably less than about 20% of non-VEGFR protein, still more preferably less than about 10% of non-VEGFR protein, and most preferably less than about 5% non-VEGFR protein.
  • non-VEGFR protein also referred to herein as a "contaminating protein”
  • the VEGFR polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of soluble VEGFR polypeptide in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of soluble VEGFR polypeptide having less than about 30% (by dry weight) of chemical precursors or non- VEGFR chemical, more preferably less than about 20% chemical precursors or non- VEGFR chemicals, still more preferably less than about 10% chemical precursors or non- VEGFR chemicals, and most preferably less than about 5% chemical precursors or non-VEGFR chemicals.
  • Biologically active portions of a soluble VEGFR polypeptide construct of the invention include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the soluble VEGFR polypeptides, e.g., the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14 that include fewer amino acids than the full length soluble VEGFR constructs described herein, and exhibit at least one activity of a soluble VEGFR polypeptide of the invention.
  • biologically active portions comprise a domain or motif with at least one activity of the soluble VEGFR polypeptide.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 1970 J MoI Biol 48: 443-453.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I, in the case of nucleic acids
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • the invention also provides for soluble VEGF receptor chimeric or fusion proteins.
  • a soluble VEGFR chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in- frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, f ⁇ lling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplif ⁇ ed to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplif ⁇ ed to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • the invention further provides vectors containing any of the nucleic acid molecules of the invention.
  • the invention also pertains to vectors, preferably expression vectors, containing a nucleic acid encoding the soluble VEGFR polypeptides of the invention, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise any of the nucleic acids of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., soluble VEGFR polypeptides, mutant forms of soluble VEGFR polypeptides, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of soluble VEGFR constructs of the invention in prokaryotic or eukaryotic cells.
  • Other suitable expression systems for both prokaryotic and eukaryotic cells are known in the art. ⁇ See, e.g., Chapters 16 and 17 of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989).
  • the invention also provides host cells or cell lines containing such vectors (or any of the nucleic acid molecules described herein).
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • the host cell may be an ARPE- 19 cell.
  • other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the soluble VEGF receptor construct or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) any of the soluble VEGFR polypeptide constructs described herein. Accordingly, the invention further provides methods for producing these soluble VEGFR polypeptides using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding soluble VEGFR has been introduced) in a suitable medium such that soluble VEGFR polypeptide is produced.
  • the method further comprises isolating soluble VEGFR from the medium or the host cell.
  • the invention also provides cell lines of ARPE- 19 cells genetically engineered to produce a soluble VEGF receptor, wherein the soluble VEGF receptor is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO: 13.
  • the invention also provides cell lines of ARPE- 19 cells genetically engineered to produce a soluble VEGF receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO: 14.
  • implantable cell culture devices containing a containing one or more ARPE- 19 cells that are genetically engineered to produce any of the polypeptides of the invention and a semipermeable membrane surrounding the core, wherein the membrane permits the diffusion of the soluble VEGF receptor there through.
  • the core additionally contains a matrix disposed within the semipermeable membrane.
  • the matrix may include a hydrogel or extracellular matrix components.
  • the hydrogel may contain alginate cross-linked with a multivalent ion.
  • the matrix includes a plurality of monofilaments, wherein the monofilaments are twisted into a yarn or woven into a mesh or are twisted into a yarn that is in non- woven strands, and wherein the cells or tissue are distributed thereon.
  • the filamentous cell-supporting matrix can be made from a biocompatible material selected from the group consisting of acrylic, polyester, polyethylene, polypropylene polyacetonitrile, polyethylene terephthalate, nylon, polyamides, polyurethanes, polybutester, silk, cotton, chitin, carbon, and/or biocompatible metals.
  • the cell encapsulation devices described herein also have a tether anchor.
  • the tether anchor may be an anchor loop that is adapted for anchoring the capsule to an ocular structure.
  • the capsules can be implanted into the vitreous, the aqueous humor, the Subtenon's space, the periocular space, the posterior chamber, and/or the anterior chamber of the eye.
  • the jackets of the devices described herein preferably are made from a permselective, immunoisolatory membrane.
  • the jackets are made from an ultrafiltration membrane or a micro filtration membrane.
  • an ultrafiltration membrane typically has a pore size of 1-100 nm
  • a micro filtration membrane typically has a pore size of 0.1-10 ⁇ m.
  • the jacket may be made from a non-porous membrane material (e.g., a hydrogel or a polyurethane).
  • the capsule can be configured as a hollow fiber or a flat sheet.
  • at least one additional biologically active molecule can be co-delivered from these devices.
  • the at least one additional biologically active molecule can be from a non-cellular or a cellular source (i.e., the at least one additional biologically active molecule is produced by one or more genetically engineered ARPE- 19 cell in the core).
  • methods for treating ophthalmic disorders by implanting the implantable cell culture devices of the invention into the eye of a patient and allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye, thereby treating the ophthalmic disorder.
  • the ophthalmic disorder to be treated can be selected from the group consisting of retinopathy of prematurity, diabetic macular edema, diabetic retinopathy, age-related macular degeneration, glaucoma, retinitis pigmentosa, cataract formation, retinoblastoma and retinal ischemia.
  • age- related macular degeneration is wet form age-related macular degeneration.
  • the ophthalmic disorder is diabetic retinopathy.
  • the invention further provides methods for inhibiting endothelial cell proliferation by implanting the implantable cell culture devices of the invention into a patient suffering from a cell proliferation disorder and allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF, wherein the binding inhibits endothelial cell proliferation in the patient.
  • the disorder is selected from the group consisting of hematologic disorders, atherosclerosis, inflammation, increased vascular permeability and malignancy.
  • Preferred target regions include the central nervous system, including the brain, ventricle, spinal cord, or the aqueous and vitreous humors of the eye.
  • the invention also provides methods for making the implantable cell culture devices of the invention.
  • at least one ARPE- 19 cell is genetically engineered to secrete a soluble VEGF receptor encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13 and the genetically modified ARPE-19 cells are encapsulated within a semipermeable membrane, wherein said membrane allows the diffusion of the soluble VEGF receptor therethrough.
  • At least one ARPE- 19 cell is genetically engineering to secrete a soluble VEGF receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, and SEQ ID NO: 14, and the genetically modified ARPE-19 cells are encapsulated within a semipermeable membrane, wherein said membrane allows the diffusion of the soluble VEGF receptor therethrough.
  • the invention also describes the use of one or more ARPE-19 cells that are genetically engineered to produce any of the polypeptides of the invention (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14) in the manufacture of any of the implantable cell culture devices according to the invention for treating ophthalmic disorders by implantation of the device into the eye of a patient and by allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye.
  • any of the implantable cell culture devices described herein can be used for treating ophthalmic disorders by implantation of the device into the eye of a patient and by allowing the soluble VEGF receptors of the invention to diffuse from the device and bind to VEGF in the eye.
  • ARPE- 19 cells that are genetically engineered to produce any of the polypeptides of the invention for treating ophthalmic disorders by implantation of any of the implantable cell culture devices of the invention into the eye of a patient and by allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye.
  • any of the isolated nucleic acid molecules described herein can also be used in the manufacture of one or more ARPE- 19 cells that are genetically engineered to produce one of the polypeptides of the invention for treating ophthalmic disorders by implantation into the eye of a patient an implantable cell culture device of the invention and by allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye.
  • any of the isolated nucleic acid molecules of the invention can also be used for treating ophthalmic disorders where treating comprises implanting into the eye of a patient an implantable cell culture device according to the instant invention and allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye, wherein said one or more ARPE- 19 cells in said device have been genetically engineered with said isolated nucleic acid molecule to thereby produce any of the isolated polypeptides described herein.
  • the ophthalmic disorder is selected from the group consisting of retinopathy of prematurity, diabetic macular edema, diabetic retinopathy, age-related macular degeneration (e.g. wet form age-related macular degeneration), glaucoma, retinitis pigmentosa, cataract formation, retinoblastoma and retinal ischemia.
  • the invention also provides for the use of one or more ARPE- 19 cells that are genetically engineered to produce a polypeptide of the invention in the manufacture of an implantable cell culture device according to the invention for inhibiting endothelial cell proliferation by implantation of the device into the eye of a patient suffering from a cell proliferation disorder and by allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye and to thereby inhibit endothelial cell proliferation in said patient.
  • the invention also provides implantable cell culture devices of the invention for inhibiting endothelial cell proliferation by implantation of the device into the eye of a patient suffering from a cell proliferation disorder and by allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye and to thereby inhibit endothelial cell proliferation in said patient.
  • the invention provides one or more ARPE- 19 cells that are genetically engineered to produce any of the polypeptides of the invention for inhibiting endothelial cell proliferation by implantation of any of the implantable cell culture devices described herein into the eye of a patient suffering from a cell proliferation disorder and by allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye and to thereby inhibit endothelial cell proliferation in said patient.
  • the invention also provides any of the isolated nucleic acid molecules according to the invention for inhibiting endothelial cell proliferation, the treating comprising implanting into the eye of a patient suffering from a cell proliferation disorder an implantable cell culture device according to the invention and allowing the soluble VEGF receptor to diffuse from the device and bind to VEGF in the eye and to thereby inhibit endothelial cell proliferation in said patient, and wherein said one or more ARPE- 19 cells in said device have been genetically engineered with said isolated nucleic acid molecule to thereby produce a polypeptide of the invention.
  • the cell proliferation disorder may be selected from the group consisting of hematologic disorders, atherosclerosis, inflammation, increased vascular permeability and malignancy.
  • any of the implantable cell culture devices of the invention can be used for delivering a soluble VEGF receptor to a recipient host by implantation of the device into a target region of the recipient host and wherein the encapsulated one or more ARPE- 19 cells secrete the soluble VEGF receptor at the target region.
  • one or more ARPE- 19 cells that are genetically engineered to produce any of the polypeptides described herein can be used for delivering a soluble VEGF receptor to a recipient host by implantation of any implantable cell culture devices of the invention into a target region of the recipient host and wherein the encapsulated one or more ARPE- 19 cells secrete the soluble VEGF receptor at the target region.
  • any of the isolated nucleic acid molecules described herein can be used in the manufacture of one or more ARPE- 19 cells that are genetically engineered to produce any of the polypeptides described herein for delivering a soluble VEGF receptor to a recipient host by implantation into a target region of the recipient host an implantable cell culture device of the invention, wherein the encapsulated one or more ARPE- 19 cells secrete the soluble VEGF receptor at the target region.
  • any of the isolated nucleic acid molecules described herein can also be used for delivering a soluble VEGF receptor to a recipient host, said delivering comprising implanting into a target region of the recipient host an implantable cell culture device of the invention, wherein the encapsulated one or more ARPE- 19 cells secrete the soluble VEGF receptor at the target region, and wherein said one or more ARPE- 19 cells in said device have been genetically engineered with said isolated nucleic acid molecule to thereby produce any of the polypeptides of the invention.
  • the target region is selected from the group consisting of the central nervous system, including the brain, ventricle, spinal cord, and the aqueous and vitreous humors of the eye.
  • the invention also provides methods of producing an isolated polypeptide, the method comprising expressing any of the isolated nucleic acid molecules described herein and harvesting the expressed polypeptide.
  • Figures IA and IB are graphs showing VEGF saturation binding of truncated VR iso forms. Using VRl D 1-3 -Fc as a positive control, the relative dissociation constant Kd, is shown in Figure 1C. Briefly, increasing amounts of VEGF receptor from condition media was incubated overnight with VEGF, and free VEGF was measured using a commercially available ELISA.
  • Figures 2 A -2C are graphs showing the results of the HUVEC Inhibition Bioassay. Increasing amounts of VEGF receptor from condition media were analyzed for their ability to inhibit human endothelial cell proliferation using a HUVEC bioassay.
  • Figure 3 is a schematic showing the pKAN3 Plasmid Map.
  • VEGF Vascular endothelial growth factor
  • VEGF is a signaling protein involved in both vasculogenesis, the formation of the embryonic circulatory system, and angiogenesis, the growth of blood vessels from pre-existing vasculature. While VEGF is mostly known for its effects on cells of the vascular endothelium, it also effects a broad range of other cells types, e.g., stimulation monocyte/macrophage migration, neurons, cancer cells, kidney epithelial cells, etc. There are a number of proteins within the VEGF family, which arise as a result of alternate splicing of mRNA. The various splice variants impact the function of VEGF, as they determine whether the resulting proteins are pro- or anti-angiogenic.
  • the splice variants also effect the interaction of VEGF with heparin sulfate proteoglycans (HSPGs) and neuripilin co-receptors on the cell surface, which, in turn, enhances the ability of VEGF to bind to and activate VEGF signaling receptors (VEGFRs).
  • HSPGs heparin sulfate proteoglycans
  • VAGFRs VEGF signaling receptors
  • VEGF glycosylated disulfide-bonded dimers.
  • VEGF belongs to the PDGF family of cysteine-knot growth factors, and, thus, several closely-related proteins exist, i.e., placenta growth factor (PlGF), VEGF-B, VEGF-C and VEGF-D, which together comprise the VEGF sub-family of growth factors.
  • PlGF placenta growth factor
  • VEGF-B VEGF-B
  • VEGF-C VEGF-C
  • VEGF-D which together comprise the VEGF sub-family of growth factors.
  • VEGF itself, is commonly referred to as VEGF-A in order to differentiate it from these other, related growth factors.
  • VEGF The VEGF family of proteins stimulates cellular response by binding to the VEGFRs or to the tyrosine kinase receptors present on a cell surface.
  • VEGF receptors have an extracellular portion consisting of seven immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a split tyrosine- kinase domain.
  • VEGF-A binds to both VEGFR-I (FIt-I) and VEGFR-2 (KDR/Flk-1).
  • VEGFRl is expressed as a full-length receptor tyrosine kinase (RTK) as well as in a soluble form, which carries only the extracellular domain.
  • VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF and is expressed in mesodermal progenitor cells that are destined to differentiate into hemangioblasts and angioblasts.
  • the function of VEGFR-I is less well-defined, although it is thought to modulate VEGFR-2 signaling.
  • VEGF-C and VEGF-D, but not VEGF-A, are also ligands for a third receptor (VEGFR-3), which mediates lymphangiogenesis.
  • a gene of interest i.e., a gene that encodes a given VEGF receptor construct
  • a gene of interest can be inserted into a cloning site of a suitable expression vector by using standard techniques.
  • the nucleic acid and amino acid sequences of the human (and other mammalian) genes encoding VEGF receptor molecules are known. See, e.g., U.S. Pat. Nos. 4,997,929; 5,141,856; 5,364,769; 5,453,361; WO 93/06116; WO 95/30686, incorporated herein by reference.
  • VEGF receptor constructs are contemplated by the instant invention.
  • the proteins described herein comprise fragments of secretory VEGF receptor 1 (sVRl), secretory VEGF receptor 2 (sVR2), and/or a chimera of VEGF binding domains of VEGF receptor 1 and secretory VEGF receptor 2. These secretory proteins bind VEGF and contain multiple Ig-like domains.
  • sVRl secretory VEGF receptor 1
  • sVR2 secretory VEGF receptor 2
  • These secretory proteins bind VEGF and contain multiple Ig-like domains.
  • Several immunoglobulin-like domains from both VRl and VR2 are used in the constructs disclosed herein.
  • Domain 2 (D2) is the VEGF -binding domain ("VBD") of sVRl, but it requires domain 3 (D3) for high affinity binding of VEGF.
  • VR2 has similar requirements for high affinity VEGF- binding but additionally must be dimeric, since the monomeric version binds with very low affinity.
  • dimeric SVR2 D2-3 may be used in the methods and compositions described herein.
  • the chimera described herein includes sVRlDl-2 or SVR1D2 along with sVR2D3. Versions may be monomeric or dimeric in form.
  • the specific VEGF receptor constructs of the invention include: 1) sVRl Dl-3 2) sVRl D2-3
  • Construct 5 is composed of VRl D 1-2 fused to VR2 D3.
  • the dimeric form includes a carboxy human IgG hinge region that is fused to VR2 D3.
  • Construct 6 is composed of VRl D2 fused to VR2 D3.
  • the dimeric form includes a carboxy human IgG hinge region fused to VR2 D3. The specific nucleotide and amino acid sequences of each of these VEGF antagonist constructs are shown below.
  • capitalized nucleotide sequence represents CDS, while lower case letters indicate intron (IgSP).
  • Underlined 5' terminus indicates leader sequence.
  • Underlined 3' terminus indicates IgG hinge region.
  • Underlined N-terminus indicates signal sequence.
  • Underlined C-terminus indicates IgG hinge region.
  • the predicted mature protein is shaded.
  • a wide variety of host/expression vector combinations may be used to express the gene encoding the growth factor, or other biologically active molecule(s) of interest.
  • Long- term, stable in vivo expression is achieved using expression vectors ⁇ i.e., recombinant DNA molecules) in which the gene encoding the VEGF receptor is operatively linked to a promoter that is not subject to down regulation upon implantation in-vivo in a mammalian host.
  • Suitable promoters include, for example, strong constitutive mammalian promoters, such as beta-actin, eIF4Al, GAPDH, etc.
  • Stress-inducible promoters such as the metallothionein 1 (MT-I) or VEGF promoter may also be suitable.
  • hybrid promoters containing a core promoter and custom 5 ' UTR or enhancer elements may be used.
  • Other known non- retroviral promoters capable of controlling gene expression, such as CMV or the early and late promoters of SV40 or adenovirus are suitable.
  • the expression vector containing the gene of interest may then be used to transfect the desired cell line.
  • Standard transfection techniques such as liposomal, calcium phosphate co- precipitation, DEAE-dextran transfection or electroporation may be utilized.
  • mammalian transfection kits such as Fugene ⁇ (Roche Applied Sciences)
  • viral vectors may be used to transducer the desired cell line.
  • An example of a suitable viral vector is the commercially available pLenti family of viral vectors (Invitrogen). Human mammalian cells can be used. In all cases, it is important that the cells or tissue contained in the device are not contaminated or adulterated.
  • Preferred promoters used in the disclosed constructs include the SV40 promoter, the
  • Amp promoter and the MTl promoter as shown in Figure 3.
  • Other useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., pUC, pBlueScriptTM plasmids from E. coli including pBR322, pCRl, pMB9 and their derivatives.
  • Expression vectors containing the geneticin (G418) or hygromycin drug selection genes are also useful.
  • These vectors can employ a variety of different enhancer/promoter regions to drive the expression of both a biologic gene of interest and/or a gene conferring resistance to selection with toxin such as G418 or hygromycin B.
  • a variety of different mammalian promoters can be employed to direct the expression of the genes for G418 and hygromycin B and/or the biologic gene of interest.
  • the G418 resistance gene codes for aminoglycoside phosphotransferase (APH) which enzymatically inactivates G418 (100-1000 ⁇ g/ ⁇ l) added to the culture medium. Only those cells expressing the APH gene will survive drug selection usually resulting in the expression of the second biologic gene as well.
  • APH aminoglycoside phosphotransferase
  • the hygromycin B phosphotransferase (HPH) gene codes for an enzyme which specifically modifies hygromycin toxin and inactivates it. Genes co-transfected with or contained on the same plasmid as the hygromycin B phosphotransferase gene will be preferentially expressed in the presence of hygromycin B at 50-200 ⁇ g/ml concentrations.
  • expression vectors examples include, but are not limited to, the commercially available pRC/CMV, pRC/RSV, and pCDNAlNEO (InVitrogen). Other suitable commercially available vectors include pBlast, pMono, or pVitro.
  • the pNUT expression vector which contains the cDNA of the mutant DHFR and the entire pUC 18 sequence including the polylinker, can be used. See, e.g., Aebischer, P., et al, Transplantation, 58, pp. 1275-1277 (1994); Baetge et al, PNAS, 83, pp. 5454-58 (1986).
  • the pNUT expression vector can be modified such that the DHFR coding sequence is replaced by the coding sequence for G418 or hygromycin drug resistance.
  • the SV40 promoter within the pNUT expression vector can also be replaced with any suitable constitutively expressed mammalian promoter, such as those discussed above.
  • any other suitable, commercially available expression vectors e.g., pcDNA family (Invitrogen), pBlast, pMono, pVitro, or pCpG-vitro (Invivogen)
  • pcDNA family Invitrogen
  • pBlast pMono
  • pVitro pVitro
  • pCpG-vitro Invivogen
  • Principal elements regulating expression are typically found in the expression cassette. These elements include the promoter, 5' untranslated region (5' UTR) and 3' untranslated region (3' UTR).
  • Other elements of a suitable expression vector may be critical to plasmid integration or expression but may not be readily apparent.
  • the skilled artisan will be able to design and construct suitable expression vectors for use in the claimed invention. The choice, design, and/or construction of a suitable vector is well within the routine level of skill in the art.
  • genes encoding the VEGFl and VEGF2 receptors have been cloned and their nucleotide sequences published. (GenBank Accession UOl 134 and AF063658). Other genes encoding the biologically active molecules useful in this invention that are not publicly available may be obtained using standard recombinant DNA methods such as PCR amplification, genomic and cDNA library screening with oligonucleotide probes. Any of the known genes coding for biologically active molecules may be employed in the methods of this invention.
  • the cell of choice is the ARPE- 19 cell line, a spontaneously arising continuous human retinal pigmented epithelial cell line.
  • suitable cells including by not limited to CHO cells, BHK cells, RPE (primary cells or immortalized cells), can also be used.
  • the choice of cell depends upon the intended application.
  • the encapsulated cells may be chosen for secretion of a particular VEGF receptor construct. Cells can also be employed which synthesize and secrete agonists, analogs, derivatives or fragments of the construct, which are active.
  • suitable cell types may also be genetically engineered to secrete any of the VEGF receptor constructs described herein.
  • the cell line should have as many of the following characteristics as possible: (1) the cells should be hardy under stringent conditions (the encapsulated cells should be functional in the avascular tissue cavities such as in the central nervous system or the eye, especially in the intra-ocular environment); (2) the cells should be able to be genetically modified (the desired therapeutic factors needed to be engineered into the cells); (3) the cells should have a relatively long life span (the cells should produce sufficient progenies to be banked, characterized, engineered, safety tested and clinical lot manufactured); (4) the cells should preferably be of human origin (which increases compatibility between the encapsulated cells and the host); (5) the cells should exhibit greater than 80% viability for a period of more than one month in vivo in device (which ensures long-term delivery); (6) the encapsulated cells should deliver an efficacious quantity of a useful biological product (which ensures effectiveness of the treatment); (7) the cells should have a low level of host immune reaction (which
  • the ARPE-19 cell line (see Dunn et al, 62 Exp. Eye Res. 155-69 (1996), Dunn et al, 39 Invest. Ophthalmol. Vis. Sci. 2744-9 (1998), Finnemann et al., 94 Proc. Natl. Acad. Sci. USA 12932-7 (1997), Handa et al., 66 Exp. Eye. 411-9 (1998), Holtkamp et al., 112 Clin. Exp. Immunol. 34-43 (1998), Maidji et al., 70 J. Virol. 8402-10 (1996); United States Patent No. 6,361,771) demonstrates all of the characteristics of a successful platform cell for an encapsulated cell-based delivery system.
  • ARPE-19 cell line is available from the American Type Culture Collection (ATCC Number CRL-2302).
  • ARPE-19 cells are normal retinal pigmented epithelial (RPE) cells and express the retinal pigmentary epithelial cell- specific markers CRALBP and RPE-65.
  • RPE-19 cells form stable monolayers, which exhibit morphological and functional polarity.
  • the devices of the invention When the devices of the invention are used, preferably between 10 2 and 10 8 engineered ARPE-19 cells, most preferably 5xlO 2 to 3xlO 5 ARPE-19 cells that have been genetically engineered to secrete one or more VEGF receptor constructs described herein are encapsulated in each device. Dosage may be controlled by implanting a fewer or greater number of capsules, preferably between 1 and 50 capsules per patient.
  • the devices described herein are capable of delivering between about 0.1 pg and 1000 ⁇ g of the soluble VEGF receptor construct(s) per eye per patient per day.
  • the cells to be isolated are replicating cells or cell lines adapted to growth in vitro, it is particularly advantageous to generate a cell bank of these cells.
  • a particular advantage of a cell bank is that it is a source of cells prepared from the same culture or batch of cells. That is, all cells originated from the same source of cells and have been exposed to the same conditions and stresses. Therefore, the vials can be treated as homogenous culture. In the transplantation context, this greatly facilitates the production of identical or replacement devices. It also allows simplified testing protocols, which insure that implanted cells are free of retroviruses and the like. It may also allow for parallel monitoring of vehicles in vivo and in vitro, thus allowing investigation of effects or factors unique to residence in vivo.
  • the terms "individual” or “recipient” or “host” are used interchangeably to refer to a human or an animal subject.
  • a “biologically active molecule” (“BAM”) is a substance that is capable of exerting a biologically useful effect upon the body of an individual in whom a device of the present invention is implanted.
  • BAM biologically active molecule
  • VEGF receptor constructs described herein are examples of BAMs.
  • capsule and “device” and “vehicle” are used interchangeably herein to refer to the ECT devices of the invention.
  • cells means cells in any form, including but not limited to cells retained in tissue, cell clusters, and individually isolated cells.
  • biocompatible vehicle means that the capsule or device or vehicle, upon implantation in an individual, does not elicit a detrimental host response sufficient to result in the rejection of the capsule or to render it inoperable, for example through degradation.
  • an “immunoisolatory capsule” or “immunoisolatory device” or “immunoisolatory vehicle” means that the capsule upon implantation into an individual, minimizes the deleterious effects of the host's immune system on the cells within its core.
  • long-term, stable expression of a biologically active molecule means the continued production of a biologically active molecule at a level sufficient to maintain its useful biological activity for periods greater than one month, preferably greater than three months and most preferably greater than six months. Implants of the devices and the contents thereof are able to retain functionality for greater than three months in vivo and in many cases for longer than a year.
  • the "semi-permeable" nature of the jacket membrane surrounding the core permits molecules produced by the cells ⁇ e.g., metabolites, nutrients and/or therapeutic substances) to diffuse from the device into the surrounding host eye tissue, but is sufficiently impermeable to protect the cells in the core from detrimental immunological attack by the host.
  • jacket nominal molecular weight cutoff (MWCO) values up to 1000 kD are contemplated.
  • the MWCO is between 50-700 kD.
  • the MWCO is between 70-300 kD. See, e.g., WO 92/19195.
  • the instant invention also relates to biocompatible, optionally immunoisolatory, devices for the delivery of one or more of the soluble VEGF receptors described herein to the eye.
  • Such devices contain a core containing living cells that produce or secrete the VEGF receptor and a biocompatible jacket surrounding the core, wherein the jacket has a molecular weight cut off (“MWCO") that allows the diffusion of the VEGF receptor into the eye and to the central nervous system, including the brain, ventricle, spinal cord.
  • MWCO molecular weight cut off
  • a variety of biocompatible capsules are suitable for delivery of molecules according to this invention.
  • Useful biocompatible polymer capsules comprise (a) a core which contains a cell or cells, either suspended in a liquid medium or immobilized within a biocompatible matrix, and (b) a surrounding jacket comprising a membrane which does not contain isolated cells, which is biocompatible, and permits diffusion of the cell-produced biologically active molecule into the eye.
  • a capsule having a liquid core comprising, e.g. , a nutrient medium, and optionally containing a source of additional factors to sustain cell viability and function.
  • the core of the devices of the invention can function as a reservoir for growth factors ⁇ e.g. , prolactin, or insulin- like growth factor 2), growth regulatory substances such as transforming growth factor ⁇ (TGF- ⁇ ) or the retinoblastoma gene protein or nutrient-transport enhancers ⁇ e.g., perfluorocarbons, which can enhance the concentration of dissolved oxygen in the core). Certain of these substances are also appropriate for inclusion in liquid media.
  • the instant devices can also be used as a reservoir for the controlled delivery of needed drugs or biotherapeutics.
  • the core contains a high concentration of the selected drug or biotherapeutic (alone or in combination with cells or tissues).
  • satellite vehicles containing substances which prepare or create a hospitable environment in the area of the body in which a device according to the invention is implanted can also be implanted into a recipient.
  • the devices containing immunoisolated cells are implanted in the region along with satellite vehicles releasing controlled amounts of, for example, a substance which down-modulates or inhibits an inflammatory response from the recipient (e.g. , anti-inflammatory steroids), or a substance which stimulates the ingrowth of capillary beds (e.g., an angiogenic factor).
  • the core may comprise a biocompatible matrix of a hydrogel or other biocompatible material (e.g. , extracellular matrix components) which stabilizes the position of the cells.
  • hydrogel herein refers to a three dimensional network of cross-linked hydrophilic polymers. The network is in the form of a gel, substantially composed of water, preferably gels being greater than 90% water. Compositions which form hydrogels fall into three classes. The first class carries a net negative charge (e.g., alginate). The second class carries a net positive charge (e.g., collagen and laminin). Examples of commercially available extracellular matrix components include Matrigel and Vitrogen . The third class is net neutral in charge (e.g., highly crosslinked polyethylene oxide, or polyvinylalcohol).
  • any suitable matrix or spacer may be employed within the core, including precipitated chitosan, synthetic polymers and polymer blends, microcarriers and the like, depending upon the growth characteristics of the cells to be encapsulated.
  • the capsule may have an internal scaffold.
  • the scaffold may prevent cells from aggregating and improve cellular distribution within the device.
  • the scaffold defines the microenvironment for the encapsulated cells and keeps the cells well distributed within the core.
  • the optimal internal scaffold for a particular device is highly dependent on the cell type to be used. In the absence of such a scaffold, adherent cells aggregate to form clusters.
  • the internal scaffold may be a yarn or a mesh.
  • the filaments used to form a yarn or mesh internal scaffold are formed of any suitable biocompatible, substantially non-degradable material. (See United States Patent Nos. 6,303,136 and 6,627,422, which are herein incorporated by reference).
  • the capsule of this invention will be similar to those described by PCT International patent applications WO 92/19195 or WO 95/05452, incorporated by reference; or U.S. Pat. Nos. 5,639,275; 5,653,975; 4,892,538; 5,156,844; 5,283,187; or 5,550,050, incorporated by reference.
  • Materials useful in forming yarns or woven meshes include any biocompatible polymers that are able to be formed into fibers such as, for example, acrylic, polyester, polyethylene, polypropylene, polyacrylonitrile, polyethylene terephthalate, nylon, polyamides, polyurethanes, polybutester, or natural fibers such as cotton, silk, chitin or carbon.
  • thermoplastic polymer thermoplastic elastomer, or other synthetic or natural material having fiber-forming properties may be inserted into a pre-fabricated hollow fiber membrane or a hollow cylinder formed from a flat membrane sheet.
  • silk, PET or nylon filaments used for suture materials or in the manufacture of vascular grafts are highly conducive to this type of application.
  • metal ribbon or wire may be used and woven.
  • Each of these filament materials has well-controlled surface and geometric properties, may be mass produced, and has a long history of implant use.
  • the filaments may be "texturized" to provide rough surfaces and "hand-holds" onto which cell projections may attach.
  • the filaments may be coated with extracellular matrix molecules or surface-treated (e.g. plasma irradiation) to enhance cellular adhesion to the filaments.
  • the filaments are twisted in bundles to form yarns of varying thickness and void volume.
  • Void volume is defined as the spaces existing between filaments.
  • the void volume in the yarn should vary between 20-95%, but is preferably between 50-95%.
  • the preferred void space between the filaments is between 20-200 ⁇ m, sufficient to allow the scaffold to be seeded with cells along the length of the yarn, and to allow the cells to attach to the filaments.
  • the preferred diameter of the filaments comprising the yarn is between 5-100 ⁇ m. These filaments should have sufficient mechanical strength to allow twisting into a bundle to comprise a yarn.
  • the filament cross-sectional shape can vary, with circular, rectangular, elliptical, triangular, and star-shaped cross-section being preferred.
  • the filaments or yarns can be woven into a mesh.
  • the mesh can be produced on a braider using carriers, similar to bobbins, containing monofilaments or multifilaments, which serve to feed either the yarn or filaments into the mesh during weaving.
  • the number of carriers is adjustable and may be wound with the same filaments or a combination of filaments with different compositions and structures.
  • the angle of the braid is controlled by the rotational speed of the carriers and the production speed.
  • a mandrel is used to produce a hollow tube of mesh.
  • the braid is constructed as a single layer, in other embodiments it is a multi-layered structure.
  • the tensile strength of the braid is the linear summation of the tensile strengths of the individual filaments.
  • a tubular braid is constructed.
  • the braid can be inserted into a hollow fiber membrane upon which the cells are seeded.
  • the cells can be allowed to infiltrate the wall of the mesh tube to maximize the surface area available for cell attachment.
  • the braid serves both as a cell scaffold matrix and as an inner support for the device. The increase in tensile strength for the braid- supported device is significantly higher than in alternative approaches.
  • the capsules are preferably immunoisolatory.
  • Components of the biocompatible material may include a surrounding semipermeable membrane and the internal cell- supporting scaffolding.
  • the transformed cells are preferably seeded onto the scaffolding, which is encapsulated by the permselective membrane, which is described above.
  • bonded fiber structures can be used for cell implantation. (See U.S. Pat. No. 5,512,600, incorporated by reference).
  • Biodegradable polymers include those comprised of poly(lactic acid) PLA, poly(lactic-coglycolic acid) PLGA, and poly(glycolic acid) PGA and their equivalents.
  • Foam scaffolds have been used to provide surfaces onto which transplanted cells may adhere (PCT International patent application Ser. No. 98/05304, incorporated by reference).
  • Woven mesh tubes have been used as vascular grafts (PCT International patent application WO 99/52573, incorporated by reference).
  • the core can be composed of an immobilizing matrix formed from a hydrogel, which stabilizes the position of the cells.
  • a hydrogel is a 3-dimensional network of cross-linked hydrophilic polymers in the form of a gel, substantially composed of water.
  • the surrounding semipermeable membrane can be used to manufacture the surrounding semipermeable membrane, including polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof.
  • the surrounding semipermeable membrane is a biocompatible semipermeable hollow fiber membrane.
  • Such membranes, and methods of making them are disclosed by U.S. Pat. Nos.
  • the surrounding semipermeable membrane is formed from a polyether sulfone hollow fiber, such as those described by U.S. Pat. No. 4,976,859 or U.S. Pat. No. 4,968,733, incorporated by reference.
  • An alternate surrounding semipermeable membrane material is poly sulfone.
  • the capsule can be any configuration appropriate for maintaining biological activity and providing access for delivery of the product or function, including for example, cylindrical, rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule can be coiled or wrapped into a mesh- like or nested structure. If the capsule is to be retrieved after it is implanted, configurations which tend to lead to migration of the capsules from the site of implantation, such as spherical capsules small enough to travel in the recipient host's blood vessels, are not preferred. Certain shapes, such as rectangles, patches, disks, cylinders, and flat sheets offer greater structural integrity and are preferable where retrieval is desired.
  • the device has a tether that aids in maintaining device placement during implant, and aids in retrieval.
  • a tether may have any suitable shape that is adapted to secure the capsule in place.
  • the suture may be a loop, a disk, or a suture.
  • the tether is shaped like an eyelet, so that suture may be used to secure the tether (and thus the device) to the sclera, or other suitable ocular structure.
  • the tether is continuous with the capsule at one end, and forms a pre-threaded suture needle at the other end.
  • the tether is an anchor loop that is adapted for anchoring the capsule to an ocular structure.
  • the tether may be constructed of a shape memory metal and/or any other suitable medical grade material known in the art.
  • the fiber will have an inside diameter of less than 1000 microns, preferably less than 750 microns.
  • the capsule will preferably be between 0.4 cm to 1.5 cm in length, most preferably between 0.4 to 1.0 cm in length. Longer devices may be accommodated in the eye, however, a curved or arcuate shape may be required for secure and appropriate placement.
  • the hollow fiber configuration is preferred for intraocular placement.
  • a hollow fiber configuration (with dimensions substantially as above) or a flat sheet configuration is contemplated.
  • the upper limit contemplated for a flat sheet is approximately 5 mm x 5 mm--assuming a square shape. Other shapes with approximately the same surface area are also contemplated.
  • Microdevices manufactured for delivery of soluble VEGFR may have a length of between 1 and 2.5 millimeters, with an inner diameter of between 300 and 500 microns and an outer diameter of between 450 and 700 microns.
  • an inner scaffolding containing between 10 and 60 monofilaments of PET can be utilized.
  • the molecular weight cut off ranges from these micronized devices are between 100 and 2000 kDa.
  • passive diffusion of a 70 kDa dextran ranges between 100 and 2000 x 10 "10 cm 2 /s.
  • any suitable membrane material(s) described herein may be used in these micronized devices, two preferred materials are polyethersulfone and/or polysulfone.
  • microdevices can be manufactured with and without anchors made of a suitable material (e.g., nitinol).
  • a suitable material e.g., nitinol
  • the open membrane contemplated for use with the VEGFR constructs described herein has an upper hydraulic permeability value of approximately 3000 mls/min/m 2 /mmHg.
  • a more "immunoisolatory" membrane will be used.
  • the hydraulic permeability will typically be in the range of 0.4-170 mls/min/m 2 /mmHg, for example, 0.5-100 mls/min/m 2 /mmHg, preferably in the range of 25 to 70 mls/min/m 2 /mmHg.
  • the glucose mass transfer coefficient of the capsule defined, measured and calculated as described by
  • the surrounding or peripheral region (jacket), which surrounds the core of the instant devices can be permselective, biocompatible, and/or immunoisolatory. It is produced in such a manner that it is free of isolated cells, and completely surrounds (i.e., isolates) the core, thereby preventing contact between any cells in the core and the recipient's body.
  • Biocompatible semi-permeable hollow fiber membranes, and methods of making them are disclosed in U.S. Pat. Nos. 5,284,761 and 5,158,881 (See also, WO 95/05452), each of which incorporated herein by reference in its entirety.
  • the capsule jacket can be formed from a poly ether sulfone hollow fiber, such as those described in U.S. Pat. Nos. 4,976,859 and 4,968,733, and 5,762,798, each incorporated herein by reference.
  • the jacket is formed in such a manner that it has a molecular weight cut off (“MWCO") range appropriate both to the type and extent of immunological reaction anticipated to be encountered after the device is implanted and to the molecular size of the largest substance whose passage into and out of the device into the eye is desirable.
  • MWCO molecular weight cut off
  • the type and extent of immunological attacks which may be mounted by the recipient following implantation of the device depend in part upon the type(s) of moiety isolated within it and in part upon the identity of the recipient (i.e., how closely the recipient is genetically related to the source of the BAM).
  • immunological rejection may proceed largely through cell-mediated attack by the recipient's immune cells against the implanted cells.
  • molecular attack through assembly of the recipient's cytolytic complement attack complex may predominate, as well as the antibody interaction with complement.
  • the jacket allows passage into the eye of substances up to a predetermined size, but prevents the passage of larger substances. More specifically, the surrounding or peripheral region is produced in such a manner that it has pores or voids of a predetermined range of sizes, and, as a result, the device is permselective.
  • the MWCO of the surrounding jacket must be sufficiently low to prevent access of the substances required to carry out immunological attacks to the core, yet sufficiently high to allow delivery of the VEGF receptor to the recipient's eye.
  • the MWCO of the biocompatible jacket of the devices of the instant invention is from about 1 kD to about 150 kD. However, if delivery of a non-truncated receptor is desired, an open membrane with a MWCO greater than 200 kD should be used.
  • biocompatible refers collectively to both the device and its contents. Specifically, it refers to the capability of the implanted intact device and its contents to avoid the detrimental effects of the body's various protective systems and to remain functional for a significant period of time.
  • protective systems refers to the types of immunological attack which can be mounted by the immune system of an individual in whom the instant vehicle is implanted, and to other rejection mechanisms, such as the fibrotic response, foreign body response and other types of inflammatory response which can be induced by the presence of a foreign object in the individuals' body.
  • biocompatible In addition to the avoidance of protective responses from the immune system or foreign body fibrotic response, the term "biocompatible”, as used herein, also implies that no specific undesirable cytotoxic or systemic effects are caused by the vehicle and its contents such as those that would interfere with the desired functioning of the vehicle or its contents.
  • the external surface of the device can be selected or designed in such a manner that it is particularly suitable for implantation at a selected site.
  • the external surface can be smooth, stippled or rough, depending on whether attachment by cells of the surrounding tissue is desirable.
  • the shape or configuration can also be selected or designed to be particularly appropriate for the implantation site chosen.
  • the biocompatibility of the surrounding or peripheral region (jacket) of the device is produced by a combination of factors. Important for biocompatibility and continued functionality are device morphology, hydrophobicity and the absence of undesirable substances either on the surface of, or leachable from, the device itself. Thus, brush surfaces, folds, interlayers or other shapes or structures eliciting a foreign body response are avoided. Moreover, the device-forming materials are sufficiently pure to insure that unwanted substances do not leach out from the device materials themselves. Additionally, following device preparation, the treatment of the external surface of the device with fluids or materials (e.g. serum) which may adhere to or be absorbed by the device and subsequently impair device biocompatibility is avoided.
  • fluids or materials e.g. serum
  • the materials used to form the device jacket are substances selected based upon their ability to be compatible with, and accepted by, the tissues of the recipient of the implanted device. Substances are used which are not harmful to the recipient or to the isolated cells.
  • Preferred substances include polymer materials, i.e., thermoplastic polymers. Particularly preferred thermoplastic polymer substances are those which are modestly hydrophobic, i.e. those having a solubility parameter as defined in Brandrup J., et al. Polymer Handbook 3rd Ed., John Wiley & Sons, NY (1989), between 8 and 15, or more preferably, between 9 and 14 (Joules/m 3 ) 1/2 .
  • the polymer substances are chosen to have a solubility parameter low enough so that they are soluble in organic solvents and still high enough so that they will partition to form a proper membrane. Such polymer substances should be substantially free of labile nucleophilic moieties and be highly resistant to oxidants and enzymes even in the absence of stabilizing agents.
  • the period of residence in vivo which is contemplated for the particular vehicle must also be considered: substances must be chosen which are adequately stable when exposed to physiological conditions and stresses. Many thermoplastics are known which are sufficiently stable, even for extended periods of residence in vivo, such as periods in excess of one or two years. The choice of materials used to construct the device is determined by a number of factors as described in detail in Dionne WO 92/19195, herein incorporated by reference.
  • Polymeric membranes forming the device and the growth surfaces therein may include polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, polymethylmethacrylate, polyvinyldifluoride, polyolefms, cellulose acetates, cellulose nitrates, polysulfones, polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof.
  • polyacrylates including acrylic copolymers
  • polyvinylidenes including acrylic copolymers
  • polyurethanes polystyrenes
  • polyamides polymethylmethacrylate
  • polyvinyldifluoride polyolefms
  • cellulose acetates cellulose nitrates
  • polysulfones polyphosphazenes
  • a preferred membrane casting solution comprises a either polysulfone dissolved in the water-miscible solvent dimethylacetamide (DMACSO) or polyethersulfone dissolved in the water-miscible solvent butyrolactone.
  • This casting solution can optionally comprise hydrophilic or hydrophobic additives which affect the permeability characteristics of the finished membrane.
  • a preferred hydrophilic additive for the polysulfone or polyethersulfone is polyvinylpyrrolidone (PVP).
  • suitable polymers comprise polyacrylonitrile (PAN), polymethylmethacrylate (PMMA), polyvinyldifluoride (PVDF), polyethylene oxide, polyolefms (e.g., polyisobutylene or polypropylene), polyacrylonitrile/polyvinyl chloride (PAN/PVC), and/or cellulose derivatives (e.g., cellulose acetate or cellulose butyrate).
  • PAN polyacrylonitrile
  • PMMA polymethylmethacrylate
  • PVDF polyvinyldifluoride
  • PAN/PVC polyacrylonitrile/polyvinyl chloride
  • cellulose derivatives e.g., cellulose acetate or cellulose butyrate
  • substances used in preparing the biocompatible jacket of the device are either free of leachable pyrogenic or otherwise harmful, irritating, or immunogenic substances or are exhaustively purified to remove such harmful substances. Thereafter, and throughout the manufacture and maintenance of the device prior to implantation, great care is taken to prevent the adulteration or contamination of the device or jacket with substances, which would adversely affect its biocompatibility.
  • the exterior configuration of the device is formed in such a manner that it provides an optimal interface with the eye of the recipient after implantation.
  • Certain device geometries have also been found to specifically elicit foreign body fibrotic responses and should be avoided.
  • devices should not contain structures having interlay ers such as brush surfaces or folds.
  • opposing vehicle surfaces or edges either from the same or adjacent vehicles should be at least 1 mm apart, preferably greater than 2 mm and most preferably greater than 5 mm.
  • Preferred embodiments include cylinders having an outer diameter of between about 200 and 350 ⁇ m and a length between about 0.4 and 6 mm.
  • the core of the devices of the invention has a volume of approximately 2.5 ⁇ l.
  • micronized devices having a core volume of less than 0.5 ⁇ l (e.g., about 0.3 ⁇ l).
  • the surrounding jacket of the biocompatible devices can optionally include substances which decrease or deter local inflammatory response to the implanted vehicle and/or generate or foster a suitable local environment for the implanted cells or tissues.
  • substances which decrease or deter local inflammatory response to the implanted vehicle and/or generate or foster a suitable local environment for the implanted cells or tissues For example antibodies to one or more mediators of the immune response could be included. Available potentially useful antibodies such as antibodies to the lymphokines tumor necrosis factor (TNF), and to interferons (IFN) can be included in the matrix precursor solution. Similarly, an anti-inflammatory steroid can be included. See Christenson, L., et al., J.
  • the jacket of the present device is immunoisolatory. That is, it protects cells in the core of the device from the immune system of the individual in whom the device is implanted. It does so (1) by preventing harmful substances of the individual's body from entering the core, (2) by minimizing contact between the individual and inflammatory, antigenic, or otherwise harmful materials which may be present in the core and (3) by providing a spatial and physical barrier sufficient to prevent immunological contact between the isolated moiety and detrimental portions of the individual's immune system.
  • the external jacket may be either an ultrafiltration membrane or a microporous membrane.
  • ultrafiltration membranes are those having a pore size range of from about 1 to about 100 nanometers while a microporous membrane has a range of between about 0.05 to about 10 microns.
  • the thickness of this physical barrier can vary, but it will always be sufficiently thick to prevent direct contact between the cells and/or substances on either side of the barrier.
  • the thickness of this region generally ranges between 5 and 200 microns; thicknesses of 10 to 100 microns are preferred, and thickness of 20 to 50 or 20 to 75 microns are particularly preferred.
  • Types of immunological attack which can be prevented or minimized by the use of the instant device include attack by macrophages, neutrophils, cellular immune responses (e.g. natural killer cells and antibody-dependent T cell-mediated cytoloysis (ADCC)), and humoral response (e.g. antibody-dependent complement mediated cyto lysis).
  • cellular immune responses e.g. natural killer cells and antibody-dependent T cell-mediated cytoloysis (ADCC)
  • humoral response e.g. antibody-dependent complement mediated cyto lysis.
  • the capsule jacket may be manufactured from various polymers and polymer blends including polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof.
  • polyacrylates including acrylic copolymers
  • polyvinylidenes including acrylic copolymers
  • polyvinyl chloride copolymers polyurethanes
  • polystyrenes polyamides
  • cellulose acetates cellulose nitrates
  • polysulfones including polyether sulfones
  • polyphosphazenes polyacrylonitriles
  • poly(acrylonitrile/covinyl chloride)
  • Capsules formed from a polyether sulfone (PES) fiber such as those described in U.S. Pat. Nos. 4,976,859 and 4,968,733, incorporated herein by reference, may also be used.
  • PES polyether sulfone
  • Tl Type 2
  • T 1/2 Type 4
  • Such membranes are described, e.g., in Lacy et al., "Maintenance Of Normoglycemia In Diabetic Mice By Subcutaneous Xenografts Of Encapsulated Islets", Science, 254, pp. 1782-84 (1991), Dionne et al., WO 92/19195 and Baetge, WO 95/05452.
  • a smooth outer surface morphology is preferred.
  • capsule jackets with permselective, immunoisolatory membranes are preferable for sites that are not immunologically privileged.
  • microporous membranes or permselective membranes may be suitable for immunologically privileged sites.
  • capsules made from the PES or PS membranes are preferred. Any suitable method of sealing the capsules know in the art may be used, including the employment of polymer adhesives and/or crimping, knotting and heat sealing.
  • any suitable "dry” sealing method can also be used. In such methods, a substantially non-porous fitting is provided through which the cell-containing solution is introduced. Subsequent to filling, the capsule is sealed.
  • Such methods are described in, e.g. , United States Patent Nos. 5,653,688; 5,713,887; 5,738,673; 6,653,687; 5,932,460; and 6,123,700, which are herein incorporated by reference.
  • VEGF receptors may be co-delivered in addition to the VEGF receptors described herein.
  • Co-delivery can be accomplished in a number of ways. First, cells may be transfected with separate constructs containing the genes encoding the described molecules. Second, cells may be transfected with a single construct containing two or more genes as well as the necessary control elements. Third, two or more separately engineered cell lines can be either co-encapsulated or more than one device can be implanted at the site of interest.
  • Multiple gene expression from a single transcript is preferred over expression from multiple transcription units. See, e.g., Macejak, Nature, 353, pp. 90-94 (1991); WO 94/24870; Mountford and Smith, Trends Genet., 11, pp. 179-84 (1995); Dirks et al, Gene, 128, pp. 247-49 (1993); Martinez- S alas et al., J. Virology, 67, pp. 3748-55 (1993) and Mountford et al., Proc. Natl. Acad. Sci. USA, 91, pp. 4303-07 (1994).
  • BAMs may be preferable to deliver BAMs to two different sites in the eye concurrently.
  • a neurotrophic factor to the vitreous to supply the neural retina (ganglion cells to the RPE) and to deliver an anti- angiogenic factor (such as one or more of the VEGF receptors of the invention) via the sub- Tenon's space to supply the choroidal vasculature.
  • This invention also contemplates use of different cell types during the course of the treatment regime.
  • a patient may be implanted with a capsule device containing a first cell type (e.g., BHK cells). If after time, the patient develops an immune response to that cell type, the capsule can be retrieved, or explanted, and a second capsule can be implanted containing a second cell type (e.g., CHO cells).
  • a first cell type e.g., BHK cells
  • a second capsule e.g., CHO cells
  • the methods and devices of this invention are intended for use in a primate, preferably human host, recipient, patient, subject or individual.
  • a primate preferably human host, recipient, patient, subject or individual.
  • implantation sites include, but are not limited to, the aqueous and vitreous humors of the eye, the periocular space, the anterior chamber, and/or the Subtenon's capsule.
  • the type and extent of immunological response by the recipient to the implanted device will be influenced by the relationship of the recipient to the isolated cells within the core. For example, if core contains syngeneic cells, these will not cause a vigorous immunological reaction, unless the recipient suffers from an autoimmunity with respect to the particular cell or tissue type within the device. Syngeneic cells or tissue are rarely available. In many cases, allogeneic or xenogeneic cells or tissue (i.e., from donors of the same species as, or from a different species than, the prospective recipient) may be available.
  • the use of immunoisolatory devices allows the implantation of allogeneic or xenogeneic cells or tissue, without a concomitant need to immunosuppress the recipient. Use of immunoisolatory capsules also allows the use of unmatched cells (allographs). Therefore, the instant device makes it possible to treat many more individuals than can be treated by conventional transplantation techniques.
  • the type and vigor of an immune response to xenografted tissue is expected to differ from the response encountered when syngeneic or allogeneic tissue is implanted into a recipient. This rejection may proceed primarily by cell-mediated, or by complement-mediated attack.
  • the exclusion of IgG from the core of the vehicle is not the touchstone of immunoprotection, because in most cases IgG alone is insufficient to produce cyto lysis of the target cells or tissues.
  • immunoisolatory devices it is possible to deliver needed high molecular weight products or to provide metabolic functions pertaining to high molecular weight substances, provided that critical substances necessary to the mediation of immunological attack are excluded from the immunoisolatory capsule.
  • These substances may comprise the complement attack complex component CIq, or they may comprise phagocytic or cytotoxic cells.
  • Use of immunoisolatory capsules provides a protective barrier between these harmful substances and the isolated cells.
  • the devices of the present invention are macrocapsules, those skilled in the art will recognize that microcapsules such as, for example those described in Rha, Lim, and Sun may also be used. (See, Rha, C. K. et al, U.S. Pat. No. 4,744,933; Methods in Enzymology 137, pp. 575-579 (1988); U.S. Patent No. 4,652,833; U.S. Patent No. 4,409,331).
  • microcapsules differ from macrocapsules by (1) the complete exclusion of cells from the outer layer of the device, and (2) the thickness of the outer layer of the device.
  • microcapsules typically have a volume on the order of 1 ⁇ l and contain fewer than 10 4 cells. More specifically, microencapsulation encapsulates approximately 500-50,000 cells, generally, per capsule.
  • Capsules with a lower MWCO may be used to further prevent interaction of molecules of the patient's immune system with the encapsulated cells.
  • any of the devices used in accordance with the methods described herein must provide, in at least one dimension, sufficiently close proximity of any isolated cells in the core to the surrounding eye tissues of the recipient in order to maintain the viability and function of the isolated cells.
  • the diffusional limitations of the materials used to form the device do not in all cases solely prescribe its configurational limits.
  • Certain additives can be used which alter or enhance the diffusional properties, or nutrient or oxygen transport properties, of the basic vehicle.
  • the internal medium of the core can be supplemented with oxygen-saturated perfluorocarbons, thus reducing the needs for immediate contact with blood-borne oxygen.
  • the thickness of the device jacket should be sufficient to prevent an immunoresponse by the patient to the presence of the devices.
  • the devices preferably have a minimum thickness of 1 ⁇ m or more and are free of the cells.
  • reinforcing structural elements can also be incorporated into the devices.
  • these structural elements can be made in such a fashion that they are impermeable and are appropriately configured to allow tethering or suturing of the device to the eye tissues of the recipient.
  • these elements can act to securely seal the jacket (e.g., at the ends of the cylinder), thereby completing isolation of the core materials (e.g., a molded thermoplastic clip).
  • the device of the present invention is of a sufficient size and durability for complete retrieval after implantation.
  • One preferred device of the present invention has a core of a volume of approximately l-3uL.
  • the internal geometry of micronized devices has a volume of approximately 0.05-0.1 uL.
  • At least one additional BAM can also be delivered from the device to the eye.
  • the at least one additional BAM can be provided from a cellular or a noncellular source.
  • the additional BAM(s) may be encapsulated in, dispersed within, or attached to one or more components of the cell system including, but not limited to: (a) sealant; (b) scaffold; (c) jacket membrane; (d) tether anchor; and/or (e) core media.
  • co-delivery of the BAM from a noncellular source may occur from the same device as the BAM from the cellular source.
  • the least one additional biologically active molecule can be a nucleic acid, a nucleic acid fragment, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an organic molecule, an inorganic molecule, a therapeutic agent, or any combinations thereof.
  • the therapeutic agents may be an anti-angiogenic drug, a steroidal and nonsteroidal anti-inflammatory drug, an anti-mitotic drug, an anti-tumor drug, an anti-parasitic drug, an IOP reducer, a peptide drug, and/or any other biologically active molecule drugs approved for ophthalmologic use.
  • Suitable excipients include, but are not limited to, any non-degradable or biodegradable polymers, hydrogels, solubility enhancers, hydrophobic molecules, proteins, salts, or other complexing agents approved for formulations.
  • Non-cellular dosages can be varied by any suitable method known in the art such as varying the concentration of the therapeutic agent, and/or the number of devices per eye, and/or modifying the composition of the encapsulating excipient.
  • Cellular dosage can be varied by changing (1) the number of cells per device, (2) the number of devices per eye, and/or (3) the level of BAM production per cell.
  • Cellular production can be varied by changing, for example, the copy number of the gene for the BAM in the transduced cell, or the efficiency of the promoter driving expression of the BAM.
  • Suitable dosages from non- cellular sources may range from about 1 pg to about 1000 ng per day.
  • the instant invention also relates to methods for making the macrocapsular devices described herein.
  • Devices may be formed by any suitable method known in the art. (See, e.g., United States Patent Nos. 6,361,771; 5,639,275; 5,653,975; 4,892,538; 5,156,844; 5,283,138; and 5,550,050, each of which is incorporated herein by reference).
  • Membranes used can also be tailored to control the diffusion of molecules, such as soluble VEGF receptor, based on their molecular weight. (See Lysaght et al., 56 J. Cell Biochem. 196 (1996), Colton, 14 Trends Biotechnol. 158 (1996)).
  • cells can be transplanted into a host without immune rejection, either with or without use of immunosuppressive drugs.
  • the capsule can be made from a biocompatible material that, after implantation in a host, does not elicit a detrimental host response sufficient to result in the rejection of the capsule or to render it inoperable, for example through degradation.
  • the biocompatible material is relatively impermeable to large molecules, such as components of the host's immune system, but is permeable to small molecules, such as insulin, growth factors, and nutrients, while allowing metabolic waste to be removed.
  • a variety of biocompatible materials are suitable for delivery of growth factors by the composition of the invention. Numerous biocompatible materials are known, having various outer surface morphologies and other mechanical and structural characteristics. If a device with a jacket of thermoplastic or polymer membrane is desired, the pore size range and distribution can be determined by varying the solids content of the solution of precursor material (the casting solution), the chemical composition of the water-miscible solvent, or optionally including a hydrophilic or hydrophobic additive to the casting solution, as taught by U.S. Pat. No. 3,615,024. The pore size may also be adjusted by varying the hydrophobicity of the coagulant and/or of the bath.
  • the casting solution will comprise a polar organic solvent containing a dissolved, water-insoluble polymer or copolymer.
  • This polymer or copolymer precipitates upon contact with a solvent-miscible aqueous phase, forming a permselective membrane at the site of interface.
  • the size of pores in the membrane depends upon the rate of diffusion of the aqueous phase into the solvent phase; the hydrophilic or hydrophobic additives affect pore size by altering this rate of diffusion.
  • the remainder of the polymer or copolymer is precipitated to form a trabecular support which confers mechanical strength to the finished device.
  • the external surface of the device is similarly determined by the conditions under which the dissolved polymer or copolymer is precipitated (i.e., exposed to the air, which generates an open, trabecular or sponge-like outer skin, immersed in an aqueous precipitation bath, which results in a smooth permselective membrane bilayer, or exposed to air saturated with water vapor, which results in an intermediate structure).
  • the surface texture of the device is dependent in part on whether the extrusion nozzle is positioned above, or immersed in, the bath: if the nozzle is placed above the surface of the bath a roughened outer skin will be formed, whereas if the nozzle is immersed in the bath a smooth external surface is formed.
  • the surrounding or peripheral matrix or membrane can be preformed, filled with the materials which will form the core (for instance, using a syringe), and subsequently sealed in such a manner that the core materials are completely enclosed.
  • the device can then be exposed to conditions which bring about the formation of a core matrix if a matrix precursor material is present in the core.
  • the devices of the invention can provide for the implantation of diverse cell or tissue types, including fully-differentiated, anchorage-dependent, fetal or neonatal, or transformed, anchorage-independent cells or tissue.
  • the cells to be isolated are prepared either from a donor (i.e., primary cells or tissues, including adult, neonatal, and fetal cells or tissues) or from cells which replicate in vitro (i.e., immortalized cells or cell lines, including genetically modified cells). In all cases, a sufficient quantity of cells to produce effective levels of the needed product or to supply an effective level of the needed metabolic function is prepared, generally under sterile conditions, and maintained appropriately (e.g.
  • the ECT devices of the invention are of a shape which tends to reduce the distance between the center of the device and the nearest portion of the jacket for purposes of permitting easy access of nutrients from the patient into the cell or of entry of the patient's proteins into the cell to be acted upon by the cell to provide a metabolic function.
  • a non-spherical shape such as a cylinder, is preferred.
  • the diffusion of critical nutrients and metabolic requirements into the cells as well as diffusion of metabolites away from the cell are critical to the continued viability of the cells.
  • the neighboring cells are able to phagocytize the dying cells and use the debris as an energy source.
  • the metabolic requirements met by diffusion of substances into the device is the requirement for oxygen.
  • the oxygen requirements of the specific cells must be determined for the cell of choice. See Methods and references for determination of oxygen metabolism are given in Wilson D. F. et al, J. Biol. Chem., 263, pp. 2712-2718, (1988).
  • the second factor cell division
  • the geometry and size of the device will be chosen so that complete filling of the device core will not lead to deprivation of critical nutrients due to diffusional limitations.
  • the third factor viscosity of core materials
  • cells in densities occupying up to 70% of the device volume can be viable, but cell solutions in this concentration range would have considerable viscosity. Introduction of cells in a very viscous solution into the device could be prohibitively difficult.
  • BAM e.g., the VEGF receptor
  • Devices can be constructed and analyzed using model systems in order to allow the determination of the efficacy of the vehicle on a per cell or unit volume basis.
  • the actual device size for implantation will then be determined by the amount of biological activity required for the particular application.
  • the number of devices and device size should be sufficient to produce a therapeutic effect upon implantation and is determined by the amount of biological activity required for the particular application.
  • standard dosage considerations and criteria known to the art will be used to determine the amount of secretory substance required. Factors to be considered include the size and weight of the recipient; the productivity or functional level of the cells; and, where appropriate, the normal productivity or metabolic activity of the organ or tissue whose function is being replaced or augmented. It is also important to consider that a fraction of the cells may not survive the immunoisolation and implantation procedures.
  • Encapsulated cell therapy is based on the concept of isolating cells from the recipient host's immune system by surrounding the cells with a semipermeable biocompatible material before implantation within the host.
  • the invention includes a device in which genetically engineered ARPE- 19 cells are encapsulated in an immunoisolatory capsule, which, upon implantation into a recipient host, minimizes the deleterious effects of the host's immune system on the ARPE- 19 cells in the core of the device.
  • ARPE- 19 cells are immunoisolated from the host by enclosing them within implantable polymeric capsules formed by a microporous membrane. This approach prevents the cell-to-cell contact between the host and implanted tissues, thereby eliminating antigen recognition through direct presentation.
  • the VEGF receptors described herein can be delivered intraocularly (e.g. , in the anterior chamber and the vitreous cavity) or periocularly (e.g., within or beneath Tenon's capsule), or both.
  • the devices of the invention may also be used to provide controlled and sustained release of the receptors to treat various ophthalmic disorders, ophthalmic diseases and/or diseases which have ocular effects.
  • Intraocular preferably in the vitreous or per ocular (preferably in the sub-Tenon's space or region) delivery of an anti-angiogenic factor, such as any of the soluble VEGF receptors described herein, in a dosage range of 0.1 pg and 1000 ⁇ g (e.g., between 0.1 pg and 500 ⁇ g; between 0.1 pg and 250 ⁇ g; between 0.1 pg and 100 ⁇ g; between 0.1 pg and 50 ⁇ g; between 0.1 pg and 25 ⁇ g; between 0.1 pg and 10 ⁇ g; between 0.1 pg and 5 ⁇ g; between 0.1 pg and 100 ng; between 0.1 pg and 50 ng; between 0.1 pg and 25 ng; between 0.1 pg and 10 ng; or between 0.1 pg and 5 ng) per eye per patient per day is contemplated.
  • an anti-angiogenic factor such as any of the soluble VEGF receptors described herein
  • Ophthalmic disorders that may be treated by various embodiments of the present invention include, but are not limited to diabetic retinopathies, diabetic macular edema, proliferative retinopathies, retinal vascular diseases, vascular anomalies, age-related macular degeneration and other acquired disorders, endophthalmitis, infectious diseases, inflammatory but non-infectious diseases, AIDS-related disorders, ocular ischemia syndrome, pregnancy-related disorders, peripheral retinal degenerations, retinal degenerations, toxic retinopathies, retinal tumors, choroidal tumors, choroidal disorders, vitreous disorders, retinal detachment and proliferative vitreoretinopathy, non-penetrating trauma, penetrating trauma, post-cataract complications, and inflammatory optic neuropathies.
  • age-related macular degeneration includes, but is not limited to, wet and dry age-related macular degeneration, exudative age- related macular degeneration, and myopic degeneration.
  • the disorder to be treated is the wet form of age- related macular degeneration or diabetic retinopathy.
  • the present invention may also be useful for the treatment of ocular neovascularization, a condition associated with many ocular diseases and disorders.
  • retinal ischemia-associated ocular neovascularization is a major cause of blindness in diabetes and many other diseases.
  • the cell lines and devices of the present invention may also be used to treat ocular symptoms resulting from diseases or conditions that have both ocular and non-ocular symptoms. Some examples include cytomegalovirus retinitis in AIDS as well as other conditions and vitreous disorders; hypertensive changes in the retina as a result of pregnancy; and ocular effects of various infectious diseases such as tuberculosis, syphilis, lyme disease, parasitic disease, toxocara canis, ophthalmonyiasis, cyst cercosis and fungal infections.
  • the devices and cell lines may also be used to treat conditions relating to other intraocular neovascularization-based diseases.
  • neovascularization can occur in diseases such as diabetic retinopathy, central retinal vein occlusion and, possibly, age-related macular degeneration.
  • Corneal neovascularization is a major problem because it interferes with vision and predisposes patients to corneal graft failure. A majority of severe visual loss is associated with disorders that result in ocular neovascularization.
  • the invention also relates to methods and the delivery of soluble VEGF receptors in order to treat cell proliferative disorders, such as, hematologic disorders, atherosclerosis, inflammation, increased vascular permeability, and malignancy.
  • the VEGF receptors can be delivered to the eye directly, which reduces or minimizes unwanted peripheral side effects and very small doses of the receptor (i.e., nanogram or low microgram quantities rather than milligrams) can be delivered compared with topical applications, thereby also potentially lessening side effects.
  • very small doses of the receptor i.e., nanogram or low microgram quantities rather than milligrams
  • these techniques should be superior to injection delivery of VEGF receptor, where the dose fluctuates greatly between injections and the receptor is continuously degraded but not continuously replenished.
  • Living cells and cell lines genetically engineered to secrete the soluble VEGF receptors of the invention can be encapsulated in the device of the invention and surgically inserted (under retrobulbar anesthesia) into any appropriate anatomical structure of the eye.
  • the devices can be surgically inserted into the vitreous of the eye, where they are preferably tethered to the sclera to aid in removal. Devices can remain in the vitreous as long as necessary to achieve the desired prophylaxis or therapy.
  • the desired therapy may include promotion of neuron or photoreceptor survival or repair, or inhibition and/or reversal of retinal or choroidal neovascularization, as well as inhibition of uveal, retinal and optic nerve inflammation.
  • the VEGF receptor may be delivered to the retina or the retinal pigment epithelium (RPE).
  • cell-loaded devices are implanted periocularly, within or beneath the space known as Tenon's capsule, which is less invasive than implantation into the vitreous. Therefore, complications such as vitreal hemorrhage and/or retinal detachment are potentially eliminated.
  • This route of administration also permits delivery of the soluble VEGF receptors described herein to the RPE or the retina.
  • Periocular implantation is especially preferred for treating choroidal neovascularization and inflammation of the optic nerve and uveal tract. In general, delivery from periocular implantation sites will permit circulation of the soluble VEGF receptors to the choroidal vasculature, retinal vasculature, and the optic nerve.
  • soluble VEGF receptors of the invention Delivery of anti-angiogenic factors, such as the soluble VEGF receptors of the invention, directly to the choroidal vasculature (periocularly) or to the vitreous (intraocularly) using the devices and methods described herein may reduce or alleviate the problems associated with prior art treatment methods and devices and may permit the treatment of poorly defined or occult choroidal neovascularization as well as provide a way of reducing or preventing recurrent choroidal neovascularization via adjunctive or maintenance therapy.
  • anti-angiogenic factors such as the soluble VEGF receptors of the invention
  • Implantation of the biocompatible devices of the invention is performed under sterile conditions.
  • the device can be implanted using a syringe or any other method known to those skilled in the art.
  • the device is implanted at a site in the recipient's body which will allow appropriate delivery of the secreted product or function to the recipient and of nutrients to the implanted cells or tissue, and will also allow access to the device for retrieval and/or replacement.
  • a number of different implantation sites are contemplated. These include, e.g., the aqueous humor, the vitreous humor, the sub-Tenon's capsule, the periocular space, and the anterior chamber.
  • the capsules are immunoisolatory.
  • any assays or diagnostic tests well known in the art can be used for these purposes.
  • an ELISA enzyme-linked immunosorbent assay
  • chromatographic or enzymatic assay or bioassay specific for the secreted product
  • bioassay specific for the secreted product can be used.
  • secretory function of an implant can be monitored over time by collecting appropriate samples (e.g. , serum) from the recipient and assaying them.
  • VEGF receptors described herein can also be used in accordance with this invention. Further, the use of active fragments of these receptors (i.e., those fragments having biological activity sufficient to achieve a therapeutic effect) is also contemplated. Also contemplated are receptor molecules modified by attachment of one or more polyethylene glycol (PEG) or other repeating polymeric moieties as well as combinations of these proteins and polycistronic versions thereof.
  • PEG polyethylene glycol
  • Therapeutic dosages may be between about 0.1 pg and 1000 ⁇ g per eye per patient per day (e.g., between 0.1 pg and 500 ⁇ g; between 0.1 pg and 250 ⁇ g, between 0.1 pg and 100 ⁇ g; between 0.1 pg and 50 ⁇ g; between 0.1 pg and 25 ⁇ g; between 0.1 pg and 10 ⁇ g; between 0.1 pg and 5 ⁇ g; between 0.1 pg and 100 ng; between 0.1 pg and 50 ng; between 0.1 pg and 25 ng; between 0.1 pg and 10 ng; or between 0.1 pg and 5 ng per eye per patient per day).
  • Each of the devices of the present invention is capable of storing between about 1,000 and about 750,000 cells, in individual or cluster form, depending on their type.
  • VEGF receptor 1 Various fragments of secretory VEGF receptor 1 (“VRl”) were expressed in NTC- 200 cells. These fragments include domain 2 ("D2"), which is the VEGF binding domain (VBD) of VRl . D2 was fused in frame with combinations of domain 1 , 3 or 4 (referred to herein as “Dl”, “D3” and “D4", respectively). In some cases, domain 3 of VEGF receptor 2 (“VR2 D3”) was used in place of VRl D3 to create a VRl /VR2 chimeric molecule. In another embodiment, dimers of domains 2 and 3 (referred to herein as "D2" and "D3”) of VEGF receptor 2 were used.
  • the constructs contained either the native or IgSP murine immunoglobulin leader sequence, the latter of which was used previously to direct secretion of CNTF from NTC-201 cells.
  • Nucleotide and amino acid sequences of intended proteins and corresponding nucleic acid sequences are described herein as follows:
  • sVRl is secretory VEGF receptor 1
  • sVR2 is secretory VEGF receptor 2
  • Dl is Domain 1
  • D2 is Domain 2
  • D3 is Domain 3
  • D4 is Domain 4 and the sVR chimeras comprise fragments of sVRl and sVR2.
  • the pKAN3 backbone is based on the pNUT-IgSP-hCNTF expression plasmid used to create the ARPE-
  • nucleotide sequence of pKAN3 is shown below:
  • GAATAGCTCA GAGGCCGAGG CGGCCTCGGC CTCTGCATAA ATAAAAAAAA TTAGTCAGCC 4141 ATGGGGCGGA GAATGGGCGG AACTGGGCGG AGTTAGGGGC GGGATGGGCG GAGTTAGGGG 420 L CGGGACTATG GTTGCTGACT AATTGAGATG CATGCTTTGC ATACTTCTGC CTGCTGGGGA 4261 GCCTGGGGAC TTTCCACACC TGGTTGCTGA CTAATTGAGA TGCATGCTTT GCATACTTCT 432' GCCTGCTGGG GAGCCTGGGG ACTTTCCACA CCCTAACTGA CACACATTCC ACAGCTGGTT
  • nucleotides 1-595 are pMTl; nucleotides 631-918 are U5 5' UTR, nucleotides 919-953 are MCS; nucleotides 954-1250 are hGH pA; nucleotides 1258-2680 are pUC 18; nucleotides 2698-2992 are SV40 pA; nucleotides 2988-3133 are SV40 Intron E19S; nucleotides 3158-3952 are NeoR; nucleotides 4030-4400 are SV40 promoter; and 4401-4534 are AmpR promoter.
  • the complementary nucleic acid sequence for the pKAN3 vector is shown in SEQ ID NO: 16: GAACCAAAAA TTTTGGTCGG ACCTCATCTC GTCTACCCAA TTCCACTCAC TGGGGAGTCG
  • TTTTCTCAAC CATCGAGAAC TAGGCCGTTT GTTTGGTGGC GACCATCGCC ACCAAAAAAA
  • Transformed recombinant clones were selected with kanamycin, and purified miniprep plasmid DNA was analyzed by restriction digestion and agarose gel electrophoresis analysis. Putative plasmid clones containing an appropriate insert were verified by automated dideoxy sequencing followed by alignment analysis using Vector NTI v7.0 sequence analysis software (Invitrogen Corp, Carlsbad, CA).
  • Verified plasmid clones were used to transfect NTC-200 cells to obtain stable polyclonal cell lines. Briefly, 200-300K cells, plated 18 hours previously, were transfected with 3.0 ug of plasmid DNA using 6.0 ul of Fugene 6 trans fection reagent (Roche Applied Science, Indianapolis IN) according to the manufacturer's recommendations. Transfections were performed in 3.0 ml of DMEM/F12 with 10% FBS, Endothelial SFM or Optimem media (Invitrogen Corp, Carlsbad, CA). Twenty four to 48 hours later cells were either fed with fresh media containing 1.0 ug/ul of G418 or passaged to a T-25 tissue culture flask containing G418. Cell lines were passaged under selection for 14-21 days until normal growth resumed, after which time drug was removed and cells were allowed to recover ( ⁇ 1 week) prior to characterization.
  • Pulse media was stored at -20 C and assayed within one week of collection as per the manufacturer's protocol.
  • Example 3 Protein Characterization Saturation binding studies of VEGF using conditioned media from transfected cell lines were performed. Briefly, titrated amounts of conditioned media were incubated overnight at room temperature in the presence of VEGF. Free VEGF was measured using a sensitive EIA (R&D Systems, Minneapolis, MN) and non-linear regression analysis was performed (GraphPad Software Inc., San Diego, CA). The results of these studies are presented in Figures IA- 1C. Figures IA and IB depict typical VEGF binding curse for the various VR truncation forms disclosed herein. Figure 1C presents the dissociation constant, Kd, of the receptor fragments relative to control (VRl D 1-3 Fc). These data demonstrate that the recombinant VEGF receptor proteins secreted from these cell lines bind VEGF with high affinity. As a negative control, it was shown that conditioned media from mock transfected parental cells (WT) did not bind VEGF.
  • WT mock transfected parental cells
  • conditioned media from transfected cells was measured using a human umbilical vein endothelial cell (HUVEC) growth inhibition assay.
  • HUVEC human umbilical vein endothelial cell

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Abstract

L'invention porte sur des séquences isolées d'acide nucléique et polypeptidiques codant pour des récepteurs du facteur de croissance endothélial vasculaire (VEGF), tels que des récepteurs de VEGF solubles. L'invention porte également sur des lignées cellulaires codant pour de tels récepteurs de VEGF. L'invention porte également sur des dispositifs de thérapie par cellules encapsulées qui sont capables d'administrer de tels récepteurs de VEGF, ainsi que sur des procédés d'utilisation de ces dispositifs pour administrer les récepteurs de VEGF à l'œil et pour traiter des troubles ophtalmiques et de prolifération cellulaire chez des patients souffrant de ceux-ci.
PCT/US2009/046163 2008-06-03 2009-06-03 Lignées cellulaires qui secrètent des récepteurs de vegf solubles et leurs utilisations Ceased WO2009149205A2 (fr)

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WO2013181424A1 (fr) * 2012-05-30 2013-12-05 Neurotech Usa, Inc. Dispositifs de culture de cellule implantables cryopréservés et leurs utilisations
US9669154B2 (en) 2010-09-27 2017-06-06 Gloriana Therapeutics, Sarl Implantable cell device with supportive and radial diffusive scaffolding
US10888526B2 (en) 2009-01-23 2021-01-12 Gloriana Therapeutics Sarl Cell lines and their use in encapsulated cell biodelivery
US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US11912784B2 (en) 2019-10-10 2024-02-27 Kodiak Sciences Inc. Methods of treating an eye disorder
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Family Cites Families (6)

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CA2158745C (fr) * 1993-03-25 2007-06-19 Richard L. Kendall Inhibiteur du facteur de croissance des cellules vasculaires endotheliales
JPH09154588A (ja) * 1995-10-07 1997-06-17 Toagosei Co Ltd Ｖｅｇｆ結合性ポリペプチド
US20070071734A1 (en) * 1999-04-06 2007-03-29 Weng Tao ARPE-19 as platform cell line for encapsulated cell-based delivery
US7303746B2 (en) * 1999-06-08 2007-12-04 Regeneron Pharmaceuticals, Inc. Methods of treating eye disorders with modified chimeric polypeptides
EP1732947B1 (fr) * 2004-03-05 2011-04-27 Vegenics Pty Ltd Matieres et procedes de constructions de liaison de facteurs de croissance
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US9669154B2 (en) 2010-09-27 2017-06-06 Gloriana Therapeutics, Sarl Implantable cell device with supportive and radial diffusive scaffolding
US10835664B2 (en) 2010-09-27 2020-11-17 Gloriana Therapeutics Implantable cell device with supportive and radial diffusive scaffolding
JP2014506119A (ja) * 2010-12-02 2014-03-13 ニューロテック ユーエスエー， インコーポレイテッド 抗血管新生抗体足場および可溶性受容体を分泌する細胞株およびその使用
EP2646007A4 (fr) * 2010-12-02 2014-12-24 Neurotech Usa Inc Lignées cellulaires secrétant des squelettes d'anticorps anti-angiogéniques et des récepteurs solubles, et utilisations de celles-ci
US9149427B2 (en) 2010-12-02 2015-10-06 Neurotech Usa, Inc. Cell lines that secrete anti-angiogenic antibody-scaffolds and soluble receptors and uses thereof
WO2012075184A2 (fr) 2010-12-02 2012-06-07 Neurotech Usa, Inc. Lignées cellulaires secrétant des squelettes d'anticorps anti-angiogéniques et des récepteurs solubles, et utilisations de celles-ci
US10004804B2 (en) 2010-12-02 2018-06-26 Neurotech Usa, Inc. Cell lines that secrete anti-angiogenic antibody-scaffolds and soluble receptors and uses thereof
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WO2013181424A1 (fr) * 2012-05-30 2013-12-05 Neurotech Usa, Inc. Dispositifs de culture de cellule implantables cryopréservés et leurs utilisations
US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
US12071476B2 (en) 2018-03-02 2024-08-27 Kodiak Sciences Inc. IL-6 antibodies and fusion constructs and conjugates thereof
US11912784B2 (en) 2019-10-10 2024-02-27 Kodiak Sciences Inc. Methods of treating an eye disorder

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