WO2002028408A2 - Compositions and methods for the transport of biologically active agents across cellular barriers - Google Patents
Compositions and methods for the transport of biologically active agents across cellular barriers Download PDFInfo
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- WO2002028408A2 WO2002028408A2 PCT/US2001/030832 US0130832W WO0228408A2 WO 2002028408 A2 WO2002028408 A2 WO 2002028408A2 US 0130832 W US0130832 W US 0130832W WO 0228408 A2 WO0228408 A2 WO 0228408A2
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4728—Calcium binding proteins, e.g. calmodulin
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- C—CHEMISTRY; METALLURGY
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- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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- 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
- C07K14/575—Hormones
- C07K14/57527—Calcitonin gene related peptide
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- C—CHEMISTRY; METALLURGY
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- 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
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
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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
- C07K14/575—Hormones
- C07K14/62—Insulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
- C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
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- C07K2319/00—Fusion polypeptide
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- 09/898,503 (attorney docket No. 057220.1401) entitled "Compositions, Compounds And Methods For The Delivery Of Monoclonal Antibodies” by Hawley, Stephen, Chapin, Steve, and Houston, L.L., filed July 2, 2001, is drawn to the use of targeting elements and ligands to deliver monoclonal antibodies and related compounds and compositions.
- compositions that pass through cellular barriers to deliver compounds into, through and out of cells, and methods of producing and using such compositions.
- Therapeutic drugs can be introduced into the body using a variety of formulations and by various of routes of administration.
- a preferred route of administration is one that is non-invasive, i.e, does not involve any physical damage to the body.
- physical damage of this type results from the use of a medical device, such as a needle, to penetrate or breach a dermal surface or other external surface of an animal.
- Invasive routes of administration include, for example, surgical implants and injections. Injections can be intravascular, intrathecal or subcutaneous, all of which have undesirable features.
- Non-invasive routes of administration include uptake from the gastrointestinal tract as well as non-invasive parenteral (i.e, other than gastrointestinal) routes such as, e.g., inhalation therapy.
- Endocytosis is a general term for the process of cellular internalization of molecules, i.e, processes in which cells takes in molecules from their environment, either passively or actively.
- Exocytosis is a general term for processes in which molecules are passively or actively moved from the interior of a cell into the medium surrounding the cell.
- Transcytosis is a general term for processes in which molecules are transported from one surface of a cell to another.
- Active endocytosis, exocytosis and transcytosis typically involve or are mediated by receptors, molecules that are at least partially displayed on the surface of cells.
- Receptors have varying degrees of specificity; some are specific for a single molecule (e.g., a receptor specific for epidermal growth factor; or a receptor that specifically recognizes Ca “1"1” ); some are semi-specific (e.g., a receptor that mediates the cellular internalization of many members of a family of cellular growth factors, or a receptor that recognizes Ca " " " " , Mg 44" and Zn 4"4" ); or of limited specificity (e.g., a receptor that mediates the cellular internalization of any phosphorylated protein, or a receptor that recognizes any divalent cation).
- cellular pores, pumps, and coated pits Other types of molecules that can cause or influence the entry of molecules into cells include, e.g., cellular pores, pumps, and coated pits. Pores such as gated channels and ionophores form a channel that extends through the cellular membrane and through which certain molecules can pass.
- Cellular pumps exchange one type of molecule within a cell for another type of molecule in the cell's environment. Coated pits are depressions in the cellular surface that are "coated” with bristlelike structures and which condense to surround external molecules; the condensed coated pits then "pinch off to form membrane-bound, coated vesicles within the cell.
- Molecules that cause, influence or undergo endocytosis, exocytosis and/or transcytosis can do so constitutively, i.e, at all times, or regulated, for example, only under certain conditions or at specific times. Some such molecules can only mediate and/or undergo endocytosis, whereas some mediate and/or undergo transcytosis as well as endocytosis. Moreover, some such molecules are present in all or most cells (i.e, are ubiquitous), or are present mostly or only in certain tissues (i.e, are tissue-specific) or particular cell types.
- compositions and methods causing, enhancing, mediating or regulating the endocytosis of therapeutic, diagnostic or analytical compounds and compositions hinders or prevents various uses of such compounds.
- the full therapeutic potential of many compounds could be realized if they were taken up by cells lining the gastrointestinal tract, as one could then formulate pills or tablets for the administration of therapeutic agents to patients.
- pills and other formulations for the oral delivery, and suppositories for the rectal delivery, of therapeutic agents to the gastrointestinal tract result in better patient compliance, and less use of medical resources, as opposed to other delivery modalities such as, e.g., intravenous administration.
- the therapeutic potential of many compounds could be realized if they were taken up by cells lining the respiratory tract, including the nasal cavity; cells lining the gastrointestinal tract; vaginal surfaces; on dermal surfaces; and ocular surfaces and buccal surfaces (see Sayani et al., Crit. Rev. Ther. Drug Carrier Systems 13:85-184, 1996) . Attempts to develop oral delivery formulations for proteins are discussed by Wang (J. Drug Targeting 4: 195-232, 1996), Sinko et al. (Pharm. Res. 16:527, 1999) and Stoll et al. (J. Controlled Release 64:217-228, 2000).
- Transcytosis is the general term given for processes whereby molecules, including biologically active molecules, move from one side or surface of a cell to another.
- plgR polymeric immunoglobulin receptor
- plgR molecules are displayed on epithelial cells.
- Epithelial cells line the interior of organs that have enclosed, semi-enclosed or compartmentalized spaces.
- the interior (e.g., canals, ducts, cavities, etc.) of such organs is generically referred to as the lumen.
- the lumen of a particular organ may have a specific name, e.g., the gastrointestinal lumen, pulmonary lumen, nasal lumen, nasopharyngeal lumen, pharyngeal lumen, buccal (within the mouth) lumen, sublingual (under the tongue) lumen, vaginal lumen, urogenital lumen, ocular lumen, or tympanic lumen.
- Adjacent epithelial cells are connected by tight junctions. Disruption of tight junctions allows agents within the lumen, which often has an opening to the external environment of an animal, to penetrate into the body. Although such agents might include therapeutic agents, entry into the body via a disrupted tight junction is not specific; undesirable agents (e.g., bacteria, viruses, toxins and the like) will also be taken into the body. Due to this lack of specificity, as well as other factors, disruption of tight junctions for drag delivery purposes is generally not feasible and would, in any event, have many potential undesirable side effects.
- Epithelial cells have two distinct surfaces: the apical side, which faces the lumen and is exposed to the aqueous or gaseous medium present therein; and an opposing basolateral (a.k.a. basal lateral) side that rests upon and is supported by an underlying basement membrane.
- the tight junctions between adjacent epithelial cells separate the apical and basolateral sides of an individual epithelial cell.
- Epithelial cells are said to have polarity, that is, they are capable of generating gradients between the compartments they separate (for reviews, see Knust, Curr. Op. Genet. Develop. 10:471-475, 2000; Matter, Curr. Op. Genet. Develop. 10:R39-R42, 2000; Yeaman et al., Physiol. Rev. 79:73-98, 1999).
- This polarity reflects that fact that the cell has distinct plasma membrane domains (apical and basolateral) having distinct transport and permeability characteristics.
- the apical side often contains microvilli for the adsorption of substances from the lumen, and, in ciliated cells, cilia are found on the apical membrane.
- the Na + /K + -ATPase pump is characteristically found only on the basolateral membrane.
- Figure 1 shows the pathways of cellular transport involving the plgR protein, which undergoes or mediates endocytosis, exocytosis as well as forward and reverse transcytosis, in epithelial cells. Molecules of plgR are typically displayed on the surfaces of epithelial cells and direct the trafficking of immunoglobulin (IgA) molecules. Other classes and species of immunoglobulins may also be trafficked.
- IgA immunoglobulin
- the right side of Figure 1 illustrates the "forward" (i.e, basolateral to apical) transcytosis of plgR molecules, whereas "reverse” (apical to basolateral) transcytosis is shown on the left side ofthe Figure.
- Forward transcytosis is the best characterized biological function of plgR, and serves to convey protective antibodies (IgA and IgM immunoglobulins) from the circulatory system to the lumen of an organ.
- plgR molecules displayed on the basolateral side ofthe cell bind IgA molecules in the bloodstream, and pIgR:IgA complexes are then endocytosed, i.e, taken up into the cell and into a vesicle.
- the plgRTgA complexes are transported to the apical side ofthe cell, where they are displayed on the cell surface.
- IgA into the lumen occurs when the plgR portion of a plgRTgA complex is cleaved, i.e, undergo proteolysis. This event separates the plgR molecule into two components: the "secretory component” (SC), which is released into the lumen, and which remains bound to IgA in order to protect IgA from degradation, and the "stalk,” which remains displayed, at least temporarily, on the apical surface ofthe cell.
- SC secretory component
- ligands bound to stalks displayed on the apical side of a cell can undergo reverse transcytosis, i.e, transcytosis in the opposite direction of forward transcytosis, i.e, from the apical side of a cell to its basolateral side.
- reverse transcytosis plgR molecules or portions thereof move from the apical surfaces of cells that line the lumen of an organ to the basolateral surfaces of these cells.
- plgR-mediated reverse transcytosis could be used to deliver agents from a lumen (e.g., the interior ofthe gut or the airways ofthe lung) to the circulatory system or some other interior system, organ, tissue, portion or fluid ofthe body including by way of non-limiting example the lymphatic system, the vitreous humor, etc.
- a compound having an element that binds to a portion of plgR that undergoes reverse transcytosis could, due to its association with the plgR stalk, be carried to the basolateral side of a cell, where it would be contacted with and/or released into the bloodstream.
- plgR polyimmunoglobulin receptor
- plgR and its Ig ligands have been investigated using molecular genetic techniques.
- Norderhaug et al. Domain deletions in the human polymeric Ig receptor disclose differences between its dimeric IgA and pentameric IgM interaction, Eur J Immunol 1999 Oct;29(10):3401-9; Crottet et al., Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity ofthe polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand, J Biol Chem 1999 Oct 29;274(44):31445-55; Breitfeld et al, Deletions in the cytoplasmic domain ofthe polymeric immunoglobulin receptor differentially affect endocytotic rate and postendocytotic traffic, J Biol Chem 1990 Aug 15;265(23): 13750-7; Casanova et al., Phosphorylation ofthe polymeric immunoglobulin receptor required for its efficient transcytosis, Science
- TCR is an abbreviation for the T cell receptor.
- the cytoplasmic domain ofthe T cell receptor-zeta chain was used as an indicator of receptor oligomerization to show that a pIgR:zeta chimeric receptor expressed in Jurkat cells initiates a zeta-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA.
- Eckman et al. Am J Respir Cell Mol Biol 1999 Aug;21(2):246-52, is stated to disclose a fusion protein consisting of a sFv directed to the secretory component (SC) of human plgR and an human alpha-(l)-antitrypsin.
- SC secretory component
- Ferkol et al. Am. J.Respir. Crit. Care Med. 161:944-951, 2000, is stated to describe the basolateral-to-apical transport ofthe fusion protein of Eckman et al. across in vitro model systems of polarized respiratory epithelial cells.
- Gupta et al., Gene Ther 8:586-92, 2001 is stated to disclose the use of a single- chain antibody directed to the secretory component (SC) of human plgR to deliver reporter genes to epithelial cells in vitro.
- SC secretory component
- the sFv is stated to be conjugated to polylysine using the cross-linker SPDP.
- U.S. Patent No. 5,484,707 to Goldblum et al. is drawn to methods for monitoring organ rejection in an animal based on the concentration ofthe free secretory component of (SC) pIgR.
- PCT patent applications WO 98/30592 and WO 99/20310, both to Hein et al., and U.S. Patent 6,045,774 to Hiatt et al. are drawn to synthetic proteins that mimic IgA molecules and are thus associated with the proteolytically generated secretory component (SC) of plgR.
- U.S. Patent No. 6,072,041 to Davis et al. is drawn to fusion proteins that comprise single-chain antibodies directed to the secretory component of plgR.
- the compositions of Davis et al. are stated to be transported specifically from the basolateral surface of epithelial cells to the apical surface.
- U.S. Patent 6,261,787 Bl to Davis et al. is drawn to bifunctional molecules comprising (1) a ligand directed to the secretory component of plgR and (2) a non-protein therapeutic molecule.
- the bifunctional molecules are said to be transported specifically from the basolateral surface of an epithelial cell to the apical surface thereof.
- U.S. Patent 6,287,817 Bl to Davis et al. is drawn to a method of delivering a therapeutic protein to an epithelial cell by using a fusion protein that comprises a single- chain antibody directed to the secretory component of plgR.
- the proteins are said to be transported specifically from the basolateral surface of an epithelial cell to the apical surface thereof.
- PCT application No. WO 00/53623 published September 14, 2000, entitled “Bifunctional Molecules for Delivery of Therapeutics” by Davis, Pamela B., Ferkol Jr., Thomas W., and Eckman, Elizabeth, is stated to disclose bifunctional molecules that specifically bind secretory component (SC) of plgR. The bifunctional molecules are said to be transported specifically from the basolateral surface of an epithelial cell to the apical surface thereof.
- PCT application No. WO 00/53623 by Ziady, Assem, Davis, Pamela B., Ferkol Jr., Thomas W., and Malouf, Alfred, was published September 14, 2000 and is entitled "Enhanced Delivery Via Serpin Enzyme Complex Receptor".
- U.S. Patent No. 6,042,833 to Mostov et al. is drawn to a method by which a ligand that binds to a portion of a plgR molecule is thereby internalized into, or transported across, a cell expressing or displaying plgR
- Serial No. 09/475,088 (attorney reference Nos. 2307E-067911US and 057220-0908) is a Divisional application of Patent 6,042,833, that was filed December 30, 1999.
- the corresponding PCT application was published as WO 97/46588, entitled "Cellular Internalization of plgR Stalk and Associated Ligands" on December 11, 1997.
- the invention provides a complex or compound comprising a biologically active portion and a targeting element directed to a ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to the ligand, wherein the targeting element is not an antibody.
- a “compound” is used herein as in the field of chemistry, i.e., a substance of two or more elements, where the elements are present in fixed proportions, having a defined chemical structure.
- a “molecular complex” or “complex” comprises at least two distinct molecules that are associated with each other by noncovalent interactions.
- association it is meant that the molecules in a complex, or moieties in a compound, are specifically bound to each other.
- a targeting element and biologically active portion have a covalent association with each other whereas, in a molecular complex ofthe invention, the targeting element and biological active portion have a non-covalent association.
- a “target molecule” or “molecular target” is a compound, a molecular complex of two or more compounds, a moiety (a portion of a compound), or an interface formed between two or more compounds, to which a targeting element or ligand is directed.
- ligand encompasses any type of composition or compound that is capable of specifically binding to a molecular target.
- targeting element encompasses any moiety or compound that is included within, respectively, a compound or a molecular complex, that is capable of specifically binding to a molecular target.
- a ligand is a targeting element when it is either covalently or non-covalently associated with another compound.
- a compound or composition comprising a targeting element directed to (capable of specifically binding) a molecular target is a ligand of that target.
- a targeting element When a ligand is incorporated into a compound or complex, it becomes a targeting element so long as retains the ability to specifically bind to the target. Conversely, when a targeting element is separated from the remainder of a compound, it becomes a ligand so long as it retains the ability to specifically bind a molecular target. Any portion or derivative of a ligand that is capable of specifically binding a molecular target is a ligand.
- biologically active indicates that a composition or compound itself has a biological effect, or that it modifies, causes, promotes, enhances, blocks, reduces, limits the production or activity of, or reacts with or binds to an endogenous molecule that has a biological effect.
- a “biological effect” may be but is not limited to one that stimulates or causes an immunoreactive response; one that impacts a biological process in an animal; one that impacts a biological process in a pathogen or parasite; one that generates or causes to be generated a detectable signal; and the like.
- Biologically active compositions, complexes or compounds may be used in therapeutic, prophylactic and diagnostic methods and compositions.
- Biologically active compositions, complexes or compounds act to cause or stimulate a desired effect upon an animal.
- desired effects include, for example, preventing, treating or curing a disease or condition in an animal suffering therefrom; limiting the growth of or killing a pathogen in an animal infected thereby; augmenting the phenofype or genotype of an animal; stimulating a prophylactic immunoreactive response in an animal; or diagnosing a disease or disorder in an animal.
- biologically active indicates that the composition, complex or compound has an activity that impacts an animal suffering from a disease or disorder in a positive sense and/or impacts a pathogen or parasite in a negative sense.
- a biologically active composition, complex or compound may cause or promote a biological or biochemical activity within an animal that is detrimental to the growth and/or maintenance of a pathogen or parasite; or of cells, tissues or organs of an animal that have abnormal growth or biochemical characteristics, such as cancer cells.
- biologically active indicates that the composition, complex or compound can be used for in vivo or ex vivo diagnostic methods and in diagnostic compositions and kits.
- a preferred biologically active composition or compound is one that can be detected, typically (but not necessarily) by virtue of comprising a detectable polypeptide.
- Antibodies to an epitope found on composition or compound may also be used for its detection.
- the term "biologically active" indicates that the composition or compound induces or stimluates an immunoreactive response.
- the immunoreactive response is designed to be prophylactic, i.e, prevents infection by a pathogen.
- the immunoreactive response is designed to cause the immune system of an animal to react to the detriment of cells of an animal, such as cancer cells, that have abnormal growth or biochemical characteristics.
- compositions, complexes or compounds comprising antigens are formulated as a vaccine.
- compositions, complex or compound may be biologically active in therapeutic, diagnostic and prophylactic applications.
- a composition, complex or compound that is described as being “biologically active in a cell” is one that has biological activity in vitro (i.e, in a cell culture) or in vivo (i.e, in the cells of an animal).
- a “biologically active component” of a composition or compound is a portion thereof that is biologically active once it is liberated from the composition or compound. It should be noted, however, that such a component may also be biologically active in the context ofthe composition or compound.
- compositions, complexes and compounds that are not biologically active include elements that have no effect on biological functions but which are incorporated for ease of manipulation ofthe conjugate or member thereof such as, e.g., poly-(L)-lysine for the in vitro chemical conjugation ofthe composition or compound to another molecule; a polypeptide derived from a phage surface protein intended for compositions or compounds to be used in vitro in phage display libraries; or a composition or compound that serves as a carrier for another composition or compound such as, e.g., KLH (keyhole limpet hemocyanin), which serves as a carrier for immunogenic compositions or compounds; or the herein-disclosed "optional fusion protein elements.”
- a "transcellular property" is an attribute that causes, promotes or enhances any type of process that results in the movement of a molecule from side of a cell to another, regardless ofthe mechanism ofthe movement.
- a "transcytotic property” is an attribute that causes, promotes or enhances endocytosis, exocytosis, transcytosis and/or intracellular delivery.
- Transcytotic properties include, by way of non-limiting example, the ability to undergo a least one process selected from the group consisting of apical endocytosis, apical exocytosis, apical to basolateral transcytosis, basolateral endocytosis, basolateral exocytosis, basolateral to apical transcytosis, and intracellular delivery.
- compositions and compounds ofthe invention include an organelle- targeting sequence for transport to selected organelles.
- organelle is a subcellular component that carries out one or more specific biological and/or biochemical functions.
- organelle-targeting sequence is an amino acid sequence that mediates the delivery of a complex or compound having the organelle targeting sequence to an organelle of interest such as, e.g., a mitochondrion, the endoplasmic reticulum, the Golgi apparatus, lysosomes, peroxisomes, endosomes, the cell membrane or any membrane contained within a cell, the nucleus, or the nucleolus.
- organelle of interest such as, e.g., a mitochondrion, the endoplasmic reticulum, the Golgi apparatus, lysosomes, peroxisomes, endosomes, the cell membrane or any membrane contained within a cell, the nucleus, or the nucleolus.
- a "paracellular transporting property” is an attribute that causes, promotes paracellular transport including, by way of non-limiting example, transport through the tight junctions found in epithelial or mucosal cell layers. Tight junctions seal adjacent epithelial cells in a narrow band just near their apical surface and, as a result, agents on one side of an epithelial layer must move through epithelial cells in order to reach the other side ofthe barrier. Such movement may result from simple diffusion (passive transport), or as a result of cellular, usually ATP-dependent, activity (active transport).
- antibody includes polyclonal, monospecific, monoclonal, camelized, humanized and single-chain antibodies; Fab, Fab' (Fab')2 fragments; CDRs; and the like.
- the targeting element that is not an antibody can be any type of molecule or moeity, regardless of chemical structure, that functions as a targeting element for the ligand of choice.
- the targeting element can be a lipid, a carbohydrate, a small molecule, or a nucleic acid. Nucleic acids, such as aptamers, that bind specifically to a preselected molecular target are used as targeting elements in the complexes and compounds ofthe invention.
- the targeting element can also be a polypeptide that is not an antibody. When both the bioactive portion of a compound are polypeptides, the compound is a fusion protein or a protein conjugate, as those terms are used herein.
- the chosen ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to the ligand is the plgR stalk or a domain, conserved sequence or region thereof.
- the ligand may be a polypeptide that corresponds to an amino acid sequence that is conserved in plgR proteins from a variety of species, e.g., the ligand is a polypeptide having an amino acid sequence selected from the group consisting of LRKED, QLFVNEE, LNQLT, YWCKW, GWYWC, STLVPL, SYRTD, and KRSSK.
- the ligand maybe a polypeptide that corresponds to an amino acid sequence present in a defined region selected from the group consisting of:
- a targeting element may, by way of non-limiting example, be a polypeptide derived from a protein that binds the plgR stalk or portion thereof.
- a polypeptide that functions as a targeting element directed to the plgR stalk may be derived from a polypeptide derived from a calmodulin, an AP-1 Golgi adaptor or a bacterial polypeptide.
- Non limiting examples of polypeptides from bacterial proteins that may be used as plgR-stalk-directed targeting elements are those amino acid sequences from CbpA that are underlined in Figure 17.
- the complexes and compounds ofthe invention further comprises a PTD or MTS.
- PTD Protein transduction domains
- MTS membrane transport signals
- the biologically active portion can be any type of molecule or moeity, regardless of chemical structure, capable of achieving the desired biological effect.
- the biologically active portion is a polypeptide including a peptidomimetic, a nucleic acid, a lipid, a carbohydrate, a compound or complex comprising a metal, a small molecule, or a functional derivative of any ofthe preceding.
- the term "functional derivative” indicates a chemically modified version, an analog, or a homolog of a compound that retains a biological function of interest of that compound for any given application.
- chemical modification may include, by way of non-limiting example, adding chemical groups to a compound (e.g., glycosylation, phosphorylation, thiolation, etc.), eliminating parts of a compound that do not impact the function of interest (preparing a truncated form of a protein that retains an activity of interest, e.g., Klenow fragment), changing sets of one or more amino acids in the polypeptide (preparing muteins); analogs are exemplified by peptidomimetics; and homologs are polypeptides from other species of animals that retain biological activity (e.g., human and porcine insulin, human and salmon calcitonin, etc.) or intraspecies isomers of a polypeptide (protein "families" such as the cytochrome P450 family).
- a compound e.g., glycosylation, phosphorylation, thiolation, etc.
- analogs are exemplified by peptidomimetics
- homologs are polypeptides
- the bioactive portion ofthe complex or compound can be a complex or compound comprising a metal.
- metals include, by way of non-limiting example, platinum(II), palladium(II), zinc, cobalt(III).
- Metal-based or comprising drugs include, but are not limited to, Cisplatin.
- the biologically active portion is a nucleic acid.
- Bioactive nucleic acids include, by way of non-limiting example, aptamers, antisense molecules including ribozymes, nucleic acids that encode therapeutic polypeptides and nucleic acids that serve as a template for the production of a biologically active nucleic acid.
- the biologically active portion ofthe complex or compound can be a polypeptide.
- a bioactive polypeptide may be a growth factor, an interleukin, an immunogen, a hormone, an enzyme, an enzyme inhibitor, an antibody, a clotting factor, a receptor, a ligand for a receptor, a kinase, a phosphoptase, a scaffold protein, an adaptor protein, a dominant negative mutant, a protease, a signaling molecule, a regulatory molecule, transporter, a transcriptional regulator, a nucleic acid binding protein, and a functional derivative of any ofthe preceding.
- Bioactive polypeptides of particular interest include insulin, IL-2, IL-4, hGH, sCT and hCT.
- the biologically active portion is a second targeting element that is directed to a molecular target other than said ligand.
- the targeting element directed to a molecular target other than the ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to said ligand, is one type of bioactive portion.
- Such compounds are bispecific (polyspecific) in that they bind more than 1 molecular target.
- Polyspecific complexes or compounds may be formulated with th. target ofthe second targeting element and then administered, in order to deliver an exogenous target molecule into the body. Altematively, polyspecific complexes or compounds may be formulated separately, i.e., without the target ofthe second targeting element.
- this aspect ofthe invention provides for the delivery of exogenous drugs and "molecular sponges" that bind, neutralize and/or sequester, endogenous molecules.
- the invention provides a polyspecific complex or compound tha further comprise a biologically active portion that is not a targeting element.
- a complex or compound ofthe invention could comprise (1) a targeting element directed to a ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to said ligand, which functions to deliver the complex or compound into the body; (2) a second targeting element that is directed to a molecular target that is found only or predominantly on a preselected cell or tissue, which functions to target the complex or compound to the cell or tissue; and (3) a bioactive portion that is preferentially delivered to the cell or tissue of interest.
- Portion (3) may, by way of non-limiting example, be a cytotoxin that is preferably delivered to diseased cells (e.g., cancer cells or virally infected cells), or a therapeutically beneficial agent that is preferable delivered to cells in need of such treatment.
- diseased cells e.g., cancer cells or virally infected cells
- therapeutically beneficial agent that is preferable delivered to cells in need of such treatment.
- the second targeting element may be an antibody or an antibody derivative.
- Antibodies per se include, but are not limited to, polyclonal, monospecific, and monoclonal antibodies.
- Antibody derivatives include those prepared by recombinant DNA technology, e.g., single-chain (sFv) antibodies, and those prepared from whole antibodies by chemical manipulation, e.g., Fab, Fab' and (Fab)2 fragments.
- Another aspect ofthe invention provides a multivalent complex or compound, i.e., a complex or compound comprising 2 or more targeting elements directed to one or more ligands that confer transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to said ligand.
- multivalent complexes or compounds ofthe invention comprise two, three, or four targeting elements directed to the ligand (dimers, trimers, tetramers, respectively).
- the targeting elements TI, T2
- the invention provides complexes or compounds ofthe invention, wherein at least one ofthe targeting elements in the complex or compound is identical or substantially identical to at least one other targeting element in the complex or compound; as well as complexes or compounds wherein at least one of said targeting elements in the complex or compound is different from at least one other targeting element ofthe second complex or compound.
- the ligand that confers transcellular, transcytotic or paracellular transporting properties to a multivalent agent specifically bound thereto is the plgR stalk or a domain, conserved sequence or region thereof.
- the ligand may be a polypeptide that corresponds to an amino acid sequence that is conserved in plgR proteins from a variety of species, e.g., a polypeptide having an amino acid sequence selected from the group consisting of LRKED, QLFVNEE, LNQLT, YWCKW, GWYWC, STLVPL, SYRTD, and KRSSK.
- the ligand may be a polypeptide that corresponds to an amino acid sequence present in a defined region, e.g., a region of a plgR, wherein said plgR can be from any animal, and wherein said region is selected from the group consisting of:
- a complex or compound directed to the plgR stalk or a portion, domain or region thereof is a cytotoxic agent, and is delivered to a cancerous or otherwise diseased cell that displays plgR or the plgR stalk.
- the invention provides a compound comprising n targeting elements directed to a ligand that confers transcellular, transcytotic or paracellular transporting properties to a compound bound to said ligand, wherein one or more of desirable attributes of said compound is enhanced as compared to a second compound having m targeting elements, wherein n and m are both whole integers, and n > m.
- enhanced transcytotic properties include an increase in the relative rate of one or more processes such as of endocytosis, transcytosis or exocytosis; an increased range of recognition, or a higher degree of specificity, for particular types and species of plgR and stalk molecules; or the ability to transcytose compounds of a larger molecular weight and/or a different composition.
- enhanced properties of paracellular transport include but are not limited to an increase in the relative rate of transport; or the ability to transport compounds of a larger molecular weight.
- the "relative rate" of a multimeric compound or complex ofthe invention refers to the number of molecules of a multimer undergoing a given process (endocytosis, transcytosis, paracellular transport, etc.) over a set period of time compared to the number of molecules of a comparable monomer undergoing the same process over the same period of time. Rates may also be expressed in absolute terms, e.g., x moles of molecules per nanosecond. Similarly, other properties of complexes and compounds maybe measured in absolute or relative terms.
- An enhanced property may also be a preference for reverse transcytosis (apical to basolateral transcytosis) as compared to forward (basolateral to apical) transcytosis.
- a preference for reverse trancytosis is desirable in aspects ofthe invention where delivery of complex and compounds from the lumen of an organ to the circulatory system is the desired goal.
- compositions and compounds ofthe invention include, by way of non-limiting example, increased stability of complexes and compounds in vitro or in vivo; increased yield or improved purity of complexes and compounds, particularly as produced by recombinant DNA expression systems; removal or reduction of one or more undesirable properties, e.g., undesired side effects; and the like.
- Desirable attributes include, but are not limited to, those related to the of transport complexes and compounds through cells, particularly epithelial cells, and epithelial or mucosal barriers, i.e., transcellular properties, endocytotic properties, transcytotic properties, exocytotic properties, and paracellular transporting properties.
- desirable attributes include, but are not limited to, an increase in the relative rate of a process such as endocytosis, transcytosis, exocytosis, transcellular and paracellular transport, or a preference for transcytosis in one direction or another, apical to basolateral transcytosis and transcellular movement being preferred.
- Another type of desirable attribute involves the binding properties ofthe complex or compound, including, by way of non-limiting example, a change in affinity or avidity for a ligand.
- Another type of desirable attribute involves pharmacological properties such as half-life, decreased secretion, efficacy, selectivity, and the like.
- the invention provides a complex or compound comprising 2 or more targeting elements directed to one or more ligands that confer transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to the ligand, and at least one biologically active portion.
- Such multivalent bioactive complexes and compounds preferably have enhancements in one or more desirable attributes as compared to similar complexes and compounds that have only 1 targeting element directed to the ligand.
- the invention provides a complex or compound comprising a biologically active portion and a targeting element directed to a ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to said ligand, wherein said targeting element is not an antibody, wherein said complex or compound, or a biologically active portion or metabolite thereof, is absorbed from the lumen of an organ into the body of an animal.
- the invention provides a complex or compound comprising a biologically active portion and a targeting element directed to a ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to said ligand, wherein said targeting element is not an antibody, and at least one biologically active portion, wherein said complex or compound, or a biologically active portion or metabolite thereof, is absorbed from the lumen of an organ into the body of an animal.
- Another aspect ofthe invention is a complex or compound comprising 2 or more targeting elements directed to one or more ligands that confer transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to said ligand, and at least one biologically active portion, wherein said complex or compound, or a biologically active portion or metabolite thereof, is absorbed from the lumen of an organ into the body of an animal.
- Epithelial cells representing a cellular barrier, line the interior of said lumen.
- Lumen of particular interest include, by way of non-limiting example, gastrointestinal lumen, the pulmonary lumen, the nasal lumen, a nasopharyngeal lumen, a pharyngeal lumen, a buccal lumen, a sublingual lumen, a vaginal lumen, a urogenital lumen, an ocular lumen, a tympanic lumen, an ocular surface, uterine, urethral, bladder, mammary, salivary, lacrimal, respiratory sinus, biliary, sweat gland.
- the complex or compound may be delivered to a fluid portion ofthe body, e.g., to the blood, lymph, interstitial fluid or amniotic fluid ofthe animal.
- the complex or compound is preferably delivered into the body with a pharmacokinetic profile that results in the delivery of an effective dose of said compound or a biologically active portion thereof.
- the complexes and compounds ofthe invention are capable of undergoing one or more of a variety of processes relating to molecular transport.
- a complex or compound will be capable of undergoing transcellular movement, and/or apical to basolateral transcytosis; apical endocytosis, basolateral exocytosis, intracellular transport can lead to delivery to an intracellular compartment, i.e., an organelle.
- the complex or compound is, preferably if need be, transported across a cellular barrier.
- the cellular barrier may be an epithelial or mucosal barrier.
- the invention further provides pharmaceutical composition comprising the complexes and compounds.
- the pharmaceutical compositions can further compromise one or more antiproteases or carrier polypeptides.
- Representative antiproteases include, but are not limited to, leupeptin, aprotinin, and chymostatin.
- Representative carrier proteins include, but are not limited to, albumin, serum albumin, ovalbumin, casein, whey, soy bean protein, hemoglobin, and gluten.
- the invention further provides a method of delivering a biologically active agent to an animal in need thereof, comprising contacting said animal with the complex or compound ofthe invention.
- the invention provides a method for transporting a biologically active agent through an epithelial or mucosal barrier, comprising contacting said epithelial or mucosal barrier with a complex or compound ofthe invention.
- the invention further provides a method of treating a disease in an animal, comprising contacting said animal with a complex or compound ofthe invention.
- the invention further provides medical devices and kits comprising the pharmaceutical compositions ofthe invention.
- the complexes and compounds ofthe invention may further comprises a detectable moiety These complexes and compounds are used in methods of identifying a disease in an animal, comprising contacting said animal with the complex or compound.
- the invention further provides a diagnostic composition comprising the detectably labeled compound or composition, and diagnostic kits comprising the diagnostic composition.
- Figure 1 shows forward and reverse transcytotic pathways ofthe polyimmunoglobulin receptor (plgR) in epithelial cells.
- plgR polyimmunoglobulin receptor
- Figure 2 shows alignments ofthe amino acid sequences of plgR homologs.
- Figure 3 shows the nucleotide sequence of pSyn5AF (SEQ ID NO:l), a plasmid that encodes single chain antibody sFv5AF.
- the emboldened nucleotide sequence indicates the reading frame (ATG, start codon; TAA, stop codon); boxed sequences indicate restriction enzyme sites (aagctt, Hind III site; gaattc, EcoRI site).
- Figure 4 shows the amino acid sequence (SEQ ID NO:2) ofthe secreted form ofthe sFv5AF encoded by pSyn5AF. Symbols: Pelb leader, a leader sequence that directs secretion from E. coli; FLAG, FLAG epitope; linker, amino acid sequence (GGGGS)3; myc, c-myc epitope; 6 HIS, 6xHis tag; CDR, complementarity-determining region; FR, framework element; and the heavy and light chains ofthe sFv are indicated.
- Pelb leader a leader sequence that directs secretion from E. coli
- FLAG FLAG epitope
- linker amino acid sequence (GGGGS)3
- myc c-myc epitope
- 6 HIS 6xHis tag
- CDR complementarity-determining region
- FR framework element
- the heavy and light chains ofthe sFv are indicated.
- the sequence of sFv5AF is similar to that of sFv5A, but a FLAG tag is present in sFv5AF, and the 5th residue in the sFv sequence is glutamine (Q) in 5A and valine (V) in 5AF.
- the amino- terminal Pelb leader sequence is MKYLLPTAAAGLLLLAAQPAMA, and the carboxy terminal sequence is AAAEQKLISEEDLNGAAHHHHHH.
- Figure 5 shows the amino acid sequence ofthe secreted form ofthe sFv5AF-Cys (SEQ ID NO: 12).
- the sFv5AF-Cys protein consists of, from an amino- to carboxy- terminal direcetion, a pelb leader (for secretion in E.
- coli a FLAG epitope tag
- a heavy chain variable region a spacer sequence [GGGGS repeated three times, i.e., (G4S)3], a light chain variable region, another (G4S)3 linker, a cysteine residue (emboldened “C") that has been introduced into the sFv relative to sFv5AF, a c-myc epitope tag, and a 6xHis tag (for purification by Immobilized Metal-ion Affinity Chromatography, MAC).
- the framework (FR) and complementarity-determining regions (CDR) ofthe heavy chain and light chain are indicated.
- the non-immuno globulin regions (Pelb leader, FLAG epitope tag, linker (G4S)3, c-myc tag and 6xHis tag) are shaded.
- the amino acid sequence of sFv5AF differs from sFv5A in that the 5th residue in the sFv sequence is changed from a glutamine (Q) to a valine (V) amino acid residue.
- Figures 6, 7 and 8 illustrate reaction schemes for of forming a disulfide bond between two different binding regions (e.g., between 2 sFv molecules, 2 Fab molecules, or a sFv and a Fab molecule).
- Figure 7 illustrates a bispecific binding molecule prepared using one sFv that has been derivatized with 2-iminothiolane, and one sFv that has been derivatized with SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate.
- SPDP N-succinimidyl-3-(2-pyridyldithio
- Figure 9 shows the nucleotide sequence of a cDNA that encodes, and the amino acid sequence of, human calcitonin (GenBank Accession No. M26095; SEQ ID NOS:7-8). The start (ATG) and stop (TAA) codons are underlined.
- Figure 10 shows the nucleotide sequence of a cDNA that encodes, and the amino acid sequence of, salmon calcitonin (GenBank Accession No. 64312; SEQ ID NOS:9-10). The start (ATG) and stop (TGA) codons are underlined.
- Figure 11 shows an amino acid sequence alignment for several representative calcitonin proteins from different species.
- Figure 12 shows the strategy and sequences used to clone mouse plgR sequences.
- Figure 13 shows the strategy and sequences used to clone human plgR sequences.
- Figure 14 shows a sfrategy and sequences for cloning rat plgR sequences.
- Figure 15 shows the chimeric rabbit/rat plgR molecule.
- Panel 15A shows the structure ofthe chimeric plgR.
- Panel 15B shows the amino acid sequence ofthe chimeric plgR (SEQ ID NO: 13).
- the transmembrane domain ofthe chimera is underlined.
- the rat portion ofthe molecule is emboldened. This segment consists of half of domain 5, domain 6, and most ofthe transmembrane domain.
- the cleavage site ofthe signal sequence is indicated by a filled circle.
- Figure 16 shows the amino acid sequences for various plgR species encoded within GST-pIgR fusion proteins. Amino acids not contained within the plgR protein are shown in bold and underlined. The most amino terminal amino acids in the sequences (GS) denote the amino acid residues glycine and serine residues contained at the carboxy terminus ofthe GST portion ofthe fusion protein. The carboxy termini ofthe fusion proteins contain additional amino acids not contained within the plgR protein; in some cases these additional residues include a "His epitope tag" (HHHHHH). A consensus amino acid sequence for this part ofthe plgR protein is shown below the sequences for cynomolgus, human, rat and rabbit sequences.
- Figure 17 shows the partial amino acid sequence of a bacterial adhesion protein, CbpA (SEQ ID NO: 14). Emboldened and underlined amino acid sequences indicate amino acid sequences that bind, or contain an element that binds, plgR.
- Figure 18 shows the transwell transcytosis assay system.
- Figure 19 shows the results of assays that compare the transcytosis of sFv5AF-Cys monomers and dimers.
- Figure 20 shows the results of assays that demonstrate sFv5AF-mediated Ml antibody transcytosis.
- Figure 21 shows the time course of transcytosis of monovalent (monomers) and multivalent (dimers) sFv5 molecules.
- Figure 22 shows the nucleotide sequence of a cDNA that encodes, and the amino acid sequence of, human growth hormone (hGH; GenBank Accession No. 4503988; SEQ ID NOS:3-4).
- the reading frame for hGH is emboldened and the start (ATG) and stop (TAG) codons thereof are underlined.
- Figure 23 shows the pharmacokinetic profile of a sFv5AF-human growth hormone fusion protein (5AF-hGH) in rats.
- Panel (A) shows the response following intravenous (IV) administration of 0.33 mg/kg of 5AF-hGH.
- Panel (B) shows the response following intrajejunal (IJ) of 2.0 mg/kg of 5AF-hGH.
- the data represent mean of two animals per dosing group.
- Figure 24 shows the sequence of a cDNA encoding human interleukin-2 (IL-2; SEQ ID NO: 11).
- Figure 25 shows the sequence of a cDNA encoding human interleukin-4 (IL-4; GenBank Accession No. M13982; SEQ ID NO: 15).
- the start (ATG) and stop codons (TGA) ofthe IL-4 reading frame are underlined.
- Figure 26 shows the nucleotide sequence of a cDNA that encodes, and the amino acid sequence of, human insulin (GenBank Accession No. 4557670; SEQ ID NOS:5-6).
- the start (ATG) and stop (TAG) codons are underlined.
- proinsulin is post-translationally cleaved into two chains (peptide A and peptide B) that are covalently linked via two disulfide bonds. Binding of this mature form of insulin to the insulin receptor (ESfSR) stimulates glucose uptake.
- ESfSR insulin receptor
- Table 12 GST-Stalk Fusion Proteins
- Table 13 Crosslinkers Used to Derivatize Salmon Calcitonin
- the inventions disclosed herein relate to complexes and compounds that pass through cellular barriers to deliver compounds into, through and out of cells, and methods of producing and using such complexes and compounds.
- the complexes and compounds ofthe invention comprise a biologically active portion and a targeting element directed to a ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to the ligand, with the proviso that the targeting element is not an antibody.
- a complex or compound ofthe invention comprises 2 or more targeting elements directed to a ligand that confers transcellular, transcytotic or paracellular transporting properties to an agent specifically bound to the ligand.
- Preferred ligands include but are not limited to the stalk of plgR, a plgR domain, an amino acid sequence that is conserved among plgR's from different animals, and one of several regions of plgR defined herein.
- a polyimmunoglobulin receptor (plgR) molecule has several structurally and functionally distinct regions that are defined as follows.
- a plgR molecule is generally described as consisting of two different, loosely defined regions called the "stalk” and the “secretory component” (SC).
- a plgR molecule binds polymeric immunoglobulins (IgA or IgM) on the basolateral side, and then transports the immunoglobulin to the apical side. Proteolyic cleavage of plgR takes place on the apical side of an epithelial cell between the SC and the stalk.
- the SC molecule is released from the cellular membrane and remains bound to and protects the immunoglobulins, whereas the stalk molecule remains bound to the cellular membrane (see "Mucosal Immunoglobulins" by Mestecky et al. in: Mucosoal Immunology, edited by P.L. Ogra, M.E. Lamm, J. Bienenstock, and J.R. McGhee, Academic Press, 1999).
- plgR molecules are those described in U.S. Patent 6,042,833, and the simian plgR described in U.S. patent application Serial No. 60/266,182 (attorney docket No. 057220.0701) entitled "Compositions and Methods for Identifying,
- plgR also refers to any of that receptor's family or superfamily members, any homolog of those receptors identified in other organisms, any isoforms of these receptors, any plgR-like molecule, as well as any fragments, derivatives, mutations, or other modifications expressed on or by cells such as those located in the respiratory tract, the gastrointestinal tract, the urinary and reproductive tracts, the nasal cavity, buccal cavity, ocular surfaces, dermal surfaces and any other mucosal epithelial cells.
- plgR and plgR-like proteins are those that direct the endocytosis or transcytosis of proteins into or across epithelial cells.
- secretory component and "SC” refers to the smallest (shortest amino acid sequence) portion of an apical proteolyzed plgR molecule that retains the ability to bind immunoglobulins (IgA and IgM). After proteolytic cleavage of plgR, some amino acid residues remain associated with SC ⁇ mmunoglobulin complexes but are eventually degaraded and/or removed from such complexes (Ahen et al., J. Clin. Invest. 77: 1841-1848, 1986). According to the defmiton ofthe secretory component used herein, such amino acids are not part ofthe SC. In certain embodiments ofthe invention, plgR- targeting elements that do not recognize or bind to the SC are preferred.
- stalk refers to a molecule having an amino acid sequence derived from a plgR, wherein the stalk sequence does not comprise amino acid sequences derived from the SC.
- a stalk molecule comprises amino acid sequences that remain bound to the apical membrane following the apical proteolytic cleavage when such cleavage occurs and amino acid sequences required for such cleavage.
- Preferred stalk molecules confer one or more transcytotic properties to a ligand bound thereto. Most preferred are stalk molecules that confer the ability to undergo apical to basolateral transcytosis to a ligand bound thereto.
- a protein "domain” is a relatively small (i.e., ⁇ about 150 amino acids) globular unit that is part of a protein.
- a protein may comprise two or more domains that are linked by relatively flexible stretches of amino acids.
- a given domain may be largely or wholly responsible for carrying out functions that are normally carried out by the intact protein.
- domains that have been determined by in vitro manipulations of protein molecules, it is understood in the art that a "domain” may also have been identified in silico, i.e, by software designed to analyze the amino acid sequences encoded by a nucleic acid in order to predict the limits of domains.
- the latter type of domain is more accurately called a "predicted” or “putative” domain but, in the present disclosure, the term domain encompasses both known and predicted domains unless stated otherwise.
- Domains of plgR molecules include a leader sequence, extracellular domains 1 through 6, a transmembrane domain and an intracellular domain (see Figure 2 herein and Figure 3 of Piskurich et al., J. Immunol. 154:1735-1747, 1995).
- the intracellular domain contains signals for transcytosis and endocytosis.
- Domains of a plgR molecule that are of particular interest in the present disclosure include but are not limited to domain 5, domain 6, the transmembrane domain and the intracellular domain. Preferred domains confer the ability to undergo apical to basolateral transcytosis to a ligand bound thereto.
- plgR molecules Another way in which different portions of a plgR molecule can be defined is by reference to amino acid sequences that are conserved between plgR homologs (i.e., plgR molecules isolated from non-human species; see below).
- conserved amino acid sequences include those found in Table 1; see also Figure 2. (For brevity's sake, the one letter abbreviations for amino acids is used in Table 1, but versions of sequences that employ the three letter amino acid designations may be found in the Sequence Listing; see also Table 2.) TABLE 1 : AMINO ACID SEQUENCES THAT ARE CONSERVED IN PIGR HOMOLOGS
- a specific internal portion of a given plgR molecule might be defined as a region that has an amino-terminal border that has the amino acid sequence EKYWCKW and a carboxy-terminal border having the amino acid sequence side having the amino acid sequence DEGWYWCG.
- the region so defined would be the amino acid sequence of residues 474 through 529.
- regions of any given plgR molecule that are of particular interest include but are not limited to the regions described in Table 4 that are not conserved between plgR homologs from different species: TABLE 4: REGIONS OF PIGR AND STALK MOLECULES
- Preferred regions confer the ability to undergo apical to basolateral transcytosis to a ligand bound thereto.
- Target molecules derived from a plgR molecule, a secretory component (SC) molecule, or a stalk molecule, or to domains, conserved sequences, and defined regions thereof, are prepared as described herein and used as target molecules for the preparation of ligands and targeting elements ofthe invention.
- Preferred target molecules do not comprise amino acid sequences derived from the SC.
- Target molecules may be chimeric, i.e., hybrid molecules derived from molecules from at least two different species.
- An example of a chimeric stalk target molecule is the rat/rabbit hybrid stalk molecule described herein.
- a target molecule may also be a fusion protein, such as the domain 6-GST fusion proteins described in the Examples.
- Preferred target molecules confer the ability to undergo apical to basolateral transcytosis to a ligand bound to a plgR molecule or a stalk molecule, wherein the ligand does not bind specifically to an SC molecule.
- Other preferred target molecules comprise sequences from a stalk molecule.
- Target molecules may be produced using suitable techniques such as recombinant gene expression systems, chemical or enzymatic digestion of plgR, SC or stalk molecules, or by in vitro synthesis of oligopeptides. Additionally or alternatively, target molecules may be genetically expressed in cells for techniques and experiments designed to assess transcytotic properties.
- Homologs of plgR are also within the scope ofthe invention.
- Homologs of plgR are plgR proteins from species other than Homo sapiens.
- plgR proteins from various species include those from humans, the rat, mouse, rabbit, cow and possum (Table 5). See also Figure 3 in Mostov and Kaetzel, Chapter 12, "Immunoglobulin Transport and the Polymeric Immunoglobulin Receptor" in Mucosal Immunity, Academic Press, 1999, pages 181-211; and Piskurich et al., J. Immunol. 154:1735-1747, 1995).
- plgR-like Proteins Also within the scope ofthe invention are plgR-like proteins.
- a "plgR-like protein” is a protein that has an amino acid sequence having homolgy to a known plgR protein. In many instances, the amino acid sequences of such plgR-like molecules have been generated by the in silico translation of a nucleic acid, wherein the nucleotide sequence ofthe nucleic acid has been determined but is not known to encode a protein.
- plgR-like proteins include PIGRLl (U.S. Patent 6,114,515); PIGR-1 (U.S.
- Patent 6,232,441 a mouse gene having an exon similar to one of plgR's (GenBank Accession No. 6826652); human proteins translated in silico that have homology to plgR proteins (GenBank Accession Nos. 1062747 and 1062741); and Digrl (Luo et al., Digrl , a novel membrane receptor ofthe immunoglobulin gene superfamily, is preferentially expressed by antigen-presenting cells, Biochem Biophys Res Commun 287(1):35-41, 2001)
- a "homolog" of a plgR protein or a plgR-like protein is an isoform or mutant of human plgR, or a protein in a non-human species that either (i) is “identical” with or is “substantially identical” (determined as described below) to an amino acid sequence in human plgR, or (ii) is encoded by a gene that is identical or substantially identical to the gene encoding human plgR.
- Non-limiting examples of types of plgR isoforms include isoforms of differing molecular weight that result from, e.g., alternate RNA splicing or proteolytic cleavage; and isoforms having different post-translational modifications, such as glycosylation; and the like.
- Two amino acid sequences are said to be “identical” if the two sequences, when aligned with each other, are exactly the same with no gaps, substitutions, insertions or deletions.
- Two amino acid sequences are defined as being “substantially identical” if, when aligned with each other, (i) no more than 30%, preferably 20%, most preferably 15% or 10%, ofthe identities ofthe amino acid residues vary between the two sequences; (ii) the number of gaps between or insertions in, deletions of and subsitutions of, is no more than 10%, preferably 5%, ofthe number of amino acid residues that occur over the length ofthe shortest of two aligned sequences; or (iii) have only conservative amino acid substitutions (in one polypeptide as compared to another) that do not significantly affect the folding or activity ofthe polypeptide.
- amino acid sequence of two proteins may be substantially identical to one another, or sequences within proteins may demonstrate identity or substantial identity with sequences of similar length in other proteins. In either case, such proteins are substantially identical to each other. Typically, stretches of identical or substantially identical sequences occur over 5 to 25, preferably 6 to 15, and most preferably 7 to 10, nucleotides or amino acids.
- nucleotide sequences encoding plgR proteins are substantially identical is if two nucleic acid molecules hybridize to each other under stringent conditions.
- Stringent conditions are sequence dependent and will be different in different circumstances.
- 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.
- Tm is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe.
- stringent conditions will be those in which the salt concentration is about 0.02 M at pH 7 and the temperature is at least about 60°C.
- sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by algorithms such as, for example, the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. U.S.A.
- plgR ligand ofthe invention ability of a plgR ligand ofthe invention to bind different plgR molecules, fragments and derivatives thereof, and to undergo endocytosis, transcytosis, and/or exocytosis is a desirable attribute of these proteins.
- the plgR-binding capacity of fusion proteins are examined using the following techniques. Non-limiting examples of such assays include the following.
- Cell lines that may be used in such assays are generally epithelial cells, particularly polarized cells having apical and basolateral surfaces. Such cells include those that naturally express plgR or the plgR stalk, preferably in response to factors and conditions that can be altered or manipulated, and cells that are transfected with nucleic acids encoding plgR molecules, stalk molecules or target molecules prepared therefrom.
- a non-limiting example ofthe former type of cells are epithelial cells isolated from human trachea, nasopharynx or bronchi. When grown on plastic, these primary cultures down-regulate expression of plgR whereas, when grown on collagen-coated porous filters, the cultures produce plgR (U.S. Patent 6,261,787 Bl and Ferkol et al., Am. J.Respir. Crit. Care Med. 161:944-951, 2000).
- T560 a mouse B lymphoma that originated in gut- associated lymphoid tissue and which expresses plgR
- the IgA/IgM receptor expressed on a murine B cell lymphoma is poly-Ig receptor, J Immunol 2000 Sep 1 ;165(5):2544-55
- Fischer rat thyroid (FRT) cells Sarnataro et al., Detergent insoluble microdomains are not involved in transcytosis of polymeric Ig receptor in FRT and MDCK cells, Traffic 2000 Oct;l(10):794-802; Aging effects on hepatic NADPH cytochrome P450 reductase, CYP2B 1 &2, and polymeric immunoglobulin receptor mRNAs in male Fischer 344 rats).
- MDCK Madin-Darby canine kidney
- the ex vivo plgR binding capacity of a plgR-targeted protein is assessed by measuring endocytosis or transcytosis of bound ligand in mammalian epithelial cells.
- Receptor-mediated endocytosis provides an efficient means of causing a cell to ingest material which binds to a cell surface receptor.
- Any number of well known methods for assaying endocytosis may be used to assess binding. For example, binding, transcytosis, and internalization assays are described at length in Breiftfeld et al. (J. Cell Biol. 109:475- 486, 1989).
- Ligand-pIgR binding is measured by a variety of techniques known in the art, e.g., immunoassays and immunoprecipitation.
- antibodies to the biologically active portion of a protein conjugate can be used to bind and precipitate detectably labeled plgR or stalk molecules; the amount of labeled material thus precipitated corresponds to the degree of plgR binding to a ligand such as, e.g., a protein conjugate having a plgR-targeting element (see Tajima, J. Oral Sci. 42:27-31, 2000).
- Apical endocytosis is conveniently measured by binding a ligand, such as sFv5 A or a derivative thereof (see Figures 3 to 5), to a stalk molecule at the apical surface of transfected Madin-Darby canine kidney (MDCK) cells at 4°C, warming to 37°C. for brief periods (0-10 min), and cooling the cells back down to 4°C.
- Ligand molecules remaining on the surface are removed by stripping at pH 2.3.
- Intracellular ligand molecules are those that remain cell-associated after the stripping, while surface-bound ligand molecules are those removed by the acid wash.
- Controls for non-specific sticking include using molecules that are structurally related to the ligand but which do not bind to a plgR or stalk molecule (e.g., an unrelated sFv in the case of sF5), and/or MDCK cells that are not transfected with genetic sequences encoding a plgR molecule or a stalk molecule.
- a plgR or stalk molecule e.g., an unrelated sFv in the case of sF5
- MDCK cells that are not transfected with genetic sequences encoding a plgR molecule or a stalk molecule.
- Apical to Basolateral transcytosis is assessed by allowing MDCK cells to bind the ligand at the apical surface at 4°C, followed by incubation at 37°C for 0 to 240 min, and then measuring the amount of ligand delivered into the basolateral medium. This basolaterally-delivered ligand is compared to the sum of ligand that remains associated with the cells (intracellular or acid-stripped) and the ligand released back into the apical medium.
- transcystosis is assessed as follows.
- the general protocol for apical to basolateral transcytosis assays was as follows.
- Non-transfected (or wild type) MDCK cells and MDCK cells that have been transfected with a gene encoding a plgR from a variety of species, or hybrid plgR molecules such as the rat/rabbit hybrid plgR described herein, are grown on the surface of a porous membrane in a transwell plate (Corning Costar, #3401, 12 mm diameter, 0.4 micrometer pore size polycarbonate membrane). The cells are grown until they are confluent and form tight junctions that do not allow leakage of substances through the cell layer. MDCK cells are polarized when grown in this manner in a transwell chamber. Cells in franswells are washed 3x with MEM BSA (Sigma No.
- M4642 with 20 mM Hepes, pH 7.4, 0.6 % BSA, containing penicillin and streptomycin
- test or control articles are placed in the upper chamber (apical surface) ofthe transwell compartment in a volume of 300 ⁇ l MEM/BSA.
- the franswells are placed in a 12 well plate with 800 ul of MEM/BSA in the basolateral compartments. After a period of time, usually 8 to 16 hours, samples of the upper and lower (basolateral) chamber are removed and analyzed for the presence of the test and control articles.
- the apical and basolateral media are adjusted to a volume of 1 ml with MEM/BSA.
- One hundred (100) ⁇ l ofthe apical media is then added to 900 1 of MEM/BSA to give a 1/10 dilution.
- the entire ⁇ volume ofthe basolateral media (100%), and the 1/10 dilution of the apical media (10%), are prepared and incubated with an appropriate affinity matrix.
- 500 ⁇ l (50%) ofthe adjusted basolateral media and 50 ul (5%) ofthe adjusted apical media can be assayed.
- protein A sepharose Pharmacia
- 100 ⁇ l of a 10% slurry of protein A sepharose is added to the samples.
- beads are pelleted by centrifugation in a Beckman Microfuge for 2 minutes at full speed. The supernatant is removed, and 1 ml PBS is added, followed by centrifugation and removal ofthe supernatant. The wash is repeated 2 more times. Beads are dried by removing the excess liquid with a Hamilton syringe.
- SDS-PAGE sample buffer Fifty (50) ⁇ l of SDS-PAGE sample buffer is added, samples are boiled for 5 minutes, centrifuged briefly, and 20 ⁇ l is analyzed by SDS-PAGE (typically on an 8-16 % Tris-HCl, 1.0 mm Criterion Precast Gel (Bio-Rad 345-0038) and ran at 150 mamps for 60- 80 minutes) followed by transfer to PVDF filters for Western blotting.
- the filters are probed with antibodies to sFv5A, the FLAG or other epitope tag, or to the biologically active portion ofthe complex or compound of interest. Lanes on the Western blot correspond to samples taken from the apical chamber or the basolateral chamber ofthe transwell compartment.
- the relative intensity ofthe staining between the apical and basolateral lanes is an indication ofthe efficiency of transcytosis. Equal intensity of these bands represents approximately 10% transcytosis, since the volume ofthe apical media that is present on the gel is tenfold less than that ofthe basolateral media.
- Basolateral Endocytosis Basolateral endocytosis is assessed by methods such as those described by Tajima (J. Oral Sci. 42:27-31, 2000).
- Non-specific transport e.g., fluid phase endocytosis and transcytosis, or paracellular leakage between cells
- MDCK cells that are not transfected with a plgR or stalk protein, and/or by the addition of antibody not directed to the plgR or stalk molecule.
- Detectably labeled ligand e.g., a radioiodinated antibody
- a "detectable label” is a composition or moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate method.
- In vivo apical to basolateral (“reverse") transcytosis is assessed by measuring the delivery of a plgR-targeting ligand into the circulation as measured by the presence of a detectable label that has been incorporated into the protein that is being tested.
- the integrity ofthe ligand recovered from the circulation can be assessed by analyzing the ligand on SDS polyacrylamide gel electrophoresis. Such assays are described in more detail in the Examples.
- the binding of a ligand is target-specific in the sense that, although other molecules may be present in a mixture in which ligands and target molecules are contacted with each other, the ligand does not appreciably bind to other (non-target) molecules.
- the strength of binding between plgR and a plgR ligand i.e., the affinity of a plgR ligand for plgR, is a matter of degree.
- target-specific means that the plgR ligand has a stronger affinity for its target molecule (plgR) than for contaminating molecules, and this difference in affinity is sufficient for a given aspect ofthe invention.
- the target specificity of a plgR ligand for plgR is comparable to the specificity of antibodies for their antigens.
- the specificity for a ligand for plgR should be at least approximately that of a single chain antibody (sFv) for plgR.
- sFv's that can be used to evaluate the target specificity of a plgR ligand include but are not limited to sFv5A and derivatives thereof, such as sFv5AF, which bind to the stalk of plgR and are described herein; and sFv's that bind to the secretory component (SC) such as, e.g., those described in U.S. Patent 6,072,041.
- the specificity ofthe binding is defined in terms ofthe values of absolute and relative binding parameters, such as the comparative dissociation constants (Kd) of a ligand for its target molecule as compared to the dissociation constant with respect to the ligand and unrelated molecules and compositions.
- Kd comparative dissociation constants
- the Kd of a ligand with respect to its target molecule will be 2-fold, preferably 5-fold, more preferably 10-fold less, than the Kd ofthe ligand for unrelated molecules and compositions. Even more preferably the Kd will be 50-fold less, more preferably 100-fold less, and more preferably 200-fold less.
- the binding affinity ofthe ligands with respect to target molecules is defined in terms ofthe dissociation constant (Kd).
- Kd dissociation constant
- the value of Kd can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci, M., et al., Byte (1984) 9:340-362. In some situations, direct determination of Kd is problematic and can lead to misleading results. Under such circumstances, a competitive binding assay can be conducted to compare the affinity of a ligand for its target molecule with the affinity of molecules known to bind the target molecule.
- the value ofthe concentration at which 50% inhibition occurs (Ki) is, under ideal conditions, roughly equivalent to Kd.
- Ki cannot be less than Kd; determination of Ki sets a maximal value for the value of Kd. Under circumstances where technical difficulties preclude accurate measurement of Kd, measurement of Ki can conveniently be substituted to provide, at the very least, an upper limit for Kd.
- Kd may be measured in solution using techniques and compositions described in the following publications. Blake, D.A.; Blake, R.C.; Khosraviani, M.; Pavlov, A.R.
- Binding constants and kinetic constants are estimated using calorimetry, equilibrium dialysis, and stopped flow methods using absorbance, fluorsescence, light scattering, turbidity, fluorescence anisotropy, and the like. Additionally or alternatively, Kd is measured using immobilized binding components on a chip, for example, on a BIAcore chip using surface plasmon resonance.
- Binding parameters are measured using surface plasmon resonance, for example, with a BIAcore® chip coated with immobilized binding components.
- Surface plasmon resonance is used to characterize the microscopic association and dissociation constants of reaction between an sFv or other ligand directed against plgG associated molecules and plgR and plgR fragments.
- Such methods are generally described in the following references which are incorporated herein by reference. Vely F. et al., BIAcore analysis to test phosphopeptide-SH2 domain interactions, Methods in Molecular Biology. 121:313-21, 2000; Liparoto et al., Biosensor analysis ofthe interleukin-2 receptor complex, Journal of Molecular Recognition.
- BIAcore® uses the optical properties of surface plasmon resonance (SPR) to detect alterations in protein concentration bound within to a dextran matrix lying on the surface of a gold/glass sensor chip interface, a dextran biosensor matrix.
- SPR surface plasmon resonance
- proteins are covalently bound to the dextran matrix at a known concentration and a ligand for the protein (e.g., antibody) is injected through the dextran matrix.
- a ligand for the protein e.g., antibody
- Near infrared light, directed onto the opposite side ofthe sensor chip surface is reflected and also induces an evanescent wave in the gold film, which in turn, causes an intensity dip in the reflected light at a particular angle known as the resonance angle.
- the refractive index ofthe sensor chip surface is altered (e.g., by ligand binding to the bound protein) a shift occurs in the resonance angle.
- This angle shift can be measured and is expressed as resonance units (RUs) such that 1000 RUs is equivalent to a change in surface protein concentration of 1 ng/mm 2 .
- targeting elements and biologically active molecules are independently small molecules, nucleic acids or polypeptides.
- Examples of compounds and moities that may be used as targeting elements in the compositions and compounds ofthe invention are as follows.
- small molecule includes any chemical or other moiety that can act to affect biological processes.
- Small molecules can include any number of therapeutic agents presently known and used, or can be small molecules synthesized in a library of such molecules for the purpose of screening for biological function(s).
- Small molecules are distinguished from macromolecules by size.
- the small molecules of this invention usually have molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, most preferably less than about 500 Da.
- organic compounds include without limitation organic compounds, peptidomimetics and conjugates thereof.
- organic compound refers to any carbon- based compound other than macromolecules such nucleic acids and polypeptides.
- organic compounds may contain calcium, chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements.
- An organic compound maybe in an aromatic or aliphatic form.
- Non-limiting examples of organic compounds include acetones, alcohols, anilines, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, nucleosides, nucleotides, lipids, retinoids, steroids, proteoglycans, ketones, aldehydes, saturated, unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters, ethers, thiols, sulfides, cyclic compounds, heterocylcic compounds, imidizoles and phenols.
- An organic compound as used herein also includes nitrated organic compounds and halogenated (e.g., chlorinated) organic compounds.
- Preferred small molecules are relatively easier and less expensively manufactured, formulated or otherwise prepared. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules may be placed in tight association with macromolecules to form molecules that are biologically active and that have improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are favorable to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy characteristics ofthe chemical entity.
- nucleic acids have long been known to specifically bind other nucleic acids (e.g., ones having complementary sequences).
- aptamers nucleic acids that bind non-nucleic target molecules have been disclosed. See, e.g., Blackwell et al., Science (1990) 250:1104-1110; Blackwell et al., Science (1990) 250:1149-1152; Tuerk et al., Science (1990) 249:505-510; Joyce, Gene (1989) 82:83-87; and U.S. Patent 5,840,867 entitled "Aptamer analogs specific for biomolecules".
- binding specifically excludes the "Watson- Crick"-type binding interactions (i.e., A:T and G:C base-pairing) traditionally associated with the DNA double helix.
- the term “aptamer” thus refers to a nucleic acid or a nucleic acid derivative that specifically binds to a target molecule, wherein the target molecule is either (i) not a nucleic acid, or (ii) a nucleic acid or structural element thereof that is bound through mechanisms other than duplex- or triplex-type base pairing. Such a molecule is called a "non-nucleic molecule" herein.
- Nucleic acids refers to nucleic acids that are isolated a natural source; prepared in vitro, using techniques such as PCR amplification or chemical synthesis; prepared in vivo, e.g., via recombinant DNA technology; or by any appropriate method. Nucleic acids may be of any shape (linear, circular, etc.) or topology (single- stranded, double-stranded, supercoiled, etc.).
- nucleic acids also includes without limitation nucleic acid derivatives such as peptide nucleic acids (PNA's) and polypeptide-nucleic acid conjugates; nucleic acids having at least one chemically modified sugar residue, backbone, internucleotide linkage, base, nucleoside, or nucleotide analog; as well as nucleic acids having chemically modified 5' or 3' ends; and nucleic acids having two or more of such modifications. Not all linkages in a nucleic acid need to be identical.
- PNA's peptide nucleic acids
- polypeptide-nucleic acid conjugates nucleic acids having at least one chemically modified sugar residue, backbone, internucleotide linkage, base, nucleoside, or nucleotide analog
- nucleic acids having chemically modified 5' or 3' ends and nucleic acids having two or more of such modifications. Not all linkages in a nucleic acid need to be identical.
- Oligonucleotides include without limitation RNA, DNA and mixed RNA-DNA molecules having sequences of lengths that have minimum lengths of 2, 4, 6, 8, 10, 11, 12, 13, 14 or 15 nucleotides, and maximum lengths of about 100, 75, 50, 40, 25, 20 or 15 or more nucleotides, irrespectively. In general, a minimum of approximately 6 nucleotides, preferably 10, and more preferably 14 or 15 nucleotides, is necessary to effect specific binding.
- the oligonucleotides may be single-stranded (ss) or double-stranded (ds) DNA or RNA, or conjugates (e.g., RNA molecules having 5' and 3' DNA “clamps") or hybrids (e.g., RNA:DNA paired molecules), or derivatives (chemically modified forms thereof).
- single-stranded DNA is preferred, as DNA is less susceptible to nuclease degradation than RNA.
- chemical modifications that enhance an aptamer' s specificity or stability are preferred.
- the base residues in aptamers may be other than naturally occurring bases (e.g., A, G, C, T, U, 5MC, and the like).
- Derivatives of purines and pyrimidines are known in the art; an exemplary but not exhaustive list mcludes aziridinylcytosine, 4-acetylcytosine, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylmosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine (5MC), N6- methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2- thiouracil
- the sugar residues in aptamers may be other than conventional ribose and deoxyribose residues.
- substitution at the 2'-position of the furanose residue enhances nuclease stability.
- An exemplary, but not exhaustive list, of modified sugar residues includes 2' substituted sugars such as 2'-O-methyl-, 2'-O-alkyl, 2'- O-allyl, 2'-S-alkyl, 2'-S-allyl, 2'-fluoro-, 2'-halo, or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside, ethyl riboside or propylriboside
- Chemically modified backbones include, by way of non-limiting example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2 '-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
- Chemically modified backbones that do not contain a phosphorus atom have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages, including without limitation morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; and amide backbones.
- nucleic acid targeting element is an aptamer.
- techniques for identifying aptamers involve incubating a preselected non-nucleic target molecule with mixtures (2 to 50 members), pools (50 to 5,000 members) or libraries (50 or more members) of different nucleic acids that are potential aptamers under conditions that allow complexes of target molecules and aptamers to form.
- different nucleic acids it is meant that the nucleotide sequence of each potential aptamer may be different from that of any other member, that is, the sequences ofthe potential aptamers are random with respect to each other.
- Randomness can be introduced in a variety of manners such as, e.g., mutagenesis, which can be carried out in vivo by exposing cells harboring a nucleic acid with mutagenic agents, in vitro by chemical treatment of a nucleic acid, or in vitro by biochemical replication (e.g., PCR) that is deliberately allowed to proceed under conditions that reduce fidelity of replication process; randomized chemical synthesis, i.e., by synthesizing a plurality of nucleic acids having a preselected sequence that, with regards to at least one position in the sequence, is random.
- mutagenesis which can be carried out in vivo by exposing cells harboring a nucleic acid with mutagenic agents, in vitro by chemical treatment of a nucleic acid, or in vitro by biochemical replication (e.g., PCR) that is deliberately allowed to proceed under conditions that reduce fidelity of replication process
- biochemical replication e.g., PCR
- the sequences are increasingly less randomized and consensus sequences may appear; in any event, it is preferred to ultimately obtain an aptamer having a unique nucleotide sequence.
- Aptamers and pools of aptamers are prepared, identified, characterized and/or purified by any appropriate technique, including those utilizing in vitro synthesis, recombinant DNA techniques, PCR amplification, and the like. After their formation, targefcaptamer complexes are then separated from the uncomplexed members ofthe nucleic acid mixture, and the nucleic acids that can be prepared from the complexes are candidate aptamers (at early stages ofthe technique, the aptamers generally being a population of a multiplicity of nucleotide sequences having varying degrees of specificity for the target).
- the resulting aptamer (mixture or pool) is then substituted for the starting apatamer (library or pool) in repeated iterations of this series of steps.
- a limited number e.g., a pool or mixture, preferably a mixture with less than 10 members, most preferably 1
- the aptamer is sequenced and characterized. Pure preparations of a given aptamer are generated by any appropriate technique (e.g., PCR amplification, in vitro chemical synthesis, and the like).
- Tuerk and Gold disclose the use of a procedure termed "systematic evolution of ligands by exponential enrichment" (SELEX).
- SELEX systematic evolution of ligands by exponential enrichment
- pools of nucleic acid molecules that are randomized at specific positions are subjected to selection for binding to a nucleic acid-binding protein (see, e.g., PCT International Publication No. WO 91/19813 and U.S. Pat. No. 5,270,163).
- the oligonucleotides so obtained are sequenced and otherwise characterized.
- Kinzler, K. W., et al. Nucleic Acids Res.
- oligonucleotide combinatorial technique disclosed by Ecker, D. J. et al. (Nuc. Acids Res. 21, 1853 (1993)) known as "synthetic unrandomization of randomized fragments" (SURF), which is based on repetitive synthesis and screening of increasingly simplified sets of oligonucleotide analogue libraries, pools and mixtures (Tuerk, C. and Gold, L. (Science 249, 505 (1990)).
- the starting library consists of oligonucleotide analogues of defined length with one position in each pool containing a known analogue and the remaining positions containing equimolar mixtures of all other analogues. With each round of synthesis and selection, the identity of at least one position ofthe oligomer is determined until the sequences of optimized nucleic acid ligand aptamers are discovered.
- nucleotide sequence can be determined (as is known in the art), and its three-dimensional molecular structure can be examined by nuclear magnetic resonance (NMR).
- NMR nuclear magnetic resonance
- Selected aptamers may be resynthesized using one or more modified bases, sugars or backbone linkages.
- Aptamers consist essentially ofthe minimum sequence of nucleic acid needed to confer binding specificity, but may be extended on the 5' end, the 3' end, or both, or may be otherwise derivatized or conjugated.
- Bioactive nucleic acids, and/or templates therefor can be a bioactive portion of a complex or compound ofthe invention.
- a bioactive nucleic acid maybe an antisense oligonucleotide, an aptamer, an antisense transcript, an enzymatic nucleic acid such as a ribozyme, a ribosomal RNA (rRNA), a transfer RNA (tRNA), or a molecular decoy.
- nucleic acids can be biologically active. These include, by ay of non-limiting example, DNA, including single-stranded (ssDNA) and double-stranded (dsRNA); RNA, including but not limited to ssRNA, dsRNA, tRNA, mRNA, rRNA, enzymatic RNA; RNADNA hybrids; triplexed DNA (e.g., dsDNA in association with a short oligonucleotide); and the like.
- DNA including single-stranded (sDNA) and double-stranded (dsRNA)
- RNA including but not limited to ssRNA, dsRNA, tRNA, mRNA, rRNA, enzymatic RNA
- RNADNA hybrids triplexed DNA (e.g., dsDNA in association with a short oligonucleotide); and the like.
- the sequence of a nucleic acid may be a template for a bioactive nucleic acid such as an antisense transcript or a ribozyme.
- the nucleic acid sequence may be an ORF (open reading frame) that encodes a polypeptide.
- ORFs of particular interest in this aspect ofthe invention include but are not limited to ones that encode a polypeptide that is absent or deficient in a cell; a polypeptide activity or expression of which increased or decreased for therapeutic benefit or diagnostic use; a dominant negative mutant of a polypeptide the activity of which is increased or decreased for therapeutic benefit or diagnostic use; and a detectable polypeptide, which may be used in diagnostic applications.
- polypeptide mcludes proteins, fusion proteins, oligopeptides and polypeptide derivatives, with the exception that peptidomimetics are considered to be small molecules herein.
- Antibodies and antibody derivatives are disclosed in a separate section, but antibodies and antibody derivatives are, for purposes ofthe invention, treated as a subclass ofthe polypeptides and derivatives.
- a "protein” is a molecule having a sequence of amino acids that are linked to each other in a linear molecule by peptide bonds.
- the term protein refers to a polypeptide that is isolated from a natural source, or produced from an isolated cDNA using recombinant DNA technology; and has a sequence of amino acids having a length of at least about 200 amino acids.
- a “fusion protein” is a type of protein that has an amino acid sequence that results from the linkage ofthe amino acid sequences of two or more normally separate polypeptides and which is encoded by a chimeric reading frame. Methods of preparing and using fusion proteins are disclosed in U.S. patent application Serial No. 60/237,929 (attorney docket No. 030854.0009 entitled "Genetic Fusions of plgR Ligands and
- a “protein fragment” is a proteolytic fragment of a larger polypeptide, which may be a protein or a fusion protein.
- a proteolytic fragment may be prepared by in vivo or in vitro proteolytic cleavage of a larger polypeptide, and is generally too large to be prepared by chemical synthesis.
- Proteolytic fragments have amino acid sequences having a length from about 200 to about 1,000 amino acids.
- oligopeptide is a polypeptide having a short amino acid sequence (i.e., 2 to about 200 amino acids).
- An oligopeptide is generally prepared by chemical synthesis.
- oligopeptides and protein fragments may be otherwise prepared, it is possible to use recombinant DNA technology and/or in vitro biochemical manipulations.
- a nucleic acid encoding an amino acid sequence may be prepared and used as a template for in vitro transcription/translation reactions.
- an exogenous nucleic acid encoding a preselected polypeptide is introduced into a mixture that is essentially depleted of exogenous nucleic acids that contains all ofthe cellular components required for transcription and translation.
- One or more radiolabeled amino acids are added before or with the exogenous DNA, and transcription and translation are allowed to proceed.
- the only nucleic acid present in the reaction mix is the exogenous nucleic acid added to the reaction, only polypeptides encoded thereby are produced, and incorporate the radiolabelled amino acid(s).
- polypeptides encoded by a preselected exogenous nucleic acid are radiolabeled.
- the preselected polypeptide is the only one that is produced in the presence ofthe radiolabeled amino acids and is thus uniquely labeled.
- polypeptide derivatives include without limitation mutant polypeptides, chemically modified polypeptides, and peptidomimetics.
- polypeptides of this invention may generally be prepared following known techniques.
- synthetic production ofthe polypeptide ofthe invention may be according to the solid phase synthetic method.
- the solid phase synthesis is well understood and is a common method for preparation of polypeptides, as are a variety of modifications of that technique [Merrifield (1964), J. Am. Chem. Soc, 85: 2149; Stewart and Young (1984), Solid Phase polypeptide Synthesis, Pierce Chemical Company, Rockford, 111.; Bodansky and Bodanszky (1984), The Practice of polypeptide Synthesis, Springer- Verlag, New York; Atherton and Sheppard (1989), Solid Phase polypeptide Synthesis: A Practical Approach, IRL Press, New York].
- polypeptides of this invention may be prepared in recombinant systems using polynucleotide sequences encoding the polypeptides.
- fusion proteins are typically prepared using recombinant DNA technology.
- polypeptide derivatives include without limitation proteins that naturally undergo post- translational modifications such as, e.g., glycosylation. It is understood that a polypeptide ofthe invention may contain more than one ofthe following modifications within the same polypeptide.
- Preferred polypeptide derivatives retain a desirable attribute, which may be biological activity; more preferably, a polypeptide derivative is enhanced with regard to one or more desirable attributes, or has one or more desirable attributes not found in the parent polypeptide. Although they are described in this section, peptidomimetics are taken as small molecules in the present disclosure.
- a polypeptide having an amino acid sequence identical to that found in a protein prepared from a natural source is a "wildtype" polypeptide.
- Mutant oligopeptides can be prepared by chemical synthesis, including without limitation combinatorial synthesis.
- Mutant polypeptides larger than oligopeptides can be prepared using recombinant DNA technology by altering the nucleotide sequence of a nucleic acid encoding a polypeptide. Although some alterations in the nucleotide sequence will not alter the amino acid sequence ofthe polypeptide encoded thereby ("silent" mutations), many will result in a polypeptide having an altered amino acid sequence that is altered relative to the parent sequence. Such altered amino acid sequences may comprise substitutions, deletions and additions of amino acids, with the proviso that such amino acids are naturally occurring amino acids.
- mutant polypeptides particularly ones having substitutions of amino acids but no deletions or insertions thereof.
- a variety of mutagenic techniques are known that can be used in vitro or in vivo including without limitation chemical mutagenesis and PCR-mediated mutagenesis. Such mutagenesis may be randomly targeted (i.e., mutations may occur anywhere within the nucleic acid) or directed to a section ofthe nucleic acid that encodes a stretch of amino acids of particular interest. Using such techniques, it is possible to prepare randomized, combinatorial or focused compound libraries, pools and mixtures.
- Polypeptides having deletions or insertions of naturally occurring amino acids may be synthetic oligopeptides that result from the chemical synthesis of amino acid sequences that are based on the amino acid sequence of a parent polypeptide but which have one or more amino acids inserted or deleted relative to the sequence ofthe parent polypeptide. Insertions and deletions of amino acid residues in polypeptides having longer amino acid sequences may be prepared by directed mutagenesis.
- polypeptide mcludes those having one or more chemical modification relative to another polypeptide, i.e., chemically modified polypeptides.
- the polypeptide from which a chemically modified polypeptide is derived may be a wildtype protein, a mutant protein or a mutant polypeptide, or polypeptide fragments thereof; an antibody or other polypeptide ligand according to the invention including without limitation single-chain antibodies, bacterial proteins and polypeptide derivatives thereof; or polypeptide ligands prepared according to the disclosure.
- the chemical modification(s) confer(s) or improve(s) desirable attributes ofthe polypeptide but does not substantially alter or compromise the biological activity thereof.
- Desirable attributes include but are limited to increased shelf-life; enhanced serum or other in vivo stability; resistance to proteases; and the like. Such modifications include by way of non-limiting example N-terminal acetylation, glycosylation, and biotinylation.
- An effective approach to confer resistance to peptidases acting on the N-terminal or C-terminal residues of a polypeptide is to add chemical groups at to one or both ofthe polypeptide termini, such that the modified polypeptide is no longer a substrate for the peptidase.
- One such chemical modification is glycosylation ofthe polypeptides at either or both termini.
- Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of polypeptides in human serum (Powell et al. (1993), Pharma. Res. 10: 1268-1273).
- N-terminal alkyl group consisting of a lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group.
- N-terminal D-amino acid increases the serum stability of a polypeptide that otherwise contains L-amino acids, because exopeptidases acting on the N- terminal residue cannot utilize a D-amino acid as a substrate.
- C-terminal D-amino acid also stabilizes a polypeptide, because serum exopeptidases acting on the C-terminal residue cannot utilize a D-amino acid as a substrate.
- amino acid sequences of polypeptides with N-terminal and or C-terminal D-amino acids are usually identical to the sequences ofthe parent L- amino acid polypeptide.
- Substitution of unnatural amino acids for natural amino acids in a subsequence of a polypeptide can confer or enhance desirable attributes including biological activity. Such a substitution can, for example, confer resistance to proteolysis by exopeptidases acting on the N-terminus.
- the synthesis of polypeptides with unnatural amino acids is routine and known in the art (see, for example, Coller, et al. (1993), cited above).
- Different host cells will contain different post-translational modification mechanisms that may provide particular types of post-translational modification of a fusion protein if the amino acid sequences required for such modifications is present in the fusion protein.
- a large number ( ⁇ 100) of post-translational modifications have been described, a few of which are discussed herein.
- One skilled in the art will be able to choose appropriate host cells, and design chimeric genes that encode protein members comprising the amino acid sequence needed for a particular type of modification.
- Glycosylation is one type of post-translational chemical modification that occurs in many eukaryotic systems, and may influence the activity, stability, pharmacogenetics, immunogenicity and or antigenicity of proteins. However, specific amino acids must be present at such sites to recruit the appropriate glycosylation machinery, and not all host cells have the appropriate molecular machinery. Saccharomyces cerevisieae and Pichia pastoris provide for the production of glycosylated proteins, as do expression systems that utilize insect cells, although the pattern of glyscoylation may vary depending on which host cells are used to produce the fusion protein.
- Another type of post-translation modification is the phosphorylation of a free hydroxyl group ofthe side chain of one or more Ser, Thr or Tyr residues.
- Protein kinases catalyze such reactions. Phosphorylation is often reversible due to the action of a protein phosphatase, an enzyme that catalyzes the dephosphorylation of amino acid residues.
- bacterial proteins are synthesized with an amino terminal amino acid that is a modified form of methionine, i.e, N-formyl-methionine (fMet).
- fMet N-formyl-methionine
- coli mutants that lack the enzymes (such as, e.g., formylase) that catalyze such post-translational modifications will produce proteins having an amino terminal fMet residue (Guillon et al., J. Bacteriol. 174:4294-4301, 1992).
- acetylation ofthe initiator methionine residue, or the penultimate residue if the initiator methionine has been removed typically occurs co- or post- translationally.
- the acetylation reactions are catalyzed by N-terminal acetyltransferases (NATs, a.k.a.
- N-alpha-acetyltransferases N-alpha-acetyltransferases
- methionine aminopeptidases for reviews, see Bradshaw et al., Trends Biochem. Sci. 23:263-267, 1998; and Driessen et al., CRC Crit. Rev. Biochem. 18:281-325, 1985.
- Amino terminally acetylated proteins are said to be "N-acetylated,” “N alpha acetylated” or simply "acetylated.”
- a polypeptide mimetic is a molecule that mimics the biological activity of a polypeptide but is no longer peptidic in chemical nature.
- a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids).
- the term peptidomimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo- peptides, semi-peptides and peptoids. Examples of some peptidomimetics by the broader definition (where part of a polypeptide is replaced by a stracture lacking peptide bonds) are described below.
- peptidomimetics Whether completely or partially non-peptide, provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the polypeptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems that are similar to the biological activity ofthe polypeptide.
- polypeptides may exhibit two undesirable attributes, i.e., poor bioavailability and short duration of action.
- Peptidomimetics are often small enough to be both orally active and to have a long duration of action.
- stability, storage and immunoreactivity for polypeptides that are not experienced with peptidomimetics.
- Candidate, lead and other polypeptides having a desired biological activity can be used in the development of peptidomimetics with similar biological activities.
- Techniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups ofthe amino acids with other chemical groups of similar stracture.
- the development of peptidomimetics can be aided by determining the tertiary stracture ofthe original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling.
- the present invention provides compounds exhibiting enhanced therapeutic activity in comparison to the polypeptides described above.
- the peptidomimetic compounds obtained by the above methods having the biological activity ofthe above named polypeptides and similar three-dimensional stracture, are encompassed by this invention. It will be readily apparent to one skilled in the art that a peptidomimetic can be generated from any ofthe modified polypeptides described in the previous section or from a polypeptide bearing more than one ofthe modifications described from the previous section. It will furthermore be apparent that the peptidomimetics of this invention can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.
- Proteases act on peptide bonds. It therefore follows that substitution of peptide bonds by pseudopeptide bonds confers resistance to proteolysis. A number of pseudopeptide bonds have been described that in general do not affect polypeptide stracture and biological activity. The reduced isostere pseudopeptide bond is a suitable pseudopeptide bond that is known to enhance stability to enzymatic cleavage with no or little loss of biological activity (Couder, et al. (1993), Int. J. Polypeptide Protein Res.
- amino acid sequences of these compounds may be identical to the sequences of their parent L-amino acid polypeptides, except that one or more ofthe peptide bonds are replaced by an isostere pseudopeptide bond.
- amino acid sequences of these compounds may be identical to the sequences of their parent L-amino acid polypeptides, except that one or more ofthe peptide bonds are replaced by an isostere pseudopeptide bond.
- the most N-terminal peptide bond is substituted, since such a substitution would confer resistance to proteolysis by exopeptidases acting on the N-terminus.
- peptide bonds may also be substituted by retro- inverso pseudopeptide bonds (Dalpozzo, et al. (1993), Int. J. Polypeptide Protein Res.
- amino acid sequences ofthe compounds maybe identical to the sequences of their L-amino acid parent polypeptides, except that one or more ofthe peptide bonds are replaced by a retro- inverso pseudopeptide bond.
- the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus.
- Peptoid derivatives of polypeptides represent another form of modified polypeptides that retain the important structural determinants for biological activity, yet eliminate the peptide bonds, thereby conferring resistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci. USA, 89:9367-9371 and incorporated herein by reference).
- Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups have been described, each conesponding to the side chain of a natural amino acid.
- antibody is meant to encompass an immunoglobulin molecule obtained by in vitro or in vivo generation of an immunogenic response, and mcludes both polyclonal, monospecific and monoclonal antibodies.
- An "immunogenic response” is one that results in the production of antibodies directed to one or more proteins after the appropriate cells have been contacted with such proteins, or polypeptide derivatives thereof, in a manner such that one or more portions ofthe protein function as epitopes.
- An epitope is a single antigenic determinant in a molecule. In proteins, particularly denatured proteins, an epitope is typically defined and represented by a contiguous amino acid sequence.
- epitopes also include structures, such as active sites, that are formed by the three-dimensional folding of a protein in a manner such that amino acids from separate portions ofthe amino acid sequence ofthe protein are brought into close physical contact with each other.
- Wildtype antibodies have four polypeptide chains, two identical heavy chains and two identical light chains. Both types of polypeptide chains have constant regions, which do not vary or vary minimally among antibodies ofthe same class (i.e, IgA, IgM, etc.), and variable regions. As is explained below, variable regions are unique to a particular antibody and comprise a recognition element for an epitope.
- Each light chain of an antibody is associated with one heavy chain, and the two chains are linked by a disulfide bridge formed between cysteine residues in the carboxy- terminal region of each chain, which is distal from the amino terminal region of each chain that constitutes its portion ofthe antigen binding domain.
- Antibody molecules are further stabilized by disulfide bridges between the two heavy chains in an area known as the hinge region, at locations nearer the carboxy terminus ofthe heavy chains than the locations where the disulfide bridges between the heavy and light chains are made.
- the hinge region also provides flexibility for the antigen-binding portions of an antibody.
- variable regions located in the amino terminal regions ofthe light and heavy chains are determined by the variable regions located in the amino terminal regions ofthe light and heavy chains.
- the variable regions of a light chain and associated heavy chain form an "antigen binding domain" that recognizes a specific epitope; an antibody thus has two antigen binding domains.
- the antigen binding domains in a wildtype antibody are directed to the same epitope of an immunogenic protein, and a single wildtype antibody is thus capable of binding two molecules ofthe immunogenicschreib protein at the same time.
- compositions of antibodies have, depending on the manner in which they are prepared, different types of antibodies.
- Types of antibodies of particular interest include polyclonal, monospecific and monoclonal antibodies.
- Polyclonal antibodies are generated in an immunogenic response to a protein having many epitopes.
- a composition of polyclonal antibodies thus mcludes a variety of different antibodies directed to the same and to different epitopes within the protein.
- Methods for producing polyclonal antibodies are known in the art (see, e.g., Cooper et al., Section HI of Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 11-37 to 11-41).
- Monospecific antibodies are generated in a humoral response to a short (typically, 5 to 20 amino acids) immunogenic polypeptide that corresponds to a few (preferably one) isolated epitopes ofthe protein from which it is derived.
- a plurality of monospecific antibodies mcludes a variety of different antibodies directed to a specific portion ofthe protein, i.e, to an amino acid sequence that contains at least one, preferably only one, epitope.
- a monoclonal antibody is a specific antibody that recognizes a single specific epitope of an immunogenic protein.
- each antibody molecule is identical to the others in the plurality.
- a clonal cell line that expresses, displays and/or secretes a particular monoclonal antibody is first identified; this clonal cell line can be used in one method of producing the antibodies ofthe invention.
- Methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are known in the art (see, for example, Fuller et al., Section II of Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 11-22 to 11-11-36).
- Variants and derivatives of antibodies include antibody and T-cell receptor fragments that retain the ability to specifically bind to antigenic determinants.
- Preferred fragments include Fab fragments (i.e, an antibody fragment that contains the antigen- binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab' (an antibody fragment containing a single anti-binding domain comprising an Fab and an additional portion ofthe heavy chain through the hinge region); F(ab')2 (two Fab' molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab' molecules may be directed toward the same or different epitopes); a bispecific Fab (an Fab molecule having two antigen binding domains, each of which maybe directed to a different epitope); a single chain Fab chain comprising a variable region, a.k.a., a sFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by
- antibody also includes genetically engineered antibodies and/or antibodies produced by recombinant DNA techniques and "humanized” antibodies.
- Humanized antibodies have been modified, by genetic manipulation and/or in vitro treatment to be more human, in terms of amino acid sequence, glycosylation pattern, etc., in order to reduce the antigenicity ofthe antibody or antibody fragment in an animal to which the antibody is intended to be administered (Gussow et al., Methods Enz. 203:99- 121, 1991).
- the antibodies and antibody fragments ofthe invention may be produced by any suitable method, for example, in vivo (in the case of polyclonal and monospecific antibodies), in cell culture (as is typically the case for monoclonal antibodies, wherein hybridoma cells expressing the desired antibody are cultured under appropriate conditions), in in vitro translation reactions, and in recombinant DNA expression systems (the latter method of producing proteins is disclosed in more detail herein in the section entitled "Methods of Producing Fusion Proteins").
- Antibodies and antibody variants can be produced from a variety of animal cells, preferably from mammalian cells, with murine and human cells being particularly preferred.
- Antibodies that include non-naturally occurring antibody and T-cell receptor variants that retain only the desired antigen targeting capability conferred by an antigen binding site(s) of an antibody can be produced by known cell culture techniques and recombinant DNA expression systems (see, e.g., Johnson et al., Methods in Enzymol. 203:88-98, 1991; Molloy et al., Mol. Immunol. 32:73-81, 1998; Schodin et al., J. Immunol. Methods 200:69-77, 1997).
- Recombinant DNA expression systems are typically used in the production of antibody variants such as, e.g., bispecific antibodies and sFv molecules.
- Preferred recombinant DNA expression systems include those that utilize host cells and expression constructs that have been engineered to produce high levels of a particular protein.
- Prefe ⁇ ed host cells and expression constructs include Escherichia coli; harboring expression constructs derived from plasmids or viruses (bacteriophage); yeast such as Saccharomyces cerevisiae or Pichia pastoris harboring episomal or chromosomally integrated expression constructs; insect cells and viruses such as Sf 9 cells and baculovirus; and mammalian cells harboring episomal or chromosomally integrated (e.g., retroviral) expression constructs (for a review, see Verma et al., J. Immunol.
- Antibodies can also be produced in plants (U.S. Patent 6,046,037; Ma et al., Science 268:716-719, 1995) or by phage display technology (Winter et al., Annu. Rev. Immunol. 12:433-455, 1994).
- XenoMouse strains are genetically engineered mice in which the murine IgH and Igk loci have been functionally replaced by their Ig counterparts on yeast artificial YAC transgenes. These human Ig transgenes can carry the majority ofthe human variable repertoire and can undergo class switching from IgM to IgG isotypes. The immune system ofthe xenomouse recognizes administered human antigens as foreign and produces a strong humoral response. The use of XenoMouse in conjunction with well-established hybridomas techniques, results in fully human IgG mAbs with sub-nanomolar affinities for human antigens (see U.S. Patents Nos. 5,770,429, entitled “Transgenic non-human animals capable of producing heterologous antibodies"; 6,162,963, entitled "Generation of
- Xenogenetic antibodies 6,150,584, entitled “Human antibodies derived from immunized xenomice”; 6,114,598, entitled Generation of xenogeneic antibodies; and 6,075,181, entitled “Human antibodies derived from immunized xenomice”; for reviews, see Green, Antibody engineering via genetic engineering ofthe mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies, J. Immunol.
- a “fusion protein” is a single protein having amino acid sequences derived from two or more normally separate proteins, and which is encoded by a chimeric reading frame.
- Polypeptides for the Delivery of Therapeutic and Diagnostic Proteins is drawn to fusion proteins comprising plgR ligands and biologically active polypeptides.
- polypeptides which are polymers of amino acids, are encoded by another class of molecules, known as nucleic acids, which are polymers of structural units known as nucleotides.
- proteins are encoded by nucleic acids known as DNA and RNA (deoxyribonucleic acid and ribonucleic acid, respectively).
- the nucleotide sequence of a nucleic acid contains the "blueprints" for a protein.
- Nucleic acids are polymers of nucleotides, four types of which are present in a given nucleic acid.
- the nucleotides in DNA are adenine, cytosine and guanine and thymine, (represented by A, C, G, and T respectively); in RNA, thymine (T) is replaced by uracil (U).
- the structures of nucleic acids are represented by the sequence of its nucleotides ananged in a 5' ("5 prime”) to 3' ("3 prime”) direction, e.g.,
- proteins are typically produced in the following manner.
- a DNA molecule that has a nucleotide sequence that encodes the amino acid sequence of a protein is used as a template to guide the production of a messenger RNA (mRNA) that also encodes the protein; this process is known as transcription.
- mRNA messenger RNA
- transcription In a subsequent process called translation, the mRNA is "read” and directs the synthesis of a protein having a particular amino acid sequence.
- Each amino acid in a protein is encoded by a series of three contiguous nucleotides, each of which is known as a codon.
- some amino acids are encoded by several codons, each codon having a different sequence; whereas other amino acids are encoded by only one codon sequence.
- An entire protein i.e., a complete amino acid sequence
- a reading frame is a continuous nucleotide sequence that encodes the amino acid sequence of a protein; the boundaries of a reading frame are defined by its initiation (start) and termination (stop) codons.
- RNA (A-U-G) - (A-A-G) - (C-C-G) - (C-U-C) - (C-C-U) - . . . (etc . )
- a chimeric reading frame encoding a fusion protein is prepared as follows.
- a "chimeric reading frame” is a genetically engineered reading frame that results from the fusion of two or more normally distinct reading frames, or fragments thereof, each of which normally encodes a separate polypeptide. Using recombinant DNA techniques, a first reading frame that encodes a first amino acid sequence is linked to a second reading frame that encodes a second amino acid sequence in order to generate a chimeric reading frame.
- Chimeric reading frames may also include nucleotide sequences that encode optional fusion protein elements (see below).
- a hypothetical example of a chimeric reading frame created from two normally separate reading frames is depicted in the following flowchart.
- a first Reading Frame and "Protein- 1" is depicted in the following flowchart.
- DNA-1 (A-T-G) - (A-A-G) - (C-C-G) - (C-T-C) - (C-C-T) - ... (etc.)
- RNA-l (A-U-G) - (A-A-G) - (C-C-G) - (C-U-C) - (C-C-U) - ... (etc.)
- DNA-2 ( ⁇ -G ⁇ G) - (G-T-T) - (A-C-T) - (C-A-C) - (T-C-A) - ... (etc.)
- RNA-2 (U-G-G) - (G-U-U) - (A-C-U) - (C-A-C) - (U-C-A) - ... (etc . )
- DNA-Chimera (A-T-G) - (A-A-G) - (C-C-G) - (C-A-C) - (T-C-A) - ... (etc . )
- RNA-Chimera (A-U-G) - (A-A-G) - (C-C-G) - (C-A-C) - (U-C-A) - ... (etc . ) J- Translation
- each normally distinct reading frame therein must be fused to all ofthe other normally distinct reading frames in a manner such that all ofthe reading frames are in frame with each other.
- in frame with each other it is meant that, in a chimeric reading frame, a first nucleic acid having a first reading frame is covalently linked to a second nucleic acid having a second reading frame in such a manner that the two reading frames are "read” (translated) in register with each other.
- the chimeric reading frame encodes one extended amino acid sequence that includes the amino acid sequences encoded by each ofthe normally separate reading frames.
- a fusion protein ofthe invention comprises a polypeptide having the amino acid sequence of a monoclonal antibody and a polypeptide that is a targeting element.
- the targeting element may be an antibody derivative, such as a single-chain antibody, or some other polypeptide capable of binding the molecular target.
- Non-limiting examples of polypeptides that are plgR-targeting elements are described in Example 1.
- the fusion proteins ofthe invention may further comprise one or more non-biologically active amino acid sequences, i.e., optional fusion protein elements.
- non biologically active elements include, but are not limited to, the following optional fusion protein elements. It is understood that a chimeric reading frame will include nucleotide sequences that encode such optional elements, and that these nucleotide sequences will be positioned so as to be in frame with the reading frame encoding the fusion protein.
- Optional fusion protein elements may be inserted between the plgR-targeting element and the biologically active polypeptide, upstream or downstream (amino proximal and carboxy proximal, respectively) of these and other elements, or within the plgR-targeting element and the biologically active polypeptide.
- a person skilled in the art will be able to determine which optional element(s) should be included in a fusion protein ofthe invention, and in what order, based on the desired method of production or intended use ofthe fusion protein.
- Protein delivery elements are optional fusion protein elements that facilitate the uptake of a protein into cells but which are not plgR targeting elements.
- the ETA (detoxified exotoxin A) protein delivery element is described in U.S. Patent No. 6,086,900 to Draper.
- the VP22 protein delivery element is derived from herpes simplex virus- 1 and vectors containing sequences encoding the VP22 protein delivery element are commercially available from Invitrogen (Carlsbad, CA; see also U.S. Patent No. 6,017,735 to Ohare et al.).
- the Tat protein delivery element is derived from the amino acid sequence ofthe Tat protein of human immunodeficiency virus (HIV). See U.S. Patents 5,804,604; 5,747,641; and 5,674,980.
- Organellar delivery elements are optional fusion protein elements that direct a fusion protein into or out of a specific organelle or organelles.
- the ricin A chain can be included in a fusion protein to mediate its delivery from the endosome into the cytosol.
- delivery elements for other organelles or subcellular spaces such as the nucleus, nucleolus, mitochondria, the Golgi apparatus, the endoplasmic reticuluHi (ER), the cytoplasm, etc.
- Mammalian expression constructs that incorporate organellar delivery elements are commercially available from Invitrogen (Carlsbad, CA: pShooterTM vectors).
- H/KDEL i.e, His /Lys-Asp-Glu-Leu sequence
- H/KDEL i.e, His /Lys-Asp-Glu-Leu sequence
- a fusion protein ofthe invention preferably at the carboxy-terminus, in order to direct a fusion protein to the ER (see Andres et al., J. Biol. Chem. 266: 14277- 142782, 1991; and Pelham, Trends Bio. Sci. 15:483-486, 1990).
- organellar delivery element is one which directs the fusion protein to the cell membrane and which may include a membrane anchoring element. Depending on the nature ofthe anchoring element, it can be cleaved on the internal or external leaflet ofthe membrane, thereby delivering the fusion protein to the intracellular or extracellular compartment, respectively.
- mammalian proteins can be linked to i) myristic acid by an amide-linkage to an N-terminal glycine residue, to ii) a fatty acid or diacyl glycerol through an amide- or thioether-linkage of an N- terminal cysteine, respectively, or covalently to iii) a phophotidylinositol (PI) molecule through a C-terminal amino acid of a protein (for review, see Low, Biochem. J. 244: 1-13, 1987).
- PI phophotidylinositol
- the PI molecule is linked to the C-terminus ofthe protein through an intervening glycan structure, and the PI then embeds itself into the phopholipid bilayer; hence the term "GPI" anchor.
- GPI anchors and C-terminal amino acid sequences have been reported (see Table 1 and Figure 4 in Low, Biochemica et Biophysica Acta, 988:427-454, 1989; and Table 3 in Ferguson, Ann. Rev. Biochem., 57:285-320, 1988).
- Incorporation of GPI anchors and other membrane- targeting elements into the amino- or carboxy-terminus of a fusion protein can direct the chimeric molecule to the cell surface.
- Detectable polypeptides are optional fusion protein elements that either generate a detectable signal or are specifically recognized by a detectably labeled agent.
- An example ofthe former class of detectable polypeptide is green fluorescent protein (GFP).
- Examples ofthe latter class include epitopes such the "FLAG tag” and the c-myc epitope. These and other epitopes can be detected using labeled antibodies that are specific for the epitope; several such antibodies are commercially available.
- Protein purification elements are amino acid sequences that can be incorporated into a fusion protein in order to facilitate the purification or isolation of a fusion protein from a mixture containing other molecules.
- Protein purification elements also include secretion sequences that direct recombinantly produced proteins out ofthe host cell and into the cellular media. Secreted proteins can then be separated from the host cells that produce them by simply collecting the media. Examples of secretion elements include those described in U.S. Patents 5,846,818; 5,212,070; 5,631,144; 5,629,172; and 6,103,495; and Hardig et al., J. Biol. Chem. 268:3033-3036, 1993; Sizmann et al., Year Immunol. 7: 119-130, 1993; and Power et al., Gene 113:95-99, 1992).
- Protein purification elements also include sequences that direct a recombinant protein to the periplasmic space of bacteria (Battistoni et al., Protein Expr. Purif. 6:579-587, 1995). Those skilled in the art will be able to determine which purification elements are desired, appropriate or necessary for a given fusion protein and/or expression system.
- purification elements that can be used to isolate a fusion protein from the host cells or media of an expression system.
- purification elements include a "His tag” (6 contiguous His residues, a.k.a. 6xHis), which binds to surfaces that have been coated with nickel; streptavidin or avidin, which bind to surfaces that have been coated with biotin or "biotinylated” (see U.S. Patent 4,839,293 and Airenne et al., Protein Expr. Purif. 17:139-145, 1999); and glutathione-s-transferase (GST), which binds glutathione (Kaplan et al., Protein Sci. 6:399-406, 1997; U.S. Patent 5,654,176). Polypeptides that bind to lead ions have also been described (U.S. Patent 6,111,079).
- Epitope tags such as the c-myc epitope or FLAG-tag can be used to purify recombinant proteins via affinity chromatography using antibodies to such epitope tags.
- protein purification element also includes elements designed to enhance the solubility and or assist in the proper folding of a protein. Such elements include GST and members ofthe 14-3-3 family of proteins (U.S. Patent 6,077,689).
- Spacers are amino acid sequences that can be included in a fusion protein in between other portions of a fusion protein (e.g., between the biologically active polypeptide and the plgR-targeting element, or between an optional fusion protein element and the remainder ofthe fusion protein). Spacers can be included for a variety of reasons. For example, a spacer can provide some physical separation between two parts of a protein that might otherwise interfere with each other via, e.g., steric hinderance.
- One example of a spacer of this type is the repeating amino acid sequence (Gly 4 -Ser) x , wherein x is 1 to 10,
- the plgR-targeted fusion proteins can be designed so as to contain a site (a "protease cleavage site” or simply “cleavage site”) that is amenable to being cleaved by agents or under conditions that cause or promote such cleavage.
- the cleavage site is contained within a spacer element, so that cleavage separates, e.g., the plgR targeting element of a fusion protein from the biologically active polypeptide thereof, which is useful for in vivo therapeutic methods; or between an optional protein purification element and the remainder ofthe fusion protein, which is useful for removing extraneous and potentially interfering purification elements in the process of purifying the fusion protein in vitro.
- cleavage site or of a spacer containing a cleavage site will depend on the nature ofthe in vivo or in vitro method(s) of interest. It is understood by those skilled in the art that the amino acids sequences of fusion proteins that one wishes to have cleaved by a protease must be designed so as to retain the protease cleavage site of choice.
- Non-limiting examples of in vitro and in vivo cleavage sites and systems are as follows.
- Polypeptide fragments derived from the spacer and other optional fusion protein elements may be independently released from the cleaved fusion protein, or may remain associated with the plgR targeting element or biologically active polypeptide.
- the cleavage reaction will predominantly occur after the fusion protein has been transported into or across an epithelial cell, or within a subcellular compartment, e.g., an organelle.
- the cleavage reaction might be carried out by a protease or esterase found in an epithelial cell, by the acidic conditions found near a tumor cell, by conditions in the blood that destabilize disulfide conjugation, or by a protease found in an organelle.
- Preferred cleavage sites for in vivo applications include but are not limited to those that are recognized by caspases, which can be used, e.g., to cleave and activate a biologically active polypeptide from a fusion protein during early events in apoptosis; proteases specific for an organelle into which it is desired to deliver a fusion protein, with one intended result being that a biologically active portion ofthe cleaved fusion protein will be retained by the organelle (i.e, organellar leader sequences).
- Caspases are intracellular cysteine proteases which have been shown to play an essential role in the initiation and execution phases of apoptotic cell death. For reviews, see Fadeel et al. (IUBMB Life 49:421-425, 2000), Anderson (Cell Death Differ. 7:589-602, 2000) and Earnshaw et al., Annu. Rev. Biochem. 68:383-424, 1999). Fusion proteins can be designed so as to require proteolytic activation before it becomes biologically active. Inclusion of a given caspase cleavage site in such a fusion protein can be used to design fusion proteins that are cleaved by a particular caspase and activated.
- the biologically active component of a fusion protein is not active until released from the fusion protein
- the latter type of fusion proteins provide biologically polypeptides that act at specific times during the apoptotic process.
- Cathepsins may be used in the same way in other vesicular compartments ofthe cell.
- Organellar leader sequences include, by way of non-limiting example, mitochondrial leader peptides that are proteolytically removed from proteins after their transport into mitochondria. Cathepsin B cleavage sites may be used.
- GFQGVQFAGV The following four peptides are known to be cleaved by cathepsin B: GFQGVQFAGV, GFGSVQFAGF, GLVGGAGAGF, GGFLGLGAGF, GFGSTFFAGF (Peterson and Meares, Bioconjugate Chem. 10:553-557, 1999).
- a peptide with any one the following sequences is modified to link a maleimido group at the amino terminal, where any one ofthe amino acids within the parenthesis may be used in combination with any one ofthe amino acids within any ofthe other amino acids in a parenthesis.
- Non-limiting examples of such sequences are GFQGVQFAGV, GFGSVQFAGF, GLVGGAGAGF, GGFLGLGAGF, and GFGSTFFAGF.
- Decapeptides having one of these amino acid combinations may be flanked with sequences at either the amino terminus or the carboxy terminus to provide additional flexiblity and spacing between the ligand, protein, peptide, or macromolecule.
- flanking sequences may be, for example, (Gly x Sery) z , where x and y may be from 0 to 4
- z may be from 1 to 4. In some instances, x is 4, y is 1 , and z is 1 to 2.
- Such peptides may be synthesized by solid phase using chemistries well known to those skilled in the art. Such a peptide contains only one amino group, which is at the amino terminus ofthe peptide.
- Cleavable spacers may also be used for other purposes, especially in protein purification schemes.
- a fusion protein that has an amino terminal 6xHis tag, and a protease cleavage site located immediately carboxy terminal from the His tag, i.e, between the His tag and the remainder ofthe fusion protein being produced.
- the fusion protein After the fusion protein has been purified using the His tag's affinity for nickel-coated surfaces, it is then cleaved with the appropriate protease in order to separate the His tag from the remainder ofthe protein. It is often desirable to remove elements such as His tags that are useful for protein purification purposes but might interfere with the biological activity ofthe fusion proteins.
- Cleavable spacers may be designed so as to regenerate the amino terminal amino acid sequence present in the original protein.
- Preferred cleavage sites for in vitro applications include but are not limited to those that recognize a cleavage site, which may be introduced into a fusion protein by genetic manipulation, that is located between a portion ofthe fusion protein that is not required for, and may even be detrimental to, the in vivo uses for which the fusion protein is intended.
- Commercially available expression systems that may be used to introduce cleavage sites include by way of non-limiting example cleavage sites that are recognized by enterokinase, trypsin, Factor Xa, Factor FXa and thrombin.
- Enterokinase may be used to cleave spacer elements (see U.S. Patent 4,745,069).
- a prefe ⁇ ed enterokinase is one that is produced via recombinant DNA techniques, as it is virtually free of other proteases and is able to efficiently cleave fusion proteins in partially purified preparations (Collins-Racie et al., Biotechnology 13:982-987, 1995).
- enterokinase is relatively permissive regarding the amino acid residue downstream ofthe recognition sequence (Hosfield et al., Anal. Bochem. 269:10-16, 1999). Trypsin may also be used in this fashion (U.S. Patent 6,037,143).
- in vivo cleavage by gastrointestinal proteases such as enterokinase or trypsin may serve as a mechanism by which a fusion protein is released from a carrier in the gut.
- Factor Xa (Peter et al., Circulation 101:1158-1164, 2000; U.S. Patent 6,010,883) and thrombin are blood coagulation factors.
- Expression vectors may comprise a sequence encoding a cleavage site for thrombin or Factor Xa that can be used to remove a purification element (such as a His tag) from the fusion protein after it has served its purification purpose.
- an expression cassette or construct capable of expressing a chimeric reading frame is introduced into an appropriate host cell to generate an expression system.
- the expression cassettes and constructs ofthe invention may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression ofthe gene may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occur through the integration ofthe introduced DNA sequence into the host chromosome.
- Host cells which may be used in the expression systems ofthe present invention are not strictly limited, provided that they are suitable for use in the expression ofthe chimeric plgR-targeting peptide of interest. Suitable hosts may often include eukaryotic cells.
- Prefened eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.
- Expression cassettes and constructs may be introduced into an appropriate host cell by any of a variety of suitable means, i.e, transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate- precipitation, direct microinjection, and the like.
- recipient cells are grown in a selective medium, which selects for the growth of vector- containing cells.
- Expression ofthe cloned gene(s) results in the production of a chimeric plgR-targeting peptide ofthe invention, or fragments thereof.
- the introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies ofthe vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
- a variety of recombinant DNA expression systems may be used to produce the fusion proteins ofthe invention.
- Expression systems of particular interest include prokaryotic systems, yeast expression systems, insect expression systems mammalian expression systems.
- Prokaryotic Expression Systems utilize plasmid and viral (bacteriophage) expression vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
- Suitable phage or bacteriophage vectors may include ⁇ gtlO, ⁇ gtl 1 and the like; and suitable viras vectors may include pMAM-neo, pKRC and the like.
- Appropriate prokaryotic plasmid vectors include those capable of replication in E. coli (such as, by way of non-limiting example, pBR322, pUCl 18, pUCl 19, ColEl, pSClOl, pACYC 184, ⁇ VX; "Molecular Cloning: A Laboratory Manual", 1989, supra).
- Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan, In: The Molecular Biology ofthe Bacilli, Academic Press, NY, pp. 307-329, 1982).
- Suitable Streptomyces plasmids include plJlOl (Kendall et al., J. Bacteriol. 169:4177-4183, 1987), and streptomyces bacteriophages such as ⁇ C31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary, pp. 45- 54, 1986).
- Pseudomonas plasmids are reviewed by John et al.
- Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Senatia, and the like. However, in such hosts, the fusion protein will not be glycosylated. In any event, the host cell must be compatible with the replicon and control sequences in the expression cassette.
- a chimeric plgR-targeting peptide ofthe invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the chimeric plgR-targeting peptide ofthe invention to a functional prokaryotic promoter.
- Such promoters may be either constitutive or, more preferably, regulatable (i.e, inducible or derepressible).
- constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter ofthe ⁇ -lactamase gene sequence of pBR322, and the cat promoter ofthe chloramphenicol acetyl fransferase gene sequence of pPR325, and the like.
- inducible prokaryotic promoters include the major right and left promoters of bacteriophage ⁇ (PL and PR), the tip, recA, lacZ, lad, and gal promoters of E. coli, the ⁇ - amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and promoters of B. subtilis (Gilman et al., Gene Sequence 32:11-20, 1984), the promoters ofthe bacteriophages of Bacillus (Gryczan, in: The Molecular Biology ofthe Bacilli, Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward et al., Mol. Gen. Genet.
- Prokaryotic promoters are reviewed by Glick (Lnd. Microbiot. 1:277-282, 1987), Cenatiempo (Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
- progeny Proper expression in a prokaryotic cell also requires the presence of a ribosome- binding site upstream ofthe gene sequence-encoding sequence.
- ribosome-binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981).
- the selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
- “cell”, “cell line”, and “cell culture” may be used interchangeably and all such designations include progeny.
- the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that ofthe originally transformed cell.
- Shuttle vectors are constructs designed to replicate in a prokaryotic host such as E. coli but which contain sequences that allow the shuttle vector and a chimeric reading frame incorporated therein to be fransfened to a eukaryotic viral vector or other vector such as baculovirus or adenovirus.
- Yeast Expression Systems can be utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes that are produced in large quantities when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. Yeast cells provide a substantial advantage over prokarytoic expression systems in that they can carry out post-translational modifications of fusion proteins. A number of recombinant DNA strategies exist utilizing strong promoter sequences and high copy number plasmids which can be utilized for production ofthe desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian genes and secretes peptides bearing leader sequences (i.e, pre-peptides).
- Prefened yeast expression vectors include those derived from the episomal element known as the 2-micron circle as well as derivatives of yeast integrating (Yip), yeast replicating (YRp), yeast centromeric (YCp), yeast episomal (YEp), and yeast linear (YLp) plasmids (Broach, in: The Molecular Biology ofthe Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.
- Insect Expression Systems utilize insect host cells, e.g., Sf9 and Sf21 cells, both of which are derived from the iplbsf-21 cell line derive from the pupal ovarian tissue ofthe fall army worm spodoptera frugiperda (O'Reilly et al., Baculovirus expression vectors: A Laboratory Manual New York, New York, W.H. Freeman and Company. See also baculovirus expression protocols in Methods in Molecular Biology Vol. 39; Richardson ed. Humana Totowa New Jersey, 1992; and Vaughn et al., In vitro 13:213-217, 1977.
- insect host cells e.g., Sf9 and Sf21 cells, both of which are derived from the iplbsf-21 cell line derive from the pupal ovarian tissue ofthe fall army worm spodoptera frugiperda
- the cell line bti-tn-5bl-4 (high 5 tm cell line), which originated from the ovarian cells ofthe cabbage luper, Trichoplusa ni (Davis et al., Biotechnology 10:1148-1150, 1992; Granados et al., J.Invertebr. Pathol. 64:260-266, 1994; Wickham et al., Biotechnology Prog. 8:391- 396, 1992; Wickham et al., Biotechnol. Prog. 9:25-30, 1993).
- Other insect cell lines that can be used to express baculovirus vectors have been described (Hink et al., Biotechnol. Prog. 7:9-14, 1991).
- baculovirus vectors can be engineered to express large amounts of chimeric plgR-targeting peptides ofthe invention in insect cells (Jasny, Science 238:1653, 1987; Miller et al., in: Genetic Engineering, Vol. 8, Plenum, Setlow et al., eds., pp. 277-297, 1986).
- Mammalian Expression Systems utilize host cells such as HeLa cells, cells of fibroblast origin such as VERO, CV-1 monkey kidney cells and COS-1 (CV-1 cells transformed with large T antigen) or CHO-K1, or cells of lymphoid origin and their derivatives.
- host cells such as HeLa cells, cells of fibroblast origin such as VERO, CV-1 monkey kidney cells and COS-1 (CV-1 cells transformed with large T antigen) or CHO-K1, or cells of lymphoid origin and their derivatives.
- Prefe ⁇ ed mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as FMR 332, which may provide better capacities for conect post-translational processing.
- transcriptional and translational regulatory sequences may be employed, depending upon the nature ofthe host.
- the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian viras, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
- promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
- Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression ofthe gene sequences can be modulated.
- regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
- Prefened eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- micron circle, and the like, or their derivatives.
- Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982; Broach, in: The Molecular Biology ofthe Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon et al., J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980).
- eukaryotic regulatory regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
- Prefened eukaryotic promoters include, for example, the promoter ofthe mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.
- One type of compound ofthe invention is a protein conjugate, i.e., a biologically active polypeptide that is covalently linked to a targeting element that is also polypeptide.
- Polypeptides that are plgR-targeting elements can be linked to bioactive compounds in a varity of ways.
- protein conjugate members are linked to other protein conjugate members.
- amino acid residues present in the natural sequence of a first protein member can be directly covalently linked to amino acid residues in the natural amino acid sequence of a second protein member as in, e.g., a disulfide bridge.
- mutant amino acids useful for covalent linkages can be introduced into one or more protein members by using molecular genetics to alter the reading frame encoding such protein members or, in the case of synthetic oliogopeptides, directly during the in vitro synthesis thereof.
- natural or mutant amino acid sequences present in isolated proteins can be "derivatized” (i.e, chemically modified in vitro) so as to include chemical groups not present in natural amino acids but useful for the chemical conjugation of oligopeptides, polypeptides, and proteins in a related methodology, unnatural amino acids having moities useful for chemical conjugation are introduced into oligopeptides or peptidomimetics during their synthesis in vitro.
- a cross-linking reagent typically a bifunctional (two-armed) chemical linker that forms covalent linkages between two or more conjugate members, can be used to covalently link conjugate members to each other.
- Such bifunctional linkers can be homobifunctional (wherein both "arms" ofthe linker are the same chemical moiety) or heterobifunctional (wherein each of the two "arms” is a different chemical moiety than the other).
- Polypeptide cross-linking agents are based on reactive functional groups that modify and couple to amino acid side chains of proteins and peptides, as well as to other side groups and other macromolecules.
- Bifunctional cross- linking reagents incorporate two or more functional reactive groups.
- the functional reactive groups in a bifunctional cross-linking reagent may be the same (homobifunctional) or different (heterobifunctional).
- Many different cross-linkers are available to cross-link various proteins, peptides, and macromolecules. Table 7 lists some ofthe cross-linkers that are available through commercial sources according to their class of chemical reactivity.
- Table 8 lists some ofthe properties of chemical cross-linkers and the types of functional groups with which they react.
- Bifunctional cross-linking reagents may be classified according to their functional groups, chemical specificity, length ofthe cross bridge that they establish, the presence of similar functional groups or dissimilar functional groups, chemical or photochemical reactivity, ability to be cleaved internally by reduction or other means, and the ability ofthe reagent to be further modified by radiolabelling (i.e. radioiodination) or addition of detectable tags or labels.
- the selective groups on the cross-linking reagent can be present in a homobifunctional anangement in which the selective groups are identical, or can be present in a heterobifunctional anangement in which the selective groups are dissimilar.
- the chemical modification may be done using cross-linking reagents containing selective groups that react with primary amines, sulfhydryl (thiol) groups, carbonyl, carboxyl groups, hydroxyl, or carbohydrates and other groups placed on a protein or peptide, especially by posttranslational modifications within the cell.
- the selective groups include, but are not limited to, imidoester, N-hydroxysuccinimide ester or sulfosuccinimidyl ester, ester of l-hydroxy-2-nitrobenzene-4-sulfonic, maleimide, pyridyl disulfide, carbodiimide, hydrazideand ⁇ -haloacetyl groups.
- Sulfhydryl reactive functional groups include maleimides, alkyl and aryl halides, ⁇ - haloacyls, and pyridyl disulfides.
- Maleimides, alkyl and aryl halides, and ⁇ -haloacyls react with thiols to form stable thioether bonds that are not reduced by reagents such as 2- mercaptoethanol and dithiothreitol.
- Pyridyl disulfides form mixed disulfides with thiol groups, mixed disulfides may be used as an intermediate for cross-linking two or more macromolecules.
- Cross-linkers that first react with a carboxyl group to form an activated intennediate and then reacts with an amino group, such as a ⁇ -amino group of lysine or an ⁇ -amino group of an amino terminal amino acid, may be used.
- a spacer arm or "cross-bridge” region consisting of a spacer group or a functional group, such as a disulfide bond or hindered disulfide bond, may be used to connect the Biologically active polypeptide to the targeting element.
- the length ofthe spacer arm may be varied. The distance between the functional groups establishes the length ofthe spacer arm. Longer spacer arms may be required to diminish or eliminate steric hindrance between two molecules that are cross-linked together. Intermolecular cross-linking is more efficient with longer spacer arms. Short spacer arms favor intramolecular cross-linking, which is preferably avoided in the present invention.
- Spacer arms may have reactive bonds within them that enable further modifications.
- internal cleavable bonds may be placed within the spacer, such as disulfides or hindered disulfides, one or more ester bonds, or vicinal hydroxyl groups. Cleavage of internal disulfide bonds may be achieved using reduction with thiol containing reagents such as 2-mercaptoethanol and dithiothreitol.
- One or more metabolizable bonds may be inserted internally in the cross-linking reagent to provide the ability for the coupled entities to separate after the bond(s) is broken after the conjugate is transported into the cell and into the body.
- Homobifunctional cross-linkers contain at least two identical functional groups. Heterobifunctional cross-linkers contain two or more functional reactive groups that react with different specificity. Because heterobifunctional cross-linkers contain different reactive groups, each end can be individually directed towards different functional groups on proteins, peptides, and macromolecules. This feature results in linking, for example, amino groups on one molecular entity to carboxyl groups on another entity, or amino groups on one entity to sulfhydryl groups on another entity.
- Functional groups include reactive portions on proteins, peptides, and macromolecules that are capable of undergoing chemical reaction. Functional groups include amino and carboxyl groups, hydroxyl groups, phenolate hydroxyl groups, carbonyl groups, guanidinyl groups, and carbon-carbon double bonds.
- photoactive reagents that become reactive when exposed to light may be used.
- arylazides may be activated to form activated intermediates, such as an aryl nitrene or a dehydroazepine intermediate, that non-selectively inserts into carbon-hydrogen bonds (i.e. by aryl nitrenes) or reacts with amines (dehydroazepines).
- Other examples include fluorinated aryl azides, benzophenones, certain diazo compounds, and diazrine derivatives.
- NHS-esters react efficiently with amino groups in aqueous buffers, preferably phosphate, bicarbonate/carbonate, HEPES, and borate buffers at concenfrations between 10 and 200 mM. Buffers should not contain primary amines. Primary amines can be added to the reaction to stop or quench the NHS-ester reaction and thereby terminate further modification of amino groups on proteins, peptides, and macromolecules. The modification or coupling is typically carried out between pH 7 and pH 9, and preferably between pH 7.5 and 8.0. The time of reaction and temperature may depend on the particular molecule that is being modified.
- the time of modification may be between 10 and 180 minutes, preferably between 30 and 60 minutes at temperatures between 4 °C and 37°C, preferably between 4°C and 25°C.
- the concentration ofthe NHS-ester may vary, but is between 1.1 - to 100-fold molar excess, and preferably between 1.1- and 10-fold molar excess.
- the protein concentration may vary between 1 ⁇ M and 100 ⁇ M, preferably between 5 ⁇ M and 100 ⁇ M.
- a maleimido-aliphatic carboxylic acid may form an ester with the hydroxyl group of l-hydroxy ⁇ 2-mtrobenzene-4-sulfonic acid.
- a maleimide group may be placed at the end of a short, intermediate, or long aliphatic acid.
- An example of this is mal-sac-HNSA (U.S. Patent No. 4,954,637).
- Mal-sac-HNSA may be used to acylate amino groups on proteins, peptides, and macromolecules.
- the maleimide may then be reacted with sulfhydryl groups on other proteins, peptides, and macromolecules to form a stable, noncleavable thioether bond.
- Aqueous buffers such as sodium phosphate, and neutral to mildly alkaline conditions, pH 6.5 to 9, and preferably pH 7 to 8, may be used at temperatures from 0°C to 37°C, and preferably from 4°C to 25°C.
- Proteins and peptides contain ⁇ - amino groups at the amino terminus, ⁇ -amino groups on lysine, ⁇ -carboxyl groups on aspartic acid, ⁇ -carboxyl groups on glutamic acid, imidazole rings on histidine, hydroxyl groups on serine and threonine, phenolate hydroxyl groups on tyrosine, sulfhydryl groups on cysteine, disulfide bonds between two cysteines, mercaptide bonds in methionine, and indole rings in tryptophan, all of which can be selectively modified by cross-linking reagents.
- Carbohydrates or carbohydrate containing macromolecules contain ketone, aldehyde, hydroxyl, amine, carboxylate, sulfate, and phosphate groups as nonlimiting examples of functional groups with which cross-linkers may react.
- Carbohydrates containing vicinal hydroxyl groups may be treated with sodium periodate so that the carbon-carbon bond is cleaved; this creates reactive formyl groups on the treated carbohydrate that may be used as a target for appropriately designed cross-linking reagents.
- Hermanson discloses other methods, which are herein incorporated by reference, for cross-linking carbohydrates.
- Hermanson discloses the major sites on nucleic acids that are susceptible to chemical modification. Compositions and methods for synthesizing and conjugating oligonucleotides comprising a Cys residue have been described by Stetsenko and Gait (Nucleosides, Nucleotides and Nucleic Acids 19:1751-1764, 2000).
- Ligands genetically fused to therapeutic and diagnostic biologically active proteins and peptides may not always produce a desired result. Genetic fusion is typically performed by attaching the ligand to either the amino or carboxy terminus ofthe biologically active protein or peptide using a spacer if necessary. Therefore, the geometrical arrangement of ligand and biologically active protein and peptide is necessarily limited. Linkage ofthe biologically active protein or peptide through surface cysteinyl groups presents more flexibility in designing a combination that allows both the ligand to recognize plgR and the biologically active protein or peptide to carry out its functions after epithelial transport.
- the present invention provides a method for substituting or inserting a cysteine into the protein or peptide and using the cysteine for chemical conjugation by cross-linking.
- An amino acid may be selected for substitution by cysteine, such selected amino acid should be on the surface ofthe molecule and positioned so as not to interfere or sterically hinder the function of any biologically active site or important site on the molecule that is required for a biological activity or function.
- the substitution of cysteine for an amino acid may be achieved by methods well-known to those skilled in the art, for example, by using methods described in Maniatis, Sambrook, and Fritsch (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, 1989). Regions within the crystallographic stracture of a polypeptide are chosen so as to minimize potential steric hindrance imposed by coupling a relatively large molecule, such as a plgR binding sFv, to the cysteinyl residue.
- any ofthe amino acids in loops or unstructured regions may be substituted with a cysteine; however, prefened positions exist. Such prefened positions are at amino acids whose side chains are not hydrogen bonded to other amino acid side chains (or backbone atoms) or do not participate or contribute to the formation of salt or charge bridges with other amino acid side chains. Amino acids within helical regions may also be substituted if their side chains are oriented away from the main body ofthe protein and do not participate in interactions with other amino acid side chains that provide stability to the structure.
- a prefened position is an amino acid side chain that is fully exposed to bulk solvent and has no significant interaction with amino acids within the polypeptide' s tertiary stracture and does not participate in the biological activity or function ofthe molecule, including receptor binding, signal transduction, and the like.
- Amino acids for possible substitution may be chosen by examining the crystallographic structure using software designed for the purpose of visualization ofthe three dimensional structure.
- Several programs are available for analysis, including Protein Explorer, Insight II, MDL, Tripos, Amber, Charm, Chem-X, Chime, DOCK, Homology, MAGE, SYBYL, Midas Plus, and others known to those skilled in the art. Both visual inspection and calculations and displays within these programs can be used by those skilled in the art to select substitution positions.
- a protein or peptide surface is examined for sites at which substitution of an amino acid by cysteine or insertion of cysteine in the protein sequence does not change or modify the activity ofthe protein in a significant way. Examination of crystallographic data ofthe protein or peptide will reveal which amino acid residues and side chains are exposed, as judged by the ability of a water molecule to contact the amino acid or its side chain. Cysteine residues are inserted or substituted into loops, preferably loops that are not defined in crystallographic structures because they are so unstructured that they move during data collection. Cysteine residues are substituted for amino acids on the solvent side of helices. Those skilled in the art will know how to use software programs to analyze the surface features of a protein for the purpose of cysteine substitution or insertion (see, e.g., U.S. Patent Nos. 4,853,871 and 4,908,773).
- any amino acid that is in contact with water is a candidate for replacement by cysteine.
- Such amino acids may be replaced by cysteine, one by one, and the effect ofthe substitution examined on biological activity. Those substitutions that do not affect biological activity more than 0 to 20 per cent, preferably 0 to 10 percent, and most preferably 0 to 5 percent may be used to cross-link to ligands that bind to plgR and plgR stalk.
- Loops formed by a small stretch of 5 to 15 amino acids on the surface of a protein or peptide are used to insert a cysteine into the protein sequence. Examination ofthe surface is expected to reveal a site that has maximum exposure to the bulk solvent. Solvent accessible side chains are identified by examining the Connolly (Connolly/Richards) surfaces ofthe protein, which are essentially defined by the ability ofthe side chain to contact a water molecule 'rolled' around the surface ofthe molecule. Insertion of a cysteine at a site accessible to bulk solvent, or within 2 to 4 amino acid residues, is performed to produce a variant ofthe protein suitable for cross-linking to ligands that bind to plgR or plgR stalk.
- Loops are also identified by performing molecular dynamic analysis on the protein. Molecular dynamic analysis carried out over 50 to 250 picoseconds is expected to reveal flexible regions within the structure ofthe protein that are used for cysteine substitution or insertion. In such analyses, Cysteine residues are substituted, one at a time, between each pair of amino acids in the flexible loop.
- Helical wheels are used to identify the side ofthe helix that faces bulk solvent. Looking down the banel of a helix, one can identify residues on one side or the other ofthe helix. Where crystallographic solutions to the protein stracture are available, residues on a helical wheel can be observed in the structure to estimate their access to bulk solvent. Residues on the bulk solvent side ofthe helical wheel often participate in receptor binding. Substitution of a cysteine for such a residue is undesirable. Substitution within a helix at a residue facing the bulk solvent is provided in this invention, provided that the residue does not participate in receptor binding or is otherwise involved in the biological activity ofthe molecule. Substitution or insertion of cysteine should not alter biological function and activity.
- the effect ofthe cysteine insertion or substitution maybe analyzed using biological assays that suitably and appropriately measure the function ofthe modified protein.
- cysteine modified protein Comparisons between the cysteine modified protein and the parent unmodified protein reveal the quantitative and qualitative effects ofthe modification on function. If data are available that identify, locate, or suggest where the important sites are located on the protein surface that contribute to biological activity, or which cannot be modified by mutagenesis, sites remote for those biologically and functionally sensitive regions may be avoided.
- the cysteine substitution or insertion may be placed on the surface ofthe protein or peptide opposite from the functionally sensitive surfaces ofthe protein; i.e., spatially as far away as possible.
- Cysteine substitutions or insertions for antibodies have been described (see U.S. Patent No. 5,219,996). Methods for introducing Cys residues into the contant region ofthe IgG antibodies for use in site-specific conjugation of antibodies are described byskyl et al. (J. Biol. Chem 275:330445-30450, 2000).
- a sulfhydryl may be introduced by chemical modification.
- the sFv or a therapeutic macromolecule can be modified so as to introduce a thiol by chemical modification.
- a cysteine amino acid can be inserted or substituted on the surface of a protein or peptide by genetic manipulation.
- Sulfhydryl groups can be added by chemical modification using 2-iminothiolane (IT), also known as Traut's reagent.
- a sulfhydryl can be introduced by incubating 0.1 to 10 mg/ml, preferably 1 to 5 mg/ml, ofthe target molecule, with a 1.1- to 100-fold, preferably 1.1- to 10-fold, molar excess of 2-iminothiolane in 50 mM triethanolamine, pH 8.0, containing 150 mM NaCl and 1 mM EDTA for three hours at 4°C.
- the excess 2- iminothiolane can then be removed by desalting on either a P10 (Bio-Rad, Hercules, CA) or G25, G-50, or G-100 (Pharmacia, Piscataway, NJ) size exclusion column equilibrated with 20 mM sodium phosphate containing 0.15 M NaCl and 1 mM EDTA, pH 7.3, (PBS- EDTA).
- P10 Bio-Rad, Hercules, CA
- G25, G-50, or G-100 Pharmacia, Piscataway, NJ
- a protected sulfhydryl group can be added which allows storage ofthe derivatized protein without self-association through disulfide bond formation.
- IT and DTNB can be reacted together to form TNB-activated IT, which can then be directly added to the target molecule.
- substituted IT's can be synthesized (Goff and Canoll, Bioconjugate Chem. 1:381-6, 1990), and using these to add sulfhydryl groups to target proteins can result in disulfide linked conjugates that exhibit increased stability in vivo (Canoll et al. Bioconjugate Chem. 5:248-56, 1994).
- Protected sulfhydryls can also be added by using a modification reagent that contains a protected thiol in addition to a group that selectively reacts with primary amines.
- a modification reagent that contains a protected thiol in addition to a group that selectively reacts with primary amines.
- SATA N-hydroxy-succinimide ester of S- acetylthioacetic acid
- SATA Pierce Chemical Co., Rockford, II
- the excess hydroxylamine can be removed by desalting on either a P10 or G25 size exclusion column equilibrated with PBS-EDTA.
- PBS-EDTA PBS-EDTA
- SATP N-succinimidyl S-acetylthiopropionate
- Sulfhydryl groups can also be added by using a modification reagent that contains a disulfide bond in addition to a group that selectively reacts with primary amines.
- a modification reagent that contains a disulfide bond in addition to a group that selectively reacts with primary amines.
- the heterobifunctional cross-linker sulfosuccinimidyl 6-[3'-(2-pyridyldithio)- propionamido] hexanoate sulfo-LC-SPDP, Pierce Chemical Co.
- Thiolation can also be performed by the addition of 300 ⁇ g sulfo-LC-SPDP per ml of 10 mg/ml sFv or therapeutic macromolecule in 20 mM sodium phosphate containing 0.15 M NaCl, pH 7.3 (PBS).
- N-succinimidyl 3-(2-pyridyldithio)propionate SPDP, Pierce Chemical Co.
- N-succinimidyl 6-[3'-(2-pyridyldithio)propionamido]hexanoate LC-SPDP, Pierce Chemical Co.
- SPDP-derivatized protein Reducing the SPDP-derivatized protein under mild conditions will release pyridine-2-thione, leaving an aliphatic thiol.
- An example of a mild reducing condition is to add 1/100th volume of 1M dithiothreitol (DTT) to the above SPDP-derivatized target protein and incubating for 30 minutes at room temperature, or incubate the SPDP-derivatized target protein with 50 mM 2-meraptoethylamine in PBS-EDTA for 90 minutes at 37°C.
- DTT dithiothreitol
- the excess SPDP, LC-SPDP or sulfo-LC-SPDP, and the pyridine-2-thione can then be removed by desalting on either a P10 (Bio-Rad, Hercules, CA) or G25 (PD10 column, Pharmacia, Piscataway, NJ) column equilibrated with PBS-EDTA.
- modification reagents may also contain groups near the added thiol such that they form a hindered disulfide when oxidized.
- These reagents such as 4- succinimidyloxycarbonyl-methyl-(2-pyridyldithio)-toluene (SMPT), may result in a conjugate that exhibits increased stability in vivo (Thorpe et al. Cancer Res. 47:5924-5931, 1987).
- Other cross-linking reagents can be used for protein thiolation and are known to those well versed in the art. Many of these reagents are described in the Pierce Chemical Co. catalog, or by Ji (Methods Enzymol. 91:580-609, 1983) and Hermanson (Bioconjugate Techniques, Academic Press, Inc., San Diego, 1-785, 1996).
- V.B.4 Chemical Addition of Primary Amine Groups to the Surface of a Polypeptide or Macromolecule
- additional primary amines need to be added to either the sFv or the therapeutic macromolecule, they can be introduced through chemical modification or genetic manipulation.
- Chemical modification to add primary amines may either be reversible or non-reversible.
- amination of cysteines can be performed using N-(iodoethyl) Trifluoroacetamide (Aminoethyl-8TM, Pierce Chemical Co.) by a reaction in which the iodoalkyl group reacts specifically with sulfhydryl groups, forming a stable thioether bond and releasing free iodine.
- the trifluoroacetate protecting group can then be hydrolyzed to expose the introduced primary amine.
- a reversible amination of cysteines can be performed by using, for example, 2-aminoethyl-2'-aminoethanethiosulfonate (Pierce Chemical Co.).
- the primary amine generated by this compound can be removed by disulfide reducing agents.
- the sFv and therapeutic macromolecule will have either sulfhydryl or primary amines as the targets ofthe cross-linking reagents, and both sulfhydryl and primary amines can either exist naturally or be the result of chemical modification as described above.
- a homobifunctional cross-linker that contains maleimide, pyridyl disulfide, or ⁇ -haloacetyl groups can be used for cross-linking.
- cross- linking reagents include, but are not limited to, bismaleimidohexane (BMH) or l,4-Di-[3'- (2'-pvridyldithio)propionamido]butane (DPDPB).
- BMH bismaleimidohexane
- DPDPB l,4-Di-[3'- (2'-pvridyldithio)propionamido]butane
- a heterobifunctional cross-linker that contains a combination of maleimide, pyridyl disulfide, or ⁇ -haloacetyl groups can be used for cross-linking.
- the cross-linking reagent can contain thiophthalimide derivatives or disulfide dioxide derivatives.
- extrinsic sulfhydryl groups can be introduced into the sFv and therapeutic macromolecule, and oxidized to cross-link by disulfide formation.
- a homobifunctional cross-linker that contains succinimide ester, imidoester, acylazide, or isocyanate groups can be used for cross-linking.
- cross-linking reagents include, but are not limited to, Disuccinimidyl glutarate (DSG), Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), Bis[2- (sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSCOCOES), Disuccinimidyl suberate (DSS), Dithiobis(succinimidylpropionate) (DSP), B ⁇ S-(Sulfosuccinimidyl) Suberate (BS3), Dithiobis(sulfosuccinimidylpropionate) (DTSSP), Disuccinimidyl tartrate (DST), Disulfosuccinimidyl tartrate (sulfo-DST), Dithio-bis-maleimidoethane (DTME), Ethylene glycolbis(succinimidyls)
- Heterobifunctional cross-linking reagents that combine selective groups against different targets are generally prefened because these allow reactions to be performed selectively and sequentially, minimizing self-association or polymerization.
- heterobifunctional reagents allow selection of chemistry appropriate for the individual molecules to be joined, minimizing inhibition of enzymatic, binding, signaling or other activities required for the sFv-therapeutic macromolecule conjugate. For example, some enzymes have a primary amine present in the active site and modification of this amine will inhibit enzymatic function. These enzymes would be suitable prospects for alternative conjugation chemistry so that a thiol group is modified rather than the amine required for therapeutic activity.
- cross-linking reagents include, but are not limited to, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 6-[3'-(2- pyridyldithio)propionamido]hexanoate (LC-SPDP), sulfosuccinimidyl 6-[3'-(2- pyridyldithio)-propionamido] hexanoate (sulfo-LC-SPDP), m-maleimidobenzoyl-N- hydoxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydoxysulfosuccinimide ester (sulfo-MBS), succinimidyl 4-[P-maleimidophenyl] butyrate (SMPB), sulfosuccinimidyl 4- [p-maleimid
- the disulfide within DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] exchanges with the thiol group of proteins and peptides and other macromolecules.
- DTNB 5-thio-2-nitrobenzoic acid
- one molecule of 5-thio-2-nitrobenzoic acid (TNB) is released.
- the absorbance at 412 nm is measured to calculate the molar concentration of TNB using an extinction coefficient of 1.36 x 104 M -1 cm-1 (at pH 8.0).
- the protein is used at a concentration of 1 to 10 mg/ml in a solution of phosphate buffered saline, pH 8.
- Ellman's reagent may be dissolved in 0.1 M sodium phosphate, pH 8, at a concentration of 4 mg/ml.
- Four (4) ⁇ l of Ellman's reagent is mixed with each 100 ⁇ l protein solution and the absorbance at 412 nm measured.
- the concentration of TNB is determined using the extinction coefficient at pH 8.
- the protein is separated as a mixed disulfide (protein -SS-TNB) from Ellman's reagent and TNB by gel filtration on a column of Sephadex G-25 or Biorad P10 in an aqueous buffer.
- protein -SS-TNB mixed disulfide
- Iodoacetate and iodoacetamide are used to react with free thiols on proteins and prevent them from forming unwanted disulfide bonds.
- Iodoacetamide is a highly reactive and selective reagent for thiols and may be used as a blocking reagent. Iodoacetamide will, however, react slowly with histidine on its imidazole side chain.
- the protein, peptide, or macromolecule containing thiol groups that are to be blocked are present at a concentration of 0.1 to 10 mg/ml, preferably 1 to 5 mg/ml, in an aqueous buffer at neutral or slightly alkaline pH, pH 6.5 to 10, preferably pH 7.0 to 8.0.
- Freshly dissolved iodoacetamide is added to a final concentration of 0.1 to 10 mM, preferably 0.5 to 2 mM and reacted at room temperature for 1 hour. The reaction is preferably carried out in the dark.
- Iodoacetamide is preferably free from free iodine, whose presence can be detected by its color (yellowish) or other methods known to those skilled in the art. V.C.3. Blocking Thiols by Maleimidation
- N-ethylmaleimide reacts rapidly with thiols to form a stable thioether bond that is not cleaved by reduction with 2-mercaptoethanol or dithiothreitol.
- the protein, peptide, or macromolecule having a thiol group that is desired to be blocked is dissolved in an aqueous buffer at pH 6.5 to 8.0, preferably 6.5 to 7.5.
- Sodium phosphate buffer (0.01 to 0.1 M) at pH 7 to 7.5 is prefened.
- the buffer may also contain 0.01 to 0.5 M NaCl, and preferably 0.01 to 0.1 M.
- N-ethylmaleimide maybe freshly dissolved in an aqueous buffer at a concentration so that after dilution the final concentration of N-ethylmaleimide is between 1.1- and 20-fold molar excess, and preferably 2- to 10-fold molar excess. After 5 to 120 minutes, and preferably 5 to 30 minutes, of reaction at room temperature, the modified protein may be separated from excess N-ethylmaleimide by gel flitration on a column of Sephadex G-25 or Biorad P10.
- compositions or compounds isolated or purified preferably substantially purified.
- isolated it is meant that the composition or compound has been separated from any molecule that interferes with the biological activity or plgR-targeting capacity thereof.
- substantially purified means at least about 95%, preferably at least about 99%, free of other components used to produce and/or modify the protein conjugate.
- purified refers to a composition or compound that has been separated from at least about 50% of undesirable elements.
- the purification ofthe sFv's and the conjugated material is achieved using any of the methods that are known by those skilled in the art to purify proteins, peptides, and macromolecules. Such methods include gel filtration, HPLC using ion exchange chromatography, immobilized metal affinity chromatography, hydrophobic interaction chromatography, selective precipitation, and crystallization.
- Chromatography methods are selected for their ability to remove unreacted reagents, including unreacted derivatized proteins, peptides, and macromolecules and unreacted plgR binding ligands. Chromatography methods are also selected for their ability to separate conjugates having different molar rations or protein, peptide, or macromolecule to plgR binding ligands. Such conjugates are often refened to as l-'mers (1:1 conjugates), 2-'mers (2:1 conjugates), 3-'mers (3:1 conjugates), etc. The production of different 'mers is a function ofthe number of reactive groups present on each molecule incubated in the conjugation mixture.
- Optional protein elements can be incorporated into a fusion protein, which may be a compound ofthe invention or a member of a protein conjugate ofthe invention, or which may be comprised in a composition ofthe invention, and used during its purification and/or preparation.
- a protein member may include a protein purification element such as, for example, a "His tag" (His6).
- His- tagged protein member or conjugate thereof can be isolated, or at least partially purified, from a composition that further comprises undesirable compounds by contacting the composition with a column of nickel immobilized on a metal-binding matrix. The His- tagged protein member or conjugate will bind to the nickel column and will thus be retained in the column; undesirable compounds pass through the column.
- various methods may be used to remove the protein purification element from the protein member or conjugate after such steps.
- Post-translational modifications to a polypeptide may be created in vitro or in vivo.
- Various chemical treatments can be used for in vitro modifications of pure or semi-pure proteins; whereas in vivo modifications result from the choice of expression system and host cells.
- Post-translational modifications include, by way of non-limiting example, glycosylation, cleavage, phophorylation, cross-linking, formation or reduction of disulfide bridges, and the like.
- Polypeptides that contain plgR-derived amino acid sequences that are identical or similar to the epitopes to which sFv molecules that bind plgR are prepared according to known methods.
- the epitope-containing polypeptides are covalently coupled to thiol Sepharose (activated thio Sepharose 4B contains a thiol group to which peptides may be attached covalently).
- a thiol containing peptide is reacted with Ellman's reagent (DTNB) to form a mixed disulfide.
- DTNB Ellman's reagent
- the TNB-peptide is separated from 2-nitro-5-thiobenzoic acid by gel sizing column chromatography.
- the TNB-peptide is reacted with thiol Sepharose to form a mixed disulfide ofthe peptide covalently bound to the resin.
- a maleimido group is placed at the amino or carboxyl terminal ofthe peptide.
- the maleimido group on the peptide is reacted with thiol Sepharose to form a thioether bond.
- the epitope-containing polypeptides are covalently coupled to activated supports that react with primary amines present on the polypeptide.
- Such supports include cross-linked agarose or acrylic matrices that have functional groups such as N-hydroxysuccinimide.
- activated supports includeAffi-Gel 10 (Bio-Rad), Affi- Gel 15 (Bio-Rad), Affi-Prep 10 (Bio-Rad) and NHS-activated Sepharose 4 Fast Flow (Pharmacia). Immobilization ofthe polypeptide may also be performed with epoxy- activated matrices such as Epoxy-activated Sepharose 6B (Pharmacia) or cyanogen bromide-activated matrices such as CnBr-activated Sepharose 4 Fast Flow (Pharmacia).
- epoxy- activated matrices such as Epoxy-activated Sepharose 6B (Pharmacia) or cyanogen bromide-activated matrices such as CnBr-activated Sepharose 4 Fast Flow (Pharmacia).
- the peptide-Sepharose resin is used to bind an sFv, or other antibody derivative that binds the epitope in plgR that is recognized by the antibody, or a conjugate comprising such an antibody.
- the amino acid sequence may be modified to provide the epitope in an amino acid sequence that inlcudes a residue that may be covalently linked to thiol Sepharose.
- the amino acid sequence is, at a minimum, DPRLF.
- the maximum epitope amino acid sequence is QDPRLF in human and LDPRLF, which suggests that the most amino-terminal residue in the epitope sequence is not required for binding to sFv5.
- sFv or conjugate After the sFv or conjugate has been applied to the column, unreactive material is washed through the column.
- the sFv's, or conjugates comprising sFv's, remain attached to the column through specific interaction with the peptide.
- the specifically bound sFv or conjugate is separated from the column by low pH (pH 3 to 4) treatment for a brief time (preferably less than 15 minutes and preferably less than 5 minutes), by passing free peptide over the column, or by reducing the covalently bound peptide with DTT or mercaptoethanol.
- the peptide When using a free peptide to obtain elution ofthe sFv or conjugate, the peptide need only contain the epitope to which the sFv binds or it may contain the same peptide sequence (without the cysteine) used to conjugate to the resin.
- the sequence within the epitope may be varied such that the interaction is weakened compared to the native epitope.
- a weaker binding peptide sequence may be identified. Weak binding to the immobilized peptide on thiol Sepharose is used to obtain some retention of sFv and conjugates on the column and to allow nonbinding components to pass straight through the column without binding. Therefore, no strenuous conditions may be required for elution and free peptide may not be required for elution.
- Tribbick et al. J. Immunol. Methods 139: 155-166, 1991
- a weak binding peptide epitope is identified by performing alanine scans on the epitope to identify the amino acid side chains that provide most ofthe binding specificity and strength.
- a peptide epitope is identified using a set of peptides designed to explore all ofthe binding regions of a protein, a general net. All overlapping peptides of a defined length, homologous with the protein, are synthesised. The offset is set from 1 to 5 residues, and preferably 3 to 4 residues in the first trials.
- the peptides should be sufficiently long so as not to miss an epitope by 'dividing it' between two peptides in the nested set.
- the peptides should be preferably 8 to 12 amino acids in length and preferably 10 to 15 amino acids in length.
- the boundaries ofthe epitope may be more precisely identified using a process that examines the linear sequence ofthe protein through a series of moving windows of a different size - a window net.
- the contributions of each amino acid side chain in the epitope are estimated by substituting each amino acid position in the epitope with all ofthe other 19 amino acids and determining the effect on the binding characteristics ofthe sFv to the peptide - a replacement net.
- Such strategies are described by Geysen et al. (Mol. Immunol. 23: 7090715, 1986), Geysen et al. (J. Immunol. Methods 102: 259-274, 1987), Tribbick et al. (J. Immunol. Methods 139: 155-166, 1991), and Geysen et al. (J. Mol. Recog. 1: 32-41, 1988).
- ion exchange chromatography charged substances are separated via column materials that cany a charge.
- the solid phase carries a negative charge whereas, in anion exchange, the stationary phase carries a positive charge.
- the solid phase ofthe columns is composed of ionic groups that are covalently bound to a gel matrix. Before a sample is passed through the column, the ionic charges in the solid phase are compensated by small concentrations of counter-ions present in the column buffer. When a sample is added to the column, an exchange occurs between the weakly bound counter-ions in the column buffer and more strongly bound ions present in the sample. Bound molecules do not elute from the column until a solution of varying pH or ionic strength is passed through the column. If desired, the degree of separation may be improved by a change in the gradient slope. If a compound of interest does not bind to the column under the selected conditions, the concentration and/or the pH value ofthe starting buffer can be changed.
- polypeptides are multivalant anions or cations.
- polypeptides are anions because the amino group is a free base and the carboxy group is dissociated.
- polypeptides are cations as a result of suppression ofthe dissociation ofthe carboxy group and protonation ofthe amino group. Due to the net charge ofthe polypeptides it is possible to bind them to a conesponding charged stationary phase as long as the salt concentration is kept low.
- Ion-exchange materials are generally water insoluble polymers containing cationic or anionic groups.
- Non-limiting examples of cation exchange matrices have anionic functional groups such as -SO 3 ⁇ , -OPO 3 ⁇ and -COO-, and anion exchange matrices may contain the cationic tertiary and quaternary ammonium groups having the general formulae -NHR ++ and -NR 4 "1" . Proteins become bound by exchange with the associated counter-ions.
- HIC hydrophobic interaction chromatography
- HIC involves sequential adsorption and desorption of protein from solid matrices mediated through non-covalent hydrophobic bonding.
- sample molecules in a high salt buffer are loaded on the HIC column.
- the salt in the buffer interacts with water molecules to reduce the solvation ofthe molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column.
- the more hydrophobic the compound the less salt needed to promote binding.
- a decreasing salt gradient may be used to elute samples from the column. As the ionic strength decreases, the exposure ofthe hydrophilic regions ofthe molecules increases, and compounds elute from the column in order of increasing hydrophobicity.
- Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer.
- HIC-immobilized functional groups that can function to separate compounds include octyl groups, such as those on Octyl Sepharose CL4B media from Pharmacia, and propyl groups, such as those on High Propyl media from Baker. Alkoxy, butyl, and isoamyl functional group resins may also be used.
- Hydrophilic interaction chromatography separates compounds by passing a hydrophobic or mostly organic mobile phase across a neutral hydrophilic stationary phase, causing solutes to elute in order of increasing hydrophilicity. For example, with neutral peptides one may use 15 mM ammonium formate and reverse organic conditions. Highly charged molecules require low amounts (e.g., 10 mM) of salt for ion suppression, and a slight perchlorate or sulfate gradient (in a high organic solvent concentration) to effect desorption. HILIC has been used successfully with phosphopeptides, crude extracts, peptide digests, membrane proteins, carbohydrates, histones, oligonucleotides and their antisense analogs, and polar lipids.
- hydrophobic-interaction chromatography compounds of relatively greater hydrophobicity are retained longer on the column relative to those compounds that are more hydrophilic. Conversely, using hydrophilic-interaction chromatography, hydrophilic compounds are retained longer on the column relative to those compounds that are more hydrophobic.
- hydrophobic interaction chromatography see Ghosh, Separation of proteins using hydrophobic interaction membrane chromatography, J Chromatogr A 923(l-2):59-64, 2001; Queiroz et al., Hydrophobic interaction chromatography of proteins, J Biotechnol 87(2): 143-59, 2001; Arakawa et al., Solvent modulation in hydrophobic interaction chromatography, Biotechnol Appl Biochem 13(2): 151-72, 1991; el Rassi et al., Reversed-phase and hydrophobic interaction chromatography of peptides and proteins, Bioprocess Technol 9:447-94, 1990; Kato, High- performance hydrophobic interaction chromatography of proteins, Adv Chromatogr 26:97- 115, 1987; Hjerten, Hydrophobic interaction chromatography of proteins, nucleic acids, viruses, and cells on noncharged amphiphilic gels, Methods Biochem Anal 27:89-108, 1981
- compositions or compounds ofthe invention may comprise a detectable polypeptide by which the protein conjugate maybe monitored.
- compositions or compounds ofthe invention Some ofthe biological activities of a composition or compound ofthe invention will vary depending on the nature ofthe biologically active polypeptide(s) included therein, and assays specific for the biological activities ofthe parent proteins are used.
- the compositions or compounds are also assayed for their ability to bind plgR and undergo various forms of cellular trafficking. Assays for these and plgR-related attributes are described herein and are applicable to any ofthe compositions or compounds ofthe invention.
- Purity can be assessed by any suitable method, including HPLC analysis and staining of gels through which an aliquot ofthe preparation containing the protein conjugate has been electrophoresed.
- suitable method including HPLC analysis and staining of gels through which an aliquot ofthe preparation containing the protein conjugate has been electrophoresed.
- Those practiced in the art will know what degree of isolation or purification is appropriate for a given application. For example, (in the U.S. at least) biologicals do not have to meet the same standard of purity for, e.g., a compound.
- the invention encompasses multivalent and polyspecific complexes and compounds.
- multivalent it is meant that the complex or compound has two or more targeting elements directed to the same target. The two or more targeting elements may, but need not, be identical.
- polyspecific it is meant that the complex or compound has at least one targeting element that is directed to a first target, and a second targeting element directed to a second target.
- methods including but not limited to the following, can be used to prepare multivalent and polyspecific complexes and compounds ofthe invention.
- Diabodies are dimeric antibody fragments (Hollinger et al., "Diabodies”: small bivalent and bispecific antibody fragments, Proc Natl Acad Sci U S A 1993 Jul 15;90(14):6444-8).
- a heavy-chain variable domain V(H) is linked to a light-chain variable domain V(L) but unlike single-chain Fv fragments, each antigen- binding site is formed by pairing of one V(H) and one V(L) domain from the two different polypeptides.
- Diabodies thus have two antigen-binding sites, and can be bispecific or bivalent. (Perisic et al., Crystal stracture of a diabody, a bivalent antibody fragment, Stracture 1994 Dec 15;2(12): 1217-26).
- VILA.1 Directing Multimerization of sFv's by Altering Linker Length in sFv Antibodies
- sFv molecules are predominantly monomeric when the V(H) and V(L) domains are joined by polypeptide linkers of at least 12 amino acid residues.
- An sFv molecule with a linker of 3 to 12 amino acid residues is less likely to fold into a monomer, i.e., a single chain Fv in which the V(H) and V(L) domains are paired intramolecularly.
- sFv's that do not easily form monomers may interact with a second sFv molecule to form a "diabody".
- Diabodies may be bispecific (Miiller et al., "A dimeric bispecific miniantibody combines two specificities with avidity", Federation of European Biochemical Societies, 432 (1998), pp. 45-49) or bivalent.
- a bivalent diabody is formed from two sFv's that are identical to, or substantially the same as, each other; it has two binding [V(H)::V(L)] regions directed to the same target molecule.
- a bispecific diabody is formed from two sFv's that are different from each other, and has two binding [V(H)::V(L)] regions, each of which is directed to a different target molecule .
- Reducing the linker length below three amino acid residues can force sFv molecules to associate to form multimers (e.g., trimers a.ka. triabodies, tetramers a.k.a., tefrabodies, etc.) depending on linker length, composition and V-domain orientation (see, e.g., U.S. Patent 5,837,242).
- multimers e.g., trimers a.ka. triabodies, tetramers a.k.a., tefrabodies, etc.
- the increased valency in sFv multimers may result in higher avidity (low off-rates) (Hudson et al., High avidity scFv multimers; diabodies and triabodies, J Immunol Methods 1999 Dec 10;231(1-2): 177-89; Todorovska et al., Design and application of diabodies, triabodies and tefrabodies for cancer targeting, J. Immunol Methods 2001 Feb l;248(l-2):47-66; Hudson et al., High avidity scFv multimers; diabodies and triabodies, J Immunol Methods 1999 Dec 10;231(l-2):177-89).
- Single-chain antibodies having V(H) and V(L) domains with 10-residue (Gly4Ser)2 or five-residue (Gly4Ser) linkers, or no linkers, have been examined.
- the zero-residue linker sFv formed a trimer with three active antigen combining sites.
- sFv molecules in which the number of amino acid residues between the V(H) and V(L) domains is 0 to 15 are less likely to form monomers and are more likely to form some type of multimer.
- linker length is 1 or 2 amino acids, trimers and/or other multimers are more likely to form.
- Linker lengths of 3 to 12 amino acids favor the formation of dimers, where sFv's having linkers of 12 or more more amino acids are more likely to form monomers.
- Higher multimers of sFv molecules may be polyvalent, polyspecific, or both (see, e.g., Miiller et al., "A dimeric bispecific miniantibody combines two specificities with avidity", Federation of European Biochemical Societies, 432 (1998), pp. 45-49).
- triabodies as a non-limiting example of higher multimers of sFv's, it can be seen that there are three possible combinations of sFv molecules.
- a triabody may comprise three identical or substantially identical sFv molecules, each of which is directed to the same target molecule, and is thus a trivalent triabody.
- a triabody may comprise three different sFv molecules, each of which is directed to a different target molecule, and is thus a trispecific triabody.
- a triabody may comprise two types of sFv molecules, a pair of which (sFvla and sFvlb) is directed to a target molecule #1, whereas the third sFv in the triabody is directed to target molecule #2.
- the latter triabody is both bispecific, as it specifically binds both target molecule #1 and target molecule #2, and bivalent, as it has two binding regions directed to target molecule #1.
- Disulfide-Stabilized Single-Chain Antibodies Disulfide-stabilized sFv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy V(H) and variable light V(L) heterodimers are stabilized by disulfide bonds engineered at specific sites that do not appreciably alter the binding activity ofthe sFv. Such sites lie between structurally conserved framework positions of V(H) and V(L).
- a cysteine residue is introduced into conserved framework regions of both the heavy and light variable domains at positions compatible with the formation of an interdomain disulfide linkage.
- a disulfide-stabilized Fv may be more resistant to denaturation by heat or urea treatment than the conesponding single-chain Fv (sFv).
- the yield of dsFv may be higher than that ofthe sFv (Webber et al., Preparation and characterization of a disulfide-stabilized Fv fragment ofthe anti-Tac antibody: comparison with its single-chain analog, Mol Immunol 1995 Mar;32(4):249-58; Reiter et al., Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized sFv fragment, Cancer Res 1994 May l5;54(10):2714-8).
- Molecular graphic modeling may be used to identify sites for the introduction of interchain disulfide bonds in the framework region of sFv molecules. Mutations that result in the Cys-modification ofthe sites are introduced in the reading frame that encodes the sFv molecule using any appropriate method, e.g., PCR-mediated mutagensis.
- the disulfide-stabilized Fv (dsFv) is expressed and tested for its binding activity (Luo et al., VI -linker- Vh orientation-dependent expression of single chain Fv-containing an engineered disulfide-stabilized bond in the framework regions, J Biochem (Tokyo) 1995 Oct; 118(4): 825-31).
- F(ab')2 assembled from Fab' fragments expressed in E. coli or isolated by limited proteolysis of a monoclonal antibody
- F(ab')2 assembled using leucine zippers
- diabodies Carter et al., Toward the production of bispecific antibody fragments for clinical applications, J Hematother 1995 Oct;4(5):463-70).
- Multivalent disulfide-stabilized sFv's can be prepared by a variety of methods, including but not limited to phage display (Brinkmann et al., Phage display of disulfide-stabilized Fv fragments, J Immunol Methods 1995 May 1 l;182(l):41-50). Improved yields of multivalent sFv's may be achieved using the P.
- Cloning strategies are known that can be used to create repertoires of diabody molecules having two different antigen binding sites (bispecific diabodies) or two ofthe same, or substantially the same, binding sites (bivalent diabodies)
- McGuinness et al. Phage diabody repertoires for selection of large numbers of bispecific antibody fragments, Nat Biotechnol 1996 Sep; 14(9): 1149-54; Pluckthun et al., New protein engineering approaches to multivalent and bispecific antibody fragments, frnmunotechnology 1997 Jun;3(2):83-105
- Poljak Production and stracture of diabodies, Stracture 1994 Dec 15;2(12):1121-3; and U.S. Patent No. 6,071,515).
- Phage displaying bivalent diabodies, or multiple copies of sFv monomers are used to identify multivalent compounds and complexes that bind domain 6, the plgR stalk, or any other portion or region of plgR. Phage displaying bivalent diabodies, or multiple copies of sFv monomers, are used to identify multivalent compounds and complexes that are more efficiently endocytosed than phage displaying monomeric sFv.
- Multivalent and/or polyspecific compounds and moieties derived from T-cell receptors may also be prepared. See, e.g., Golden et al., High-level production of a secreted, heterodimeric alpha beta murine T-cell receptor in Escherichia coli, J Immunol Methods 1997 Aug 7;206(l-2): 163-9. VII.B .2. Production by Chemical Treatment of Single Chain Antibodies
- sFv molecules that contain an genetically inserted cysteine either at the amino or carboxy terminus ofthe sequence or at some place on the surface ofthe sFv that does not interfere with its ability to recognize its antigen
- a bispecific sFv can be formed using the reactions 3 and 4 in Figure 6.
- a sFv that recognizes plgR can be genetically modified to contain a cysteine residue.
- This conjugate can be reacted in a second reaction with another sFv, having a different recognition specificity, that also contains a cysteine, which can be at the amino or carboxy terminus or on the surface ofthe sFv.
- cysteines at the amino or carboxy terminus can be genetically attached using a spacer that provides sufficient distance between the surface ofthe sFv and the cysteine to facilitate chemical reactivity and to allow flexibility between the two conjugated sFv moieties.
- Either ofthe sFvs can be in any orientation, VL-linker-VH or VH-linker-VL.
- Either ofthe sFvs may contain the cysteine on a spacer (or within the spacer sequence) at the amino or carboxy terminus or on the surface ofthe sFv.
- the spacer can be comprised of (Gly4Ser)x, where x may be 1 to 5 and preferably 2 or 3. Other spacers may also be used, but the spacer should not be immunogenic.
- FIG. 7 illustrates linkage using one sFv that has been derivatized with 2-iminothiolane, a reagent that reacts with amino groups (lysines predominantly and the alpha-amino group of proteins and peptides), and one sFv that has been derivatized with SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate, a reagent that also reacts with amino groups.
- SPDP N-succinimidyl-3-(2-pyridyldithio
- Figure 8 illustrates another method of forming a disulfide bond between two different sFv molecules.
- One sFv is modified with SPDP.
- a disulfide interchange is carried out by the addition of a sFv with a cysteine added to its surface by cysteine substitution or by addition of a spacer containing a cysteine.
- F(ab')2 fragments made from the bispecific antibody can be produced using conventional approaches. Enzymatic cleavage of Ig molecules depends on the characteristics ofthe individual molecule. For example, pepsin is not always successful in producing F(ab')2 fragments, and the digestion conditions frequently needed optimization before acceptable yields are produced. In addition to pepsin, ficin can be used for F(ab')2 production.
- F(ab')2 fragments are held together by disulfide bond(s) between the H chains of the divalent molecule.
- the Fab' fragments which are monovalent, can be released.
- 2-Mercaptoethylamine and other educing agents are used for this purpose.
- Fab Fab fragments are monovalent antibody fragments that can be produced by papain digestion.
- a convenient method is to use papain immobilized on a resin so that the enzyme can be easily removed and the digestion terminated.
- Fab fragments do not have the disulfide bond(s) that are present in F(ab')2 between the H chains.
- Fab'-SH The reduction of a F(ab')2 molecule, which can be formed by pepsin digestion or ficin digestion in the presence of 1 mM cysteine of an intact antibody, produces Fab'-SH.
- Fab'-SH can be converted to a mixed disulfide with Ellman's Reagent.
- An Fab'-SH that has a different specificity may then be used in a disulfide interchange to form a Fab bispecific entity that contains combining sites directed at two different antigens.
- VII.C.Another method of linking Fab's uses a nonreducible covalent bond.
- One of the Fab'-SH partners can be modified with a bifunctional reagent, such as a reagent having two reactive maleimido groups (a bis-maleimido compound). If the maleimido reagent is in large molar excess over the Fab-SH, then only ofthe maleimido groups on the bis- maleimido will react to form a thioether bond. Therefore, a derivative such as Fab'-S- maleimide-spacer-maleimido group will be isolated.
- This derivative can be reacted with a nearly equal molar amount of another Fab'-SH (having a different antigen recognition specificity) to form another thioether bond.
- the product of this series of reactions is a divalent molecule that has two different recognition specificities that are held together by thioether bonds.
- the spacer may be relatively short, as in bis-maleimidohexane, or may be longer and more hydrophilic, as in (Gly4Ser)3.
- one or more tandem repeats ofthe DNA sequence that encode the [V(H)-V(L)] domains are introduced into the chimeric reading frame that encodes the fusion protein.
- V(H) and V(L) are combined in a single chain construct (see, e.g., U.S. Patent No. 6,121,424).
- intramolecularly folded bivalent diabodies are prepared, preferably from soluble periplasmic extracts.
- Fusion proteins comprising tetravalent single-chain antibodies, e.g., ⁇ (N(H)- V(L)]2 ⁇ 2 wherein each V(H) and V(L) can combine to form a sFv, may be prepared using similar strategies (Booth et al., Genetically Engineer Tetravalent Single-Chain Fv ofthe Pancarcinoma Monoclonal Antibody CC49: Improved Biodistribution and Potential for Therapeutic Application, Cancer Research 60, 6964-6971, December 15, 2000). See also U.S. Patents os.; 5,869,620; 5,877,291; and 5,892,020.
- fusion proteins comprising single chain Fv (sFv) fragments
- the orientations of the V(H) and V(L) domains relative to each other, and other fusion protein elements influences the expression and activity ofthe sFv portion (Luo et al., Vl-linker-Vh orientation-dependent expression of single chain Fv-containing an engineered disulfide- stabilized bond in the framework regions, J Biochem (Tokyo) 1995 Oct; 118(4): 825-31).
- Multivalent fusion proteins can comprise other polypeptidic targeting elements.
- fusion proteins may comprise polypeptides derived from bacterial proteins that bind to plgR and/or plgR stalk molecules.
- Derivatives of monoclonal antibodies directed to plgR and/or plgR stalk molecules, e.g., complementary determining sequences (CDR), (Fab)2 molecules and the like, may be prepared and incorporated into fusion proteins.
- Cysteine and other reactive amino acid residues that are naturally present or artificially introduced into a monomer molecule may be reacted in order to create chemically linked multimers.
- intermolecular disulfide (- S-S - ) bonds may be formed to link monomers together.
- Such intermolecular disulfide bridges may be eliminated or reduced by addition of reducing agents, e.g., DTT.
- Chemical cross linkers e.g., bifunctional linkers, can be used to form chemical bonds between monomers.
- multivalent compounds may be prepared by the chemical linkage of two monovalent molecules, each of which comprises a targeting element.
- the multivalent conjugate may then be covalently or non-covalently associated with a bioactive molecule.
- multivalent bioactive compounds may be prepared by chemically conjugating two monovalent bioactive molecules (i.e., molecules comprising a bioactive moiety and a single targeting element) to each other. This is one way in which the ratio of bioactive molecules to targeting elements may be controlled; in the former case, the conjugate has 2 targeting elements and 1 bioactive moiety, whereas the latter conjugate comprises 2 targeting elements and 2 bioactive moieties.
- Leucine Zippers A number of eukaryotic transcription factors comprise a dimerization motif called the "leucine zipper". These leucine zipper proteins form homodimers and/or heterodimers with another protein containing a leucine zipper motif.
- Proteins that dimerize due to the presence or introduction of leucine zippers are said to be "leucme zipped.” See, Rieker et al., Molecular applications of fusions to leucine zippers, Methods Enzymol 2000;328:282- 96; Hoyne et al., High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper, FEBS Lett 2000 Aug 1 l;479(l-2): 15-8; Behncken et al., Growth hormone (GH)-independent dimerization of GH receptor by a leucine zipper results in constitutive activation, J Biol Chem 2000 Jun 2;275(22): 17000-7; Busch et al., Dimers, leucine zippers and DNA-binding domains, Trends Genet 1990 Feb;6(2):36-40; Riley et al., Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucme zipper is
- GST sequences can be used as dimerization sequences.
- Tudyka, Glutathione S- transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli, Protein Sci 1997 Oct;6(10):2180-7.
- a fusion protein in which multiple V(H) regions are covalently bonded, may be non-covalently associated with a second fusion protein having multiple V(L) regions that are covalently linked to each other (see, e.g., U.S. Patent No. 6,239,259), a complex that has the structure found in the following diagram.
- Calcitonin is a biologically active protein that is discussed in the Examples.
- Calcitonin is a polypeptide hormone having 32 amino acids. Efforts to create a formulation, such as those that are oil-based or polymer-based delivery systems, appropriate for delivery of calcitonin via nasal, oral, vaginal and rectal routes are reviewed by Tones-Lugo et al. (Biomaterials 21:1191-1196, 2000). Despite such efforts, there is no widely used and approved non-invasive method for administering calcitonin. Nevertheless, calcitonin is of great interest as a potential therapeutic agent for diseases such as osteoporosis and osteoarthritis (Milot et al., Comp. Ther. 26:183-189, 2000; Kenny, Rheum. Dis. Clin. North Am. 26:569-591, 2000).
- Calcitonin is a polypeptide hormone that is primarily produced and secreted by the parafollicular cells ofthe thyroid gland in mammals and by the ultimobranchial gland of birds and fish, but is also synthesized in a wide variety of other tissues, including the lung and intestinal tract.
- Calcitonin is a hormone known to participate in calcium and phosphorus metabolism. Calcitonin controls the activity of osteoclasts (the cells that break down old and weakened bone), so it can be replaced by new bone. It has been shown that the calcitonins reduce calcium concentration in blood (Hirsch et al., Science Vol. 146, page 412, 1963), and inhibit feeding (Freed et al., Science Vol. 206, page 850, 1979) and gastric secretion (Hesch et al., Horm. Metab. Res. Vol. 3, page 140 (1971).
- Calcitonin Stracture Structural features of calcitonins include a constant chain length of 32 amino acids, a disulfide bridge between the cysteine residues in positions 1 and 7, forming a ring of seven amino acid residues at the N-terminal, and a carboxy terminal proline amide. Amino acid residues common to all calcitonins are those in the 1st, 4th-7th, 28th and 32nd positions (see Figures 9-11).
- full length calcitonins may be characterized for example by a bridge generally between positions 1 and 7 ofthe polypeptide chain and, alternatively or additionally, by a leucine residue in position 9, and/or a glycine residue in position 28 and/or a proline residue in position 32.
- proline amide at C-terminal is indispensable for the biological activities thereof (Potts et al., Calcium, Parathyroid Hormone and the Calcitonins, page 121 printed by Excerpta Medica, Amsterdam (1971); Sieber, Calcitonin 1969, page 28, Proc. 2nd Symp., printed by Medical Books, London (1970); and Rittel et al., Experientia, Vol. 32, page 246 (1976).
- calcitonins include but are not limited to calcitonin structures that have been altered relative to the natural protein, e.g., (a) one or more amino acid radicals are replaced by one or more other amino acid radicals (natural or synthetic) and/or (b) the disulfide bridge is replaced by an alkylene bridge and/or is open, and/or (c) one or several amino acid radicals are omitted (desaminoacyl derivatives).
- Calcitonin homologs i.e, polypeptides derived from non-human species that have amino acid sequences that are related to, but different from, the sequence of human calcitonin, are also calcitonin derivatives within the scope ofthe invention.
- Non-limiting examples of calcitonin homologs are listed in Table 9 and described in Figures 9-11.
- One skilled in the art will be able to select the appropriate source of DNA, sequence of primers, PCR conditions, etc. for each particular genetic sequence encoding an calcitonin homolog to generate other calcitonin fusion proteins.
- Calcitonin analogs are also calcitonin derivatives are also within the scope ofthe invention. Such analogs may be, for example, polypeptides having amino acid sequences derived from a calcitonin protein. Calcitonin genes and proteins that are the combination of calcitonin sequences from one species to another are also calcitonin analogs as that term is used herein.
- An example ofthe latter type of calcitonin analog is one which has an amino-terminal human calcitonin precursor fused to a salmon calcitonin (Takahashi et al., Peptides 18:439-444, 1997). See also U.S. Patents 6,265,534; 6,251,635; 6,124,299; 6,107,277; 5,977,298; 5,962,270; 5,710,244; and 5,541,159.
- Hybrid or chimeric calcitonin polypeptides may also be prepared and calcitonin derivatives within the scope ofthe invention. See, e.g., Takahashi et al., Peptides 18:439- 44 (1997); U.S. Patent No. 5,831,000; and Japanese Patent JP 1993255391-A 4.
- calcitonin polypeptide embraces calcitonin derivatives having one or more biological activities of calcitonin.
- a variety of methods are known in the art that may be used to evaluate the biological activity of calcitonin derivatives.
- the hypocalcemic rat model can be used to determine the effect of synthetic calcitonin mimetics on serum calcium
- the ovariectomized rat or mouse can be used as a model system for osteoporosis. Bone changes seen in these models and in humans during the early stages of estrogen deficiency are qualitatively similar.
- Calcitonin has been shown to be an effective agent for the prevention of bone loss in ovariectomized humans and also in rats (Mazzuoli, et al., Calcif. Tissue Int. 47:209-14, 1990; Wronski, et al., Endocrinology 129:2246-50, 1991).
- Calcitonin acts directly on osteoclasts via a cell surface receptor, the calcitonin receptor (CRE).
- CRE is a member ofthe G-protein receptor family and transduces signal via activation of adenylate cyclase, leading to elevation of cellular cAMP levels (Lin, et al., Science 254: 1022-4, 1991).
- Calcitonin-mediated receptor activation can be detected by: (1) measurement of adenylate cyclase activity (Salomon, et al., Anal. Biochem. 58:541-8, 1974; Alvarez and Daniels, Anal. Biochem.
- Calcitonin inhibits bone resorption through binding and activation of a specific calcitonin receptor on osteoclasts (The Calcitonins-Physiology and Pharmacology Azria
- Calcitonin is also used in the treatment of Paget's disease where it provides rapid relief from bone pain, which is frequently the primary symptom associated with this disease.
- This analgesic effect has also been demonstrated in patients with osteoporosis or metastatic bone disease and has been reported to relieve pain associated with diabetic neuropathy, cancer, migraine and post-hysterectomy. Reduction in bone pain occurs before the reduction of bone resorption.
- calcitonin uses include but are not limited to treatment of hypercalcemia and Paget's disease, counteracting vasospasms, ischemia, renal failure, and treating male impotence.
- hiterleukins are a class of biologically active proteins, certain members of which are discussed in the Examples. Interleukins are released from helper T-cells that promote lymphocyte proliferation. Interleukins of particular interest that can be adapted to the methods and compositions ofthe invention include interleukins- 1, -2, -3, -4 and -5 (IL-1, IL-2, IL-3, IL-4 and IL-5, respectively).
- IX.A Interleukin-2 (IL-2)
- IL-2 is a central regulator of immune response that mediates proliferation of activated B cells and T cells, including anti-tumor T cells, and plays a role in anti- inflammatory reactions.
- Jnterleukin-2 (IL-2) is a lymphokine secreted by certain T lymphocytes after antigenic or mitogenic stimulation. The actions of IL-2 are mediated through the binding ofthe IL-2 protein to specific high affinity receptors which are present in the membranes of activated, but not resting, lymphocytes.
- Trinchieri Blood 84:4008-4027, 1994.
- IX.A.2. Structure of IL-2 Human IL-2 is synthesized as a precursor protein of 153 amino acids, which includes a 20 amino acid hydrophobic leader sequence.
- the IL-2 molecule has a molecular weight of about 15.4 kD and a slightly basic pi.
- the protein comprises a single intramolecular disulfide bond (Cys58-Cysl05) that is necessary for the biological activity of IL-2 (Yamada et al., Importance of disulfide linkage for constructing the biologically active human interleukin-2, Arch Biochem Biophys 257:194-199, 1987).
- IL-2 Some forms of IL-2 comprise chemical modifications. It has been reported that O- glycosylation occurs at Thr3 of bovine IL-2, and that variants with different masses due to glycosylation exist. However, non-glycosylated IL-2 remains biologically active (Kuhnle et al., Bovine interleukins 2 and 4 expressed in recombinant bovine herpesvirus 1 are biologically active secreted glycoproteins, J Gen Virol 77( Pt 9):2231-2240, 1996).
- IX.B Interleukin-4 (IL-4)
- Interleukin 4 is a potent and pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. IL-4 performs important functions as a major regulator ofthe immune response and plays a role in allergy and asthma by directing the induction of TH2 phenofype in T-cells, activating B-cells, and stimulating the synthesis of IgE antibodies.
- the IL-4 receptor alpha chain (IL-4-BP, IL-4 binding protein) demonstrates a high affinity and specificity for IL-4.
- the crystal stracture of interleukin -4 (IL-4) and its complex with the IL-4 receptor alpha chain has been published by Hage et al.
- the region of IL-4 that binds to the receptor alpha chain is the AC helix face (Krase et al., EMBO J. 12:5121-5129, 1993).
- the functional amino acids that participate in contacts with IL-4 BP have been identified by Wang et al. (Proc. Natl. Acad. Sci. 94: 1657- 1662, 1997).
- the stracture of IL-4 is very similar between the receptor bound and unbound form, but small, significant differences exist.
- Hage et al. disclosed that II 1, N15, and Y124 are main contacts in receptor interaction. Morrison and Leder (J. Biol. Chem.
- E12, 114, L104, D106, F107, and LI 11 in murine IL-4 are important for function; the conesponding residues either homologous or identical in human IL-4 are E9, II 1, L109, Nl 11, FI 12, and LI 17, respectively.
- substitutions of cysteine within the human IL-4 sequence may be made for any residues contained in the segments 19-30, 31-40, 60-69, 95-103, and 104-109 (see Table 6).
- amino acids within helical regions may also be substituted if their side chains are oriented away from the main body ofthe protein and do not participate in interactions with other amino acid side chains that provide stability to the stracture. Furthermore, these side chains should not participate in the biological activity or function ofthe molecule. Examples of amino acid positions for cysteine substitution include His 58, Arg 81, His 74, Gin 71, and Arg 53.
- the identification of specific residues within the IL-4 structure are not meant to be limiting, as other substitutions are possible. IX.D. OTHER INTERLEUKINS
- proteins and peptides show homology with other proteins and peptides. Such homology is the basis for classification of proteins and peptides into families and subfamilies. Homology may be based on sequences or the proteins and peptides or on three dimensional stracture ofthe proteins and peptides, or on a combination of both. It is likely that the same general areas and surface regions of proteins and peptides that are members ofthe same family or subfamily interact with receptors or are involved in other biological functions and activities. Therefore, identification of residues that maybe substituted by cysteine in one family member may be translated to residues that may be substituted in other family members.
- a part ofthe present invention is directed to using cysteine substitution and insertion sites in one of these cytokines to predict equivalent cysteine substitution and insertion sites in the remaining cytokines using their homologous structures to identify those sites.
- Cysteine substitution or insertion derivatives of IL-2 and IL-3 have been described (U.S. Patents Nos. 5,206,344 and 5,166,322, respectively).
- Functional amino acids and domains may be identified by various means, including chemical modification, site specific mutation, deletion analysis, alanine scans and so on.
- Calcitonin is a polypeptide hormone having 32 amino acids (see above).
- Other hormones of interest include other calcintonins including but not limited to human salmon calcitonin, insulin; growth hormone (somatotropin); parathyroid hormone; leptin; melanocyte stimulating hormone; orexin; neuropeptide Y; adrenocorticostimulating hormone; corticotropin like intermediate lobe peptide; melanin; concentrating hormone; opioid peptides including but not limited to endorphins, enkaphalins, and dynomorphins; urotensins including but not limited to urotensin II; amylin related peptides including but not limtied to amylin; gonadotrophin-releasing hormone; follicle stimulating hormone, luteinizing hormone, any follicle stimulating hormone, and parathyroid hormone.
- Growth factors are proteins that induce, promote and otherwise mediate the growth, organization, differentation and/or maintenance of cells. Growth factors are often specific for a given tissue or cell type, and may be named accordingly. Examples of growth factors include but are not limited to various species of growth factor is selected from the group consisting of various species of NT3; various species of fibroblast growth factors, such as, but not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6 and FGF-7; platelet derived growth factor (PDGF); epidermal growth factor (EDGF); endothelial growth factor; various species of vascular endothelial growth factor (VEGF) and vascular permeability factors, including but not limited to VEGF-1, VEGF-2, VEGF-121, VEGF- 165, VEGF-189; nerve growth factor (NGF), placenta growth factors (PGF) including but not limited to PGF-1 and PGF2; hepatocyte growth factor; hepatocyte growth factor/scatter
- ILGF ILGF
- macrophage stimulating protein including but not limited to ILGF-1 and ILGF-2
- macrophage stimulating protein including but not limited to ILGF-1 and ILGF-2
- oncostatin M milk derived peptide growth factors
- eye derived growth factors various forms of transforming growth factor (TGF), including but not limited to TGF-alpha and TGF-beta
- latent transforming growth factor beta binding protein various forms of transforming growth factor (TGF) including but not limited to TGF-beta 1, TGF-beta 2,
- TGF-beta 3 various forms of bone morphogenetic protein (BMP) including but not limited to BMP-1 and BMP-7; osteogenic protein 1; endostatin; angiostatin; and ciliary neurofropic factor.
- BMP bone morphogenetic protein
- Enzymes are proteins that catalyze biochemical reactions and may serve as biologically active proteins. Enzymes of interest include but are not limited to glucocerbrosidase, for the management of Type 1 Gaucher disease; Alglucerase, which is a modified form of glucocerbrosidase; ⁇ glucocerebrosidase ( ⁇ -D-glucosyl-N- acylsphingosine glucohydrolase); intracellular molecules (synthases, phosphatases, kinases such as MAP kinases, glycogen synthase kinase); membrane-bound growth factor receptor (such as the CD45 receptor) kinases and phosphatases; nucleotide exchange factors (mSOS). Examples of inhibitors of enzymes include inhibitors of caspases, kinases, phosphatases and the like.
- Non-limiting examples of such proteins include death domain proteins such as TNF Receptor associated death domain (TRAAD); FAS associated death domain (FADD); TNF associated factor 1 to 3 (TRAF 1 to 3); leukemia inhibitory factor; receptor interacting proteins including receptor interacting protein associated Ich-l/CED-3; caspases; cathepsins; members ofthe bcl-2 family of proteins; and nucleases.
- death domain proteins such as TNF Receptor associated death domain (TRAAD); FAS associated death domain (FADD); TNF associated factor 1 to 3 (TRAF 1 to 3); leukemia inhibitory factor; receptor interacting proteins including receptor interacting protein associated Ich-l/CED-3; caspases; cathepsins; members ofthe bcl-2 family of proteins; and nucleases.
- a biologically active protein ofthe invention may also be an anti-proliferation agent or an anticancer agent.
- Non-limiting examples of such proteins include internal kringle fragments of plasminogen including kringle 1 to 3 (angiostatin) and kringle 1 to 4; amino terminal fragment of urokinase; fragments of basement membrane collagen XVIII including endostatin; soluble FLT-1 receptor; and interferon alpha inducible protein 10.
- a biologically active polypeptide may also be a receptor or fragment thereof.
- Non- limiting examples of such receptors include a TNF-alpha receptor; a cytokine receptor; and a hormone receptor.
- VEGF vascular endothelial growth factor
- VEGF-1 vascular endothelial growth factor-1
- VEGF-2 vascular endothelial growth factor-2
- VEGF-121 vascular endothelial growth factor-121
- VEGF-165 vascular endothelial growth factor-189
- metaloproteases antibodies to integrins
- plasminogen plasminogen activator
- urokinase vascular endothelial growth factor
- Cytokines are proteins involved in signaling between cells during an immune response or involved in an inflammatory response. Lymphokines are a class of cytokines produced by lymphocytes. Representative cytokines and growth factors include, for example, interferons (IFNs; e.g., IFN ⁇ , IFN ⁇ , and IFN ⁇ ); interleukins (including IL-1 through IL-15); and colony stimulating factors (e.g., those involved in the division and differentiation of bone manow stem cells and their progeny, for example, stem cell factor (SCF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), granulocyte macrophage colony stimulating factor (GM-SCF), fibroblast growth factors (e.g. FGFl and FGF2), PDGF, EDGF, various species of VEGF, NT3, andNGF, BDNF, factor VIII, factor IX and insulin-like growth factor.
- IFNs interferons
- IFN ⁇ inter
- a biologically active protein maybe an antigenic polypeptide, i.e, one designed to elicit an immune response.
- a biologically active antigenic protein may be an antigen, super-antigen, epitope or other polypeptide that is derived from a protein that is a part of, is derived from or is associated with a pathogen.
- the term biologically active antigenic protein also encompasses proteins produced by cells ofthe body in response to a pathogenic infection. The term further encompasses proteins produced by cells ofthe body that have an inappropriate pattern of growth or undesirable activity such as, e.g., cancer cells and cells that mediate autoimmune diseases.
- Fusion proteins ofthe invention that include an antigenic portion are intended to elicit an immune response once introduced into the body of an animal; if the response provides a prophylactic effect, the fusion protein may be formulated into a vaccine.
- antigenic polypeptides include a protein from a pathogen including but not limited to a bacterium, a viras, a rickettsial species or a chlamydia species; a protein that is a tumor antigen; and a protein that is required for reproduction.
- viral proteins from human immunodeficiciency viras, respiratory synctial viras, parainfluenza viras, influenza virus, hepatitis A virus, hepatitis B viras, hepatitis C.
- a biologically active protein may also be an antiviral protein.
- antiviral proteins include peptides that inhibit HLV replication and infection by human immunodeficiciency viras, respiratory synctial viras, parainfluenza viras, influenza viras, hepatitis A viras, hepatitis B viras, hepatitis C and fusion of human immunodeficiency virus infected cells including peptides related to amino acid sequences in HIV-1 glycoprotein 41 and glycoprotein gpl20.
- the monoclonal antibody is directed to a cytokine.
- a "cytokine” is a protein, generally having a molecular weight in the range of 5 to 20 kD, that is released by cells and that affect the behavior of other cells.
- cytokines are hormones, but the term tends to be used as a convenient generic shorthand for interleukins, lymphokines and several related signalling molecules such as TNF and interferons. Generally growth factors would not be classified as cytokines, though TGF is an exception.
- Chemokines are a subset of cytokines.
- compositions include but not limited to pharmaceutical compositions.
- a “composition” refers to a mixture comprising at least one carrier, preferably a physiologically acceptable carrier, and one or more compositions or compounds ofthe invention.
- carrier defines a chemical compound that does not inhibit or prevent the incorporation ofthe compositions or compounds into cells or tissues.
- a carrier typically is an inert substance that allows an active ingredient to be formulated or compounded into a suitable dosage form (e.g., a pill, a capsule, a gel, a film, a tablet, a microparticle (e.g., a microsphere), a solution; an ointment; a paste, an aerosol, a droplet, a colloid or an emulsion etc.).
- a suitable dosage form e.g., a pill, a capsule, a gel, a film, a tablet, a microparticle (e.g., a microsphere), a solution; an ointment; a paste, an aerosol, a droplet, a colloid or an emulsion etc.
- a “physiologically acceptable carrier” is a carrier suitable for use under physiological conditions that does not abrogate (reduce, inhibit, or prevent) the biological activity and properties ofthe composition or compound ofthe invention.
- dimethyl sulfoxide is a carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.
- the carrier is a physiologically acceptable carrier, preferably a pharmaceutically or veterinarily acceptable carrier, in which the composition or compound ofthe invention is disposed.
- Drags are agents (compounds and complexes) that are administered to (brought into contact with) an animal, including a human, in any of a variety of therapeutic modalities.
- the term "therapeutic” encompasses modalities including, but not limited to, prophylactic uses that prevent disease; curative uses that eliminate a disease; palliative and ameliorative uses that alleviate, make better, or more tolerable, but do not cure a disease; regressive uses that slow, prevent, or reverse the progress of a disease; and remissive uses that cause a temporary or permanent decrease of a manifestion ofthe disease.
- Therapeutic uses also include those that do not involve a disease per se but nonetheless effect the health of an animal. Examples of such agents are antitoxins, analgesics, anesthesia-inducing agents, agents that are used to treat physical and emotional trauma, and psychoactive drags, e.g., antidepressants, mood stabilizers, and anxiolytic agents.
- a therapeutically effective amount indicates the amount of drug which is effective to achieve an intended purpose without undue undesirable side effects (such as toxicity, irritation or allergic response). What constitutes a therapeutically effective amount of a drug will depend on a variety of factors which the knowledgeable practitioner will take into account in arriving at the desired dosage regimen.
- Prophylactic uses of drags include, but are not limited to, the prevention of infections due to bacteria, viruses, and other infective agents, the prevention or inhibition of further hyperproliferation of cells (i.e., the regression of tumors), and prevention of recunence of diseases that have been treated but may recur.
- Prophylactic effects may, but need not, result from the induction of an immune response to the drag, i.e., the drug is an immunogen.
- Prophylactic drags can be administered to a population or targeted to a subpopulation of high risk individuals.
- the term "high risk individual” is meant to refer to an individual for whom it has been determined, via, e.g., individual or family history or genetic testing, has a significantly higher than normal probability of being susceptible to the onset or recunence of a disease or disorder.
- the individual can be prophylactically treated to prevent the onset or recunence ofthe disease or disorder.
- prophylactically effective amount is meant to refer to an amount of drag that produces an effect observed as the prevention of the onset or recunence of a disease or disorder.
- Prophylactically effective amounts of a pharmaceutical composition are typically determined by the effect they have compared to the effect observed when a second pharmaceutical composition lacking the active agent is administered to a similarly situated individual.
- prodrug is meant to encompass prodrags as well.
- a “prodrug” is a drag that is administered in a form or as a compound that has little or none ofthe desired biological activity.
- a prodrug is altered by processes in vivo to produce a more active agent.
- a prodrug is typically metabolized in vivo in order to product an active agent.
- the active agent is thus a metabolite ofthe compound that has been administered to a patient.
- a well-known example of a prodrag is AZT.
- a compound may be inactive when held in a complex. However, a portion of the complex (a compound) separates from the complex in vivo, thereby generating the active agent.
- a common example is a salt of a drag wherein the drag becomes active upon dissolution ofthe salt in vivo.
- drag thus encompasses agents that are active in vitro as well as those that become active in vivo.
- Drags are typically administered parenterally or enterally.
- Enteral refers to the administration ofthe drug into the gastrointestinal tract, preferable via oral administration.
- Parenteral administration is the admimstration ofthe drag via any other route, e.g., intravenous injection directly into the bloodstream.
- the goal ofthe drug administration is to move the drug from the site of administration to the site in the body where the drug acts to produce its effect, or to administer a systemic therapeutically effective amount ofthe drug.
- Drag absorption usually occurs due to the transport across the membranes ofthe epithelial cells within the gastrointestinal tract. Absorption after oral administration is confounded by numerous factors that vary along the length ofthe gastrointestinal (GI) tract, including but not limited to the luminal pH, surface area per luminal volume, perfusion of tissue, bile and mucus flow, and the epithelial barrier.
- GI gastrointestinal
- Pulmonary administration of drugs i.e., delivery via the respiratory system, is also known.
- parenteral administration does provide a method for eliminating a number ofthe variables that are present with oral administration
- parenteral administration is not a preferable route. This is because parenteral administration usually requires the use of medical personnel and is not practical for the administration of many drags. Even when required, parenteral administration is not prefened due to concerns such as patient discomfort, risk of infection, etc., as well as the equipment and costs involved.
- certain therapies require parenterally delivered drugs. Such drugs include polypeptides and other macromolecules that are degraded in the body, which occurs to a large degree in the GI tract.
- Epithelial barriers must be overcome to achieve non-paxenteral routes of admimstration, such as oral and pulmonary administration.
- a drag must traverse several semipermeable cell membranes before reaching general circulation or their targeted site of action. These membranes act as a biological barrier that inhibits the passage of drug molecules.
- the barrier comprises epithelial cells and is thus an epithelial barrier.
- Epithelial barriers include, by way of non-limiting example, those that line the lumen of an organ.
- Epithelial barriers thus include, but are not limited to, surfaces that line the gastrointestinal lumen, the pulmonary lumen, the nasal lumen, the nasopharyngeal lumen, the pharyngeal lumen, the buccal lumen, the sublingual lumen, the vaginal lumen, a urogenital lumen, an ocular lumen, a tympanic lumen, and an ocular surface.
- a “pharmaceutical composition” refers to a composition comprising a drag wherein the carrier is a pharmaceutically acceptable carrier, while a “veterinary composition” is one wherein the carrier is a veterinarily acceptable carrier.
- pharmaceutically acceptable carrier or “veterinarily acceptable carrier” mcludes any medium or material that is not biologically or otherwise undesirable, i.e, the carrier may be administered to an organism along with a composition or compound ofthe invention without causing any undesirable biological effects or interacting in a deleterious manner with the complex or any of its components or the organism.
- the drag is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the patient.
- the pharmaceutical compositions ofthe invention can further comprise other chemical components, such as diluents and excipients.
- a "diluent” is a chemical compound diluted in a solvent, preferably an aqueous solvent, that facilitates dissolution ofthe drag in the solvent, and it may also serve to stabilize the biologically active form ofthe drag or one or more of its components. Salts dissolved in buffered solutions are utilized as diluents in the art. For example, prefened diluents are buffered solutions containing one or more different salts.
- a prefened buffered solution is phosphate buffered saline (particularly in conjunction with compositions intended for pharmaceutical admimstration), as it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a biologically active peptide.
- excipient is any more or less inert substance that can be added to a composition in order to confer a suitable property, for example, a suitable consistency or to form a drag.
- Suitable excipients and carriers include, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol cellulose preparations such as, for example, maize starch, wheat starch, rice starch, agar, pectin, xanthan gum, guar gum, locust bean gum, hyaluronic acid, casein potato starch, gelatin, gum tragacanth, polyacrylate, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpynolidone (PVP).
- fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol cellulose preparations such as, for example, maize starch, wheat starch, rice starch,
- disintegrating agents can also be included, such as cross-linked polyvinylpynolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- suitable excipients and carriers include hydrogels, gellable hydrocolloids, and chitosan. Chitosan microspheres and microcapsules can be used as carriers.
- WO 98/52547 which describes microsphere formulations for targeting compounds to the stomach, the formulations comprising an inner core (optionally including a gelled hydrocoUoid) containing one or more active ingredients, a membrane comprised of a water insoluble polymer (e.g., ethylcellulose) to control the release rate ofthe active ingredient(s), and an outer layer comprised of a bioadhesive cationic polymer, for example, a cationic polysaccharide, a cationic protein, and/or a synthetic cationic polymer; U.S. patent no. 4,895,724.
- chitosan is cross-linked using a suitable agent, for example, glutaraldehyde, glyoxal, epichlorohydrin, and succinaldehyde.
- a suitable agent for example, glutaraldehyde, glyoxal, epichlorohydrin, and succinaldehyde.
- Compositions employing chitosan as a carrier can be formulated into a variety of dosage forms, including pills, tablets, microparticles, and microspheres, including those providing for controlled release ofthe active ingredient(s).
- bioadhesive cationic polymers include acidic gelatin, polygalactosamine, polyamino acids such as polylysine, polyhistidine, polyomithine, polyquatemary compounds, prolamine, polyimine, diethylaminoethyldextran (DEAE), DEAE-imine, DEAE-methacrylate, DEAE-acrylamide, DEAE-dextran, DEAE- cellulose, poly-p-aminostyrene, polyoxethane, copolymethacrylates, polyamidoamines, cationic starches, polyvinylpyridine, and polythiodiethylaminomethylethylene.
- XI.D Formulation of Pharmaceutical Compositions
- compositions and compounds ofthe invention can be formulated in any suitable manner.
- the compositions or compounds may be uniformly (homogeneously) or non-uniformly (heterogenously) dispersed in the carrier.
- Suitable formulations include dry and liquid formulations. Dry formulations include freeze dried and lyophilized powders, which are particularly well suited for aerosol delivery to the sinuses or lung, or for long term storage followed by reconstitution in a suitable diluent prior to administration.
- Other prefened dry formulations include those wherein a pharmaceutical composition according to the invention is compressed into tablet or pill form suitable for oral administration or compounded into a sustained release formulation.
- the pharmaceutical compositions ofthe invention can be placed into any suitable dosage form. Pills and tablets represent some of such dosage forms.
- the pharmaceutical compositions can also be encapsulated into any suitable capsule or other coating material, for example, by compression, dipping, pan coating, spray drying, etc. Suitable capsules include those made from gelatin and starch. In rum, such capsules can be coated with one or more additional materials, for example, and enteric coating, if desired.
- Liquid formulations include aqueous formulations, gels, and emulsions.
- compositions that comprise a bioadhesive, preferably a mucoadhesive, coating.
- a “bioadhesive coating” is a coating that allows a drag to adhere to a biological surface or substance better than occurs absent the coating.
- a “mucoadhesive coating” is a prefened bioadhesive coating that allows a substance, for example, a composition according to the invention, to adhere better to ucosa occurs absent the coating.
- micronized particles e.g., particles having a mean diameter of about 5, 10, 25, 50, or 100 ⁇ m
- the coated particles can then be assembled into a dosage form suitable for delivery to an organism.
- the dosage form is then coated with another coating to protect the formulation until it reaches the desired location, where the mucoadhesive enables the formulation to be retained while the compositions or compounds ofthe invention interact with the target cell surface fransport moiety.
- compositions ofthe invention facilitate administration of monoclonal antibodies to an organism, preferably an animal, preferably a mammal, bird, fish, insect, or arachnid.
- Prefened mammals include bovine, canine, equine, feline, ovine, and porcine animals, and non-human primates. Humans are particularly prefened.
- Multiple techniques of administering or delivering a compound exist in the art including, but not limited to, oral, rectal (e.g., an enema or suppository) aerosol (e.g., for nasal or pulmonary delivery), parenteral, and topical administration.
- sufficient quantities ofthe composition or compound ofthe invention are delivered to achieve the intended effect.
- compositions or compound to be delivered will depend on many factors, including the effect to be achieved, the type of organism to which the composition is delivered, delivery route, dosage regimen, and the age, health, and sex ofthe organism. As such, the particular dosage of a composition or compound ofthe invention included in a given formulation is left to the ordinarily skilled artisan's discretion.
- a suitable pharmaceutical carrier for pharmaceutical compositions ofthe present invention are administered as agents to achieve a particular desired biological result, which may include a therapeutic or protective effect(s) (including vaccination), it may be necessary to combine the composition or compound ofthe invention with a suitable pharmaceutical carrier.
- suitable pharmaceutical carrier and the preparation ofthe composition or compound as a therapeutic or protective agent will depend on the intended use and mode of administration. Suitable formulations and methods of administration of therapeutic agents include, but are not limited to, those for oral, pulmonary, nasal, buccal, ocular, dermal, rectal, or vaginal delivery.
- the context-dependent functional entity can be delivered in a variety of pharmaceutically acceptable forms.
- the context-dependent functional entity can be delivered in the form of a solid, solution, emulsion, dispersion, micelle, liposome, and the like, incorporated into a pill, capsule, tablet, suppository, areosol, droplet, or spray.
- Pills, tablets, suppositories, areosols, powders, droplets, and sprays may have complex, multilayer structures and have a large range of sizes. Aerosols, powders, droplets, and sprays may range from small (1 micron) to large (200 micron) in size.
- compositions ofthe present invention can be used in the form of a solid, a lyophilized powder, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, wherein the resulting composition contains one or more ofthe compounds of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications.
- the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
- the carriers which can be used include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, com starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form.
- a stabilizing dry agent includes triulose, preferably at concentrations of 0.1% or greater (See, e.g., U.S. Patent No. 5,314,695).
- Dosing of therapeutic compositions is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution ofthe disease state is achieved.
- Optimal dosing schedules can be calculated from measurements of drag accumulation in the body ofthe patient.
- the term "patient” is intended to encompass animals (e.g., cats, dogs and horses) as well as humans. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual therapeutic agents, and can generally be estimated based on EC50 found to be effective in in vitro and in vivo animal models.
- dosage is from 0.01 ug to 100 g per kg of body weight, preferably 0.01 ug to 10 g/kg of body weight, 0.01 ug to 1000 mg/kg of body weight, 0.01 ug to 100 mg/kg of body weight, 0.01 ug to 10 mg/kg of body weight, 0.01 ug to 1 mg/kg of body weight, 0.01 ug to to 100 ug/kg of body weight, 0.01 ug to to 10 ug/kg of body weight, 0.01 ug to 1 ug/kg of body weight, 0.01 ug to 10 ug/kg of body weight, 0.01 ug to 1 ug/kg of body weight, 0.01 ug to 10 ug/kg of body weight, 0.01 ug to 1 ug/kg of body weight, 0.01 ug to 0.1 ug/kg of body weight, and ranges
- Doses may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations ofthe drag in bodily fluids or tissues.
- it may be desirable to have the patient undergo maintenance therapy to prevent the recunence ofthe disease state, wherein the therapeutic agent is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
- Some drugs, such as vaccines may be administered once in a lifetime, or with booster shots only as circumstances wanant. The specific dose is calculated according to the approximate body weight or surface area ofthe patient.
- determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity ofthe disease, the route of administration, and the age, sex and medical condition ofthe patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light ofthe dosage information and assays disclosed herein. The dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data.
- An individual patient's dosage can be adjusted as the progress ofthe disease is monitored. Blood levels ofthe drag in a patient can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration.
- Pharmacogenomics may be used to determine which drugs and dosages thereof are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).
- the pharmaceutical compositions ofthe invention facilitate administration of biologically active complexes and compounds to an organism, preferably an animal, preferably a mammal, bird, fish, insect, or arachnid.
- Prefened mammals include bovine, canine, equine, feline, ovine, and porcine animals, and non-human primates. Humans are particularly prefened.
- Multiple techniques of administering or delivering a pharmaceutical composition exist in the art including, but not limited to, oral, aerosol (e.g., for nasal or pulmonary delivery), parenteral, and topical administration.
- a sufficient quantity ofthe biologically active complex or compound, or a bioactive portion or metabolite thereof, ofthe pharmaceutical composition is delivered to achieve the intended effect.
- the particular amount ofthe biologically active complex or compound to be delivered will depend on many factors, including the effect to be achieved, the type of organism to which the pharmaceutical composition is delivered, delivery route, dosage regimen, and the age, health, and sex ofthe organism. As such, the particular dosage of composition or compound ofthe invention included in a given formulation is left to the ordinarily skilled artisan's discretion.
- the pharmaceutical compositions ofthe invention allow a biologically active complex or compound, or a bioactive portion or metabolite thereof, to be efficaciously delivered as part of a plgR-targeting composition or compound. Because plgR-ligands are delivered into cells by active transport, the instant pharmaceutical compositions afford better control over bioavailability of monoclonal antibodies as compared to passive transport mechanisms. As such, the plgR-targeting protein conjugates and compositions ofthe invention enable improved uptake and utilization ofthe monoclonal antibody.
- compositions and compounds ofthe invention are also useful in diagnostic and related applications.
- One aspect ofthe invention involves the diagnosis and monitoring of certain diseases, preferably in kit form. This aspect is useful for assaying and monitoring the course ofthe diagnosis and prognosis of disease, for monitoring the effectiveness and/or distribution of a therapeutic agent or an endogenous compound, in a patient as well as other related functions.
- a patient's plgR-displaying cells are capable of, or presently are, endocytosing a detectably labeled composition or compound ofthe invention.
- Such methods are used in a variety of systems depending on the nature ofthe plgR-targeting element(s) of a given protein conjugate.
- the degree to which a patient, or a biological sample therefrom, endocytoses a composition or compound that has a plgR-targeting element derived from a bacterial protein that binds plgR is a measure of a patient's susceptibility to infection by bacteria having that element. A higher degree or rate of uptake ofthe detectable label indicates that the patient is more susceptible to such infection.
- the activity, distribution and/or concentration of endogenous plgR proteins may be altered in various ways during the course of a disease or disorder.
- the plgR proteins in a patient are measured over the course of a disease for diagnostic and prognostic purposes, as well as over the course of treatment of a disease or disorder, in order to monitor the effects on plgR proteins.
- Diseases to which this aspect ofthe invention can be applied include but are not limited to diseases that involve the respiratory system, such as lung cancer and tumors, asthma, pathogenic infections, allergy-related disorders, and the like; the gastrointestinal tract, including cancers, tumors, pathogenic infections, disorders relating to gastroinstestinal hormones, Chron's disease, eating disorders, and the like; and any disease or disorder that is known or suspected to involve plgR-displaying cells.
- compositions and compounds ofthe invention may be detectably labeled by virtue of comprising a detectable polypeptide such as, e.g., a green fluorescent protein (GFP) or a derivative thereof.
- a detectable polypeptide such as, e.g., a green fluorescent protein (GFP) or a derivative thereof.
- GFP green fluorescent protein
- the protein conjugate comprises an epitope for which antibodies are available (including but not limited to commercially available ones such as c-myc epitope and the FLAG-tag)
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- a drag that is designed to be specifically or preferentially delivered to its intended site of action is said to be targeted. That is, such drugs comprise targeting elements directed to the desired site of action.
- Antibodies, particularly single-chain antibodies, directed to surface antigens specific for a particular cell type have been associated with and used to target drags. See, for example, Kuroki et al., "Specific Targeting Strategies of Cancer Gene Therapy Using a Single-Chain Variable Fragment (scFv) with a High Affinity for CEA," Anticancer Res., pp. 4067-71, 2000; U.S.
- Targeting elements directed to the plgR stalk or other plgR domains and regions described herein may serve an additional purpose beyond penetrating epithelial barriers. Some cancer cells abenantly express plgR (Phillips-Quagliata et al., J. Immunol. 165:2544- 2555, 2000). Targeting elements directed to the plgR stalk or other plgR domains and regions serve as targeting elements to such cancer cells, or other cells that abenantly express plgR. In these instances, targeting elements directed to the plgR stalk or other plgR domains and regions can be associated with cytotoxins and delivered to the cancer cells for therapeutic benefit.
- the absorption rate constant expresses the speed of drag absorption.
- Drug absorption refers to the process of drag movement from the site(s) of administration ofthe drag into the body of an animal.
- Various factors, including the formulation ofthe drag influence the efficacy of rate of absorption of a drag.
- most orally administered drags are in the form of tablets or capsules, for reasons such as convenience, economy, stability, and patient acceptance and compliance. These capsules or tablets must disintegrate or dissolve before absorption ofthe drag can occur.
- the absorption of some drags is further influenced by factors that result from the consumption of food.
- the presence of fiber or other substances in the GI fract may limit the absorption of drugs, and the secretion of fluids that occur in response to ingestion or during digestion may also impact their abso ⁇ tion.
- Once such fluid is bile, which enhances abso ⁇ tion of many substances, including some drugs.
- the release of digestive enzymes may be induced by ingestion, and these enzymes may effect the rate of dissolution of pills, tablets, and the like, and/or degrade the drag.
- Bioavailability of a drug is another pharmacological property.
- Bioavailability is defined as the rate at which and the extent to which a drag, or a biologically active metabolite or portion thereof, enters the general circulation and/or its targeted site of action. Bioavailability is influenced by a number of factors, including how the drag product is designed and manufactured, its physicochemical properties, the rate at which the drug is eliminated from the body, and factors that relate to the physiology and pathology ofthe patient. Reactions that compete with abso ⁇ tion can reduce bioavailability.
- peak time is often not a good statistical measure because it is a discrete value that depends on frequency of blood sampling and, in the case of relatively flat concentrations near the peak, on assay reproducibility.
- AUC is a more reliable measure of bioavailability, as it is directly proportional to the total amount of unchanged drag that reaches the systemic circulation.
- the rate of elimination of a drag from the body varies and effects its efficacy. A higher rate of elimination conesponds to decreased bioavailability. Thus, lower rates of elimination are generally prefened, although higher rates may be preferable for drags having undesirable effects, such as toxicity.
- One parameter relating elimination rate to plasma concentration is total clearance, which equals renal clearance plus extrarenal (metabolic) clearance.
- the elimination rate constant is a function of how a drug is cleared from the blood by the eliminating organs and how the drug distributes throughout the body.
- Another factor relating to elimination is the fraction excreted unchanged, which reflects the amount of drag that is excreted relative to the amount that is metabolized. A low fraction indicates that hepatic metabolism is the likely mechanism of elimination, whereas higher fractions indicate that renal excretion is the predominant form of drug elimination.
- the rate of elimination is desirably increased or decreased depending on the nature and use ofthe drug in question. Often, a decreased rate of elimination is desirable, as this increases bioavailability. However, in the case of some agents, an increased rate of elimination may be preferable. For example, not every molecule of a targeted drug that is introduced will find its intended site of action, and it maybe desirable to remove these molecules from the body before they cause an undesirable effect at some other site in the body.
- the therapeutic index is a measure ofthe relative desirability of a drag for the attaining of a particular therapeutic result.
- the therapeutic index is usually expressed as the ratio ofthe largest dose producing no toxic symptoms to the smallest dose that results in a desired therapeutic result. Higher therapeutic indicia are prefened and an index of ⁇ 1 is unacceptable, except in the case of some terminal diseases.
- first-pass effects may so greatly limit the bioavailability of an orally administered drug that alternative routes of administration must be employed in order to achieve a therapeutically effective dose ofthe drag.
- Drugs transported through epithelial tissues may bypass first- pass effects, which is a pharmacological property that is a desirable attribute.
- the half-life of a drag is the time required for drag concentration or the amount of drag in the body to decrease by 50%. For most drags, half-life remains the same regardless of how much drag is in the body, but there are exceptions (e.g., phenytoin, theophylline, and heparin). Generally, a higher half-life is prefened, as this reduces the amount and lowers the frequency of administration ofthe drug necessary to achieve its intended therapeutic effect. However, there are times when a decreased half life is prefened, particularly when the drug has undesirable side-effects, e.g., toxicity.
- polyclonal antibodies directed to sFv5AF are used to simultaneously detect the single-chain antibodies sFv5 AF and sFv5 AF-Cys, and conjugates comprising these sFv's.
- the anti-sFv5AF polyclonal antibodies were prepared as follows.
- FLAG-tagged sFv5AF was used as an immunogen for the production of antisera (polyclonal antibodies).
- the antisera was commercially prepared by HTI Bio-Products (Ramona, CA).
- 200 ⁇ g of FLAG-tagged sFv5AF was used for the initial injection (Day 1) with Complete Freund's Adjuvant, followed by boosts of 200 ⁇ g fusion protein with Incomplete Freund's Adjuvant every 2 weeks.
- the injections were subcutaneous. Bleeds were taken at approximately 7 weeks and 9 weeks.
- the sera was screened for reactivity with sFv5AF using an ELISA.
- Sera that tested positive in the ELISA were examined by Western blot to confirm the presence of polyclonal antibodies reactive with sFv5AF.
- Rhesus and Cynomolgus monkey intestinal tissue was obtained from Yerkes Regional Primate Center (Atlanta, GA). At least 30 grams of tissue specimens were each prepared from ileum and colon sections where the tissue was excised within one-half hour postmortem, rinsed free of feces with PBS, and then rapidly frozen using liquid nitrogen, shipped overnight on dry ice and stored frozen at -80°C.
- a section of cynomolgus colon weighing 5.3 grams (wet weight) was placed in a 50 ml conical tube and rapidly washed 3-5 times with approximately a 30 ml volume of PBS to remove residual fecal material.
- the colon segment was removed to a very small plastic weigh boat and a longitudinal incision was made exposing the luminal surface, which was quickly and gently rinsed with -50 mis of PBS.
- One (1) ml of TRIzol reagent (Life Technologies) was layered and massaged on the luminal surface, collected in a 15 ml conical tube, and total cellular RNA isolated as per manufacturer's instructions.
- RNA solution was centrifuged at 12,000 x g to remove insoluble cellular debri, and 700 uls of total solution transfened to an microfuge tube. 140 uls of chloroform was added the solution centrifuged at 14,000 rpms for 15 minutes at 4°C. 430 uls of aqueous phase was collected, 215 uls of isopropanol added, incubated at room temperature for 10 minutes, and the RNA precipitated by centrifugation at 14,000 rpms for 10 minutes at 4°C. The white pellet was washed with 1 ml of 75% ethanol, air dried for 5-10 minutes, and the RNA pellet resuspended in 50 uls of DEPC-treated water. Quantitation of total RNA was determined
- sequences ofthe synthetic degenerate DNA primers (prepared by Genset, Inc., Paris, France) that were used in the first strand cDNA synthesis (RT-PCR) and PCR amplification ofthe cynomolgus monkey partial cDNA are as follows.
- RT-PCR primer EPKKAKRS-Low Reverse primer (SEQ ID NO:
- R designates either an A or G purine base
- Y designates either an C or
- N designates either ofthe A, C, G or T bases
- R designates either an A
- oligonucleotide primer (SEQ ID: RT-PCR primer) was used together with the Superscript First Strand Synthesis Kit (Life Technologies) to synthesize the first strand cDNA from 5 ug of total cynomolgus monkey RNA as per manufacturer's instructions. Briefly, 100 pmols of primer (SEQ ID: _ RT-PCR primer) and 5 ug of total RNA was included in a 10 ul RT-PCR reaction, heated to 70°C for 10 minutes, then cooled to 4°C. A 9 ul 10X RT-buffer mixture was then added to the RT-PCR reaction and incubated at 42°C for 2 minutes, followed by the addition of 1 ul of Superscript II enzyme to each reaction.
- the reverse transcription reaction was allowed to proceed at 42°C for 50 minutes. Proper control reactions were also assembled and run simultaneously. The reactions were terminated by heating to 70°C for 15 minutes. To prevent interference ofthe RNA in the subsequent PCR amplification step, 1 ul of RNase H was added and the reaction incubated at 37°C for 20 minutes before storing the single stranded cDNA material at -20°C. 2.2. Isolation, Identification and Sequencing of Simian plgR Sequences
- the resulting PCR product was ligated into the pCR-II vector (Invitrogen) as per manufacturer's instructions and the ligation reactions transformed into TOPO One-shot competent cells (Invitrogen). Colonies were selected and 3 ml mini- cultures grown, miniprep DNA prepared using the Qiagen Miniprep Kit (Qiagen), and positive clones identified by an Eco RI restriction enzyme analysis.
- oligonucleotides were used to clone a 730 nucleotide region of cynomolgus monkey plgR cDNA from monkey intestinal tissue.
- This partial cDNA sequence encodes for most of domain 5 through the cytoplasmic domain (homologous to a region ofthe human plgR molecule conesponding to amino acids Glu474 through Ser717).
- Detailed sequence and alignment analysis comparing the human and cynomolgus monkey plgR cDNAs demonstrate that the sequences differ in 18 amino acids within this 242 amino acid region (Glu474 through Ser717).
- the amino acid sequences for a simian plgR are shown in Figure 2B.
- Nucleic acids and polypeptides having nucleotide and amino acid sequences, respectively, ofpIgR from cynomolgus, or portions of these sequences, are inco ⁇ orated into a variety of methods and compositions.
- the cynomolgus cDNA is used to exemplify the production and uses of these methods and compositions.
- Nucleic acids are used to produce plgR polypeptides via recombinant DNA technology.
- the nucleic acids are used to generate chimeric reading frames that inco ⁇ orate plgR or a portion, domain or region thereof.
- Chimeric reading frames encode fusion proteins (e.g., a GST-domain 6 fusion protein), chimeric proteins (e.g., a rat/rabbit hybrid plgR), amino acid substituted polypeptides and other derivatives of plgR, and polypeptides that have a therapeutic benefit when expressed within the cells of an animal, including a human.
- nucleic acids or synthetic oligonucleotides having sequences derived therefrom, are used as probes for the identification and/or amplification of plgR- encoding nucleic acids from other species, and other members ofthe plgR family of proteins that are present in the genome ofthe same species from which the nuclic acids originated.
- Nucleic acids having a sequence, or a portion of a sequence, that is the reverse complement ofthe sense strand ofthe nucleic acids can be used as antisense molecules.
- Derivatives of a polypeptide that has the amino acid sequence set forth including without limitation oligopeptides, proteolytic fragments, fusion proteins, and peptiomimetics. These polypeptides are therapeutic agents and/or are used as target molecules in the methods of the invention.
- a rat liver cDNA library (Clontech) was used as a source for template for the amplification of rat plgR sequences.
- the plgR cDNA was amplified as 5 separate fragments which can be combined to regenerate the entire rat plgR sequence (see Figure 14).
- the sequences contained within separately cloned cDNA's may be used as a source for sequences that encode a rat stalk molecule or sequences derived therefrom.
- the primers used to amplify the rat cDNA regenerated or introduced restriction enzyme sites into the cDNA for ease of subcloning and other subsequent manipulations.
- Each fragment was treated with the appropriate restriction enzymes and ligated into a cloning vector (e.g., pBluescript from Sfratagene or pUC19 from NEB) in order to generate an "intermediate vector".
- the sequence ofthe inserted cDNA was determined in order to confirm the sequence ofthe amplified DNA.
- a mouse liver cDNA library (Clontech) is used as a source for template for the amplification of mouse plgR sequences.
- the mouse cDNA is amplified as 5 separate fragments which can be combined to regenerate the entire mouse plgR sequence (see Figure 12).
- the sequences contained within separately cloned cDNA's maybe used as a source for sequences that encode a mouse stalk molecule or sequences derived therefrom.
- the primers used to amplify the mouse cDNA are designed to introduce restriction enzyme sites into the cDNA for ease of subcloning and other subsequent manipulations. Each fragment is treated with the appropriate restriction enzymes and ligated into a cloning vector in order to generate an "intermediate vector".
- the sequence ofthe cDNA in the intermediate vector was determined in order to confirm the sequence ofthe amplified DNA.
- a human colon cDNA library (Clontech) was used as a source for template for the amplification of human plgR sequences.
- the human cDNA sequences were amplified as 3 separate fragments which were inserted into intermediate vectors and assembled as described above (see Figure 13).
- plgR Madin-Darby canine kidney (MDCK) cells using retroviral vectors has been described by Breitfeld et al. (Methods Cell Biol 32:329-37, 1989). The expression of rabbit and human plgR in MDCK cells has been described, respectively, by Banoso et al. (J Cell Biol 1994 124:83-100) and Tamer et al. (J. Immunol 1995 155:707- 14, 1995). Because rats are useful for in vivo assays, initial in vitro transcytosis assays used MDCK cells transfected with rat plgR.
- rat plgR in transfected MDCK cells was reduced relative to results obtained with rabbit plgR transfected MDCK cells.
- the relatively reduced expression of rat plgR may be a consequence of an unusual stracture in the 5' untranslated region ofthe rat plgR cDNA (Fabregat et al., Physiol Genomics 5:53- 65, 2001; Fodor et al., DNA Cell Biol 16:215-25, 1997; Koch et al., Nucleic Acids Res 23:1098-112, 1995).
- a chimeric protein was produced via PCR using primers to rat and rabbit plgR cDNA sequences and methods known in the art.
- the chimeric protein consists of amino acids 1-554 of rabbit plgR, followed by amino acids 553-645 of rat plgR, then amino acids 651-756 of rabbit plgR.
- This chimeric protein contains the transmembrane and membrane proximal regions of rat plgR, whereas the remainder ofthe molecule is derived from rabbit plgR.
- the chimeric plgR has the same activity as wild type rabbit plgR in plgR assays such as transcytosis of IgA from the basolateral to the apical surface (forward transcytosis).
- the stracture and amino acid sequence ofthe chimeric plgR protein is shown in Figures 15 A and 15B, respectively.
- the chimeric protein was expressed from an expression construct comprising the expression vector pCB7.
- GST-stalk fusion proteins are one type of plgR target molecule.
- the GST (glutathionine-S-fransferase, from Schistosomajaponica, unless otherwise indicated) polypeptide has several illustrative desirable attributes. It specifically binds glutathione, and with a sufficiently high affinity that it can be used to attach fusion proteins to solid surfaces coated with glutathione, and many such surfaces are commercially available; detectably labeled antibodies directed to GST epitopes are commercially available; and the GST amino acid sequences allow some fusion proteins to have enhanced attributes such as, e.g., enhanced solubility, biologically active conformations, and the like.
- GST fusion proteins may optionally comprise elements useful for the detection, isolation, purification and manipulation ofthe GST fusion protein.
- elements include elements such as a 6xHis tag, a FLAG tag, a c-myc epitope, a fluorescent polypeptide (e.g., GFP), a detectable enzymatic polypeptide (e.g. horse radish peroxidase, beta-galactosidase), or a biotin-binding polypeptide (e.g., avidin or streptavidin) polypeptide.
- GST fiision proteins are expressed in E.
- Non-limiting examples of GST fusion proteins include those that comprise a portion ofthe stalk that contains the desired sites of reaction, e.g., domain 5 and domain 6, domain 6, or smaller portions of domain 6; or of any other regions of plgR and stalk molecules such as those described herein in Tables 1 and 4.
- the fragment of plgR or stalk molecule used in a GST fusion protein may change depending on the nature of a particular use ofthe GST fusion protein, but those skilled in the art will know what amino acid sequences are appropriate to include in a given GST fusion protein.
- Table 12 summarizes the general characteristics of GST-stalk fusion proteins that are described in more detail in the subsequent subsections and in Figure 16. TABLE 12: GST-STALK FUSION PROTEINS
- a plasmid comprising cynomolgus monkey plgR sequences (“pTA-CynMonk- plgR," which is a derivative ofthe pCR-II plasmid (Invitrogen) having simian plgR sequences) was used as a template for PCR amplification ofthe cynomolgus monkey plgR stalk region using the CynMpIgRstalk-5'FOR and CynMpIgRstalk-3 'REV sequencing primers.
- primers allow for the use of a directional cloning strategy (Bglll to EcoRI ligation) and result in the inco ⁇ oration of a C-terminal 6xHis tag that can be used to isolate or attach the fusion protein to a solid surface.
- CynMpIgRstalkGST 5 'FOR a 5 '-Forward PCR primer containing a Bglll site (underlined) and having the sequence (SEQ ID NO: ):
- CynMpIgRstalkGST 3 'REV a 3 '-Reverse PCR primer containing an in-frame 6xHis tag and an Eco RI site (underlined) (SEQ ID NO: ): 5" -CGGAATTCCTAGTGATGGTGATGGTGATGTTTGGAGCTCCCAC-
- the 309 bp PCR fragment was gel-purified and subjected to restriction digestion using Bglll and EcoRI enzymes, and the resulting 305 bp fragment was gel-purified.
- the purified Bglll-EcoRI fragment was cloned into BamHI- and EcoRI-digested pGEX-2TK (Amersham Pharmacia), a plasmid that has a GST-encoding nucleic acid sequence that can be fused in-frame with a cloned DNA.
- the resulting plasmid was subjected to DNA sequence analysis to confirm the absence of any PCR-induced mutations and to verify that the GST and plgR sequences were linked in-frame with each other.
- GST fusion proteins derived from human, rat and rabbit stalk sequences were prepared essentially according to the methods and methods used in the preceding subsections the preparation of a GST-(Cynmonkey stalk) fusion protein.
- stalk sequences were, in some cases, amplified from the above-described cDNA intermediate vectors comprising fragments ofthe plgR human, rat and rabbit sequences, respectively.
- a plasmid comprising rabbit plgR sequences (“pGST-RabpIgRStalk”) was digested with BamHI and EcoRI, which liberates a 312 bp fragment.
- the 312 bp fragment was cloned into BamHI- EcoRI-treated pGEX-2TK vector, a plasmid that has a GST-encoding nucleic acid sequence that can be fused in-frame with a cloned DNA.
- the resulting plasmid was subjected to DNA sequence analysis to confirm the absence of any PCR-induced mutations and to verify that the GST and plgR sequences were linked in-frame with each other.
- EXAMPLE 5 PREPARATION OF LIGANDS DIRECTED TO DOMAIN 6 AND
- An assay is prepared by applying purified plgR stalk molecules or GST-pIgR stalk molecules, or any other plgR target, to multiwell (48-well, 96-well and other size plates and allowing the protein to adhere to the wells ofthe plates during overnight incubation. The plates are washed to remove unbound proteins. Samples ofthe serum from the immunized mice are incubated with the plgR or GST-pIgR coated plates. After 1 to 2 hours of incubation (gentle shaking at room temperature), the plate is washed free of unreacted immune serum proteins.
- Mouse antibodies that react with an immobilized GST-pIgR protein are detected by adding to each well a sample of a goat antibody that has been raised against and is directed to mouse immunoglobulin, i.e., all subclasses of murine immunoglobulins.
- the goat antibody is conjugated to an enzyme that is used for detection; non-limiting examples include horse radish peroxidase and alkaline phosphatase. After unreacted horse radish peroxidase or alkaline phosphatase conjugated goat anti-mouse immunoglobulin has been washed from the wells, the substrate of horse radish peroxidase or alkaline phosphatase is added.
- the reaction is stopped and quantitated using a spectrophotometer.
- antibodies against the GST-pIgR protein will be present. Some of these antibodies are directed to the GST portion ofthe protein if GST-pIgR is used.
- This assay is also used to identify antibody producing cells and clones in 96-well plates that are part ofthe process of isolating clones of hybridomas that produce the desired monoclonal antibody. Beads that bind GST moieties on GST-fusion proteins are also used for assays. GST-pIgR bound to beads is reacted with sera that contain antibodies directed against plgR.
- the antibodies that react with and bind to plgR can then be detected by an anti- antibody conjugated to horse radish peroxidase or alkaline phosphatase. If the antibodies that react with plgR are derived from mice, then the antibodies that detected the presence ofthe mouse antibody is obtained from another animal species, such as goat or sheep. Those skilled in the art will know how to adjust the source and specificity ofthe detecting antibody conjugates (i.e. horse radish peroxidase or alkaline phosphatase conjugated to anti-FLAG tag antibody) to obtain the desired results.
- the detecting antibody conjugates i.e. horse radish peroxidase or alkaline phosphatase conjugated to anti-FLAG tag antibody
- Monoclonal antibodies are created by immunizing mice with portions of plgR, generally prepared as oligopeptides having defined amino acid sequences.
- plgR portions of plgR, generally prepared as oligopeptides having defined amino acid sequences.
- a nucleic acid encoding an amino acid sequence found in a conserved region of plgR, such as those described in Table 1, or an amino acid sequence that varies between homologs, such as, e.g., RI, R2a, R2b, R3a, R3b, R3c (etc.) (Table 4) is used to create a plgR-target-GST fusion protein that is expressed in a host cell such as E. coli.
- the GST portion ofthe fusion protein is used to isolate the fusion protein, and the purified GST-pIgR protein is mixed with adjuvant and injected into mice to produce an immune response.
- the extent ofthe immune response is measured over time by removing blood from the immunized mice at regular intervals and measuring the level of antibodies directed to the GST-pIgR fusion protein using an immunoassay, e.g., an ELISA.
- the spleen ofthe mouse is harvested, and cells therefrom are prepared for fusion with immortalized fusion partners, such as the NS/1 cell line, according to Kohler and Milstein, in order to create Mab-producing hybridoma cell lines.
- immortalized fusion partners such as the NS/1 cell line, according to Kohler and Milstein, in order to create Mab-producing hybridoma cell lines.
- Independently isolated clones and subclones are grown to an appropriate density, the cell supernatant is assayed using an ELISA to determine if antibodies that react with the GST- pIgR fusion proteins are produced by each clone or subclone. Positive wells are assayed using limiting dilution, and clonal and subclonal cell lines are eventually obtained that produce Mabs against either the GST-pIgR fusion protein.
- Mabs that either are plgR specific or are specific to an epitope not present in either plgR or GST but which occurs at the junction thereof.
- the Mabs can additionally be tested for specificity using MDCK cells and MDCK cells that have been transfected with different species of plgR (human, rat, mouse, pig, rabbit, monkey, etc.).
- Each ofthe sFvs and the Mabs are epitope mapped using the nested set of overlapping oligopeptides (each comprising 5 to 20 amino acids). Linear epitopes and conformational epitopes are identified on the strength of their binding and the location ofthe peptides in the nested set.
- plgR-targeting element is an antibody, or an antibody derivative, directed to a transcytotic molecule such as the plgR stalk.
- a transcytotic molecule such as the plgR stalk.
- sFv single chain Fv antibody fragments
- Non-limiting examples of such sFv antibodies are shown in Figures 3 to 5.
- sFv5AF A derivative of sFv5A that inco ⁇ orates an epitope known as a "FLAG tag” is designated “sFv5AF” ( Figure 3). Due to the way in which it was constructed, the amino acid sequence of sFv5AF has a mutation relative to sFv5A that is denoted “Q5V” (Gin at position 5 changed to Val).
- sFv5 AF-Cys A derivative of sFv5AF that contains a cysteinyl residue near its carboxyl terminus is designated "sFv5 AF-Cys" ( Figure 5). This derivative of sFv5AF has a cysteine residue at the carboxy terminal region was introduced into the reading frame encoding sFv5AF by PCR mutagenesis (see Example 5).
- Another source of amino acid sequences that provide ligands for plgR are targeting elements that bind intracellular portions, regions or domains ofthe plgR stalk.
- One source of such targeting elements is a protein known as calmodulin.
- calmodulin binds plgR and it is thus expected that amino acid sequences within calmodulin interact with plgR and may be isolated and used to prepare polypeptide ligands to plgR (Enrich et al., Hepatology 24:226-232; 1996; Chapin et al., J. Biol. Chem. 271: 1335-1342; 1996).
- the AP-1 clathrin adaptor complex ofthe trans-Golgi network is another protein that has been reported to bind an intracellular part ofthe plgR stalk and can thus serve as a source of targeting elements (Orzech et al., Interactions ofthe AP-1 Golgi adaptor with the polymeric immunoglobulin receptor and their possible role in mediating brefeldin A- sensitive basolateral targeting from the trans-Golgi network, J Biol Chem 274(4):2201-15, 1999). Because these targeting elements are directed to an intracellular portion ofthe plgR stalk (the intracellular or cytoplasmic domain), another element that facilitates cellular uptake may be needed in order to direct complexes or compounds comprising them to these portions ofthe plgR stalk. Once inside the cell, the targeting elements are able to bind the intiacerllular portion of plgR and thus be transported with it. Exemplary examples of such cellular uptake elements include, but are not limited to, PTD and MTS sequences.
- PTD protein transduction domains
- MTS membrane transport signals
- the MTS are very hydrophobic peptides derived from secretory signal sequences, which may be able to spontaneously partition into the hydrophobic region of membrane lipid bilayers (Rojas et al., Genetic engineering of proteins with cell membrane permeability, Nat Biotechnol 16(4):370-5, 1998; Rojas et al., Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor, J Biol Chem, 1996. 271(44):27456-61, 1996). In some cases PTD and MTS peptides are able to confer membrane permeability to proteins that would otherwise not enter cells by cloning them together as a fusion construct.
- Cloning vectors that simplify the inco ⁇ oration of PTD and MTS sequences into recombinantly produced proteins are commercially available (invitrogen). Synthetic versions of these sequences may also be used and covalently or non-covalently associated with a complex or compound ofthe invention; in the case of TAT, these include, but are not limited to, poly- Arg, poly-Lys, poly-omithine, and polymers of Arg, Lys and omithine.
- CpbA interacts with human plgR (hpIgR) as either a part ofthe outer surface of a bacterial cell or as a free molecule.
- the regions of CpbA:hpIgR interaction were mapped using a series of large peptide fragments derived from CpbA.
- CpbA (Swiss-Prot Accession No. O30874) contains a choline binding domain containing residues 454-663 and two N-terminal repetitive regions called RI and R2 (SEQ ID NOS: and , respectively) that are contained in residues 97-203 and 259-365, respectively.
- plgR targeting elements are utilized as plgR targeting elements in the present invention.
- Such polypeptides may include those identified by phage display of disulfide constrained peptides as described above or polypeptides including but are not limited to the CbpAl, CbpA2, and CbpA3 polypeptides described by Zhang et al.
- other polypeptides from bacterial proteins homologous with CpbA, the pneumococcal adhesin protein in Streptococcus pneumoniae studied by Zhang et al. are part ofthe present invention.
- homologous proteins are present in virtually all pneumococcal serotypes.
- Those skilled in the art will be able to identify additional homologous proteins from genomic and protein databases such as Swiss-Prot, Entrez, and GenBank.
- Smaller polypeptides comprising portions ofthe entire sequence of CbpA and proteins homologous to CbpA, and preferably portions of RI and R2 and polypeptides homologous to RI and R2, are identified based on their ability to bind to animal species of plgR, preferably human plgR.
- An overlapping, nested set of peptides can be synthesized and their ability to interact with plgR can be tested to identify peptides that may be used to transport biologically active polypeptides, including vaccines, into (apical and basolateral endocytosis) and across (forward or reverse transcytosis) epithelial cell barriers.
- the peptides may be tested for their ability (i) to prevent SC binding to plgR coated beads or (ii) to prevent adherence, invasion, or transmigration by S. pneumoniae R6x to Detroit cells, both methods being described by Zhang et al.
- the peptides may be from 5 to 100 amino acids long, preferably from 6 to 50, and most preferably from 6 to 20.
- An offset of 1 to 5 amino acids and preferably 3 to 4 amino acids may be used.
- the core linear sequence that is required for binding to plgR may be identified.
- a large peptide may be systematically reduced in size until the smallest peptide that produces a positive binding to plgR is identified.
- Methods for identifying the core linear sequence have been described by Geysen et al. (J. Immunol. Methods 102:259- 274, 1987), Tribbick et al. (J. Immunol. Methods 139:155-166, 1991), Geysen et al. (J. Molecular Recognition 1 :32-41, 1988), Tainer et al. (Mol. Immunol. 23:709-715, 1986).
- EXAMPLE 6 GENETIC MANIPULATION OF A TRANSCYTOSING SINGLE
- sFv5 AF-Cys is a derivative of sFv5AF into which a reactive Cys residue has been inserted, which also has one GGGGS linker between the newly introduced Cys residue and the sFv portion ofthe polypeptide (see Figures 3 to 5).
- the Cys residue contains a side group, -SH, that can react with the -SH side group of another Cys residue to form a disfulfide bond (-S-S-) that links the two Cys residues and the amino acids to which each Cys is attached.
- a Cys residue in a sFv derivative influences whether it will react with a Cys residue in the same molecule (thus producing a monomer having an intramolecular disulfide bond) or a Cys residue in another sFv molecule (thus producing a multimeric sFv molecule having an intermolecular disulfide bond).
- the sFv single-chain molecule sFv5AF was altered via PCR mutagenesis in order to inco ⁇ orate a cysteine residue at the carboxy terminal region.
- the template, a pSyn expression vector encoding sFv5 AF was amplified using a first oligonucleotide primer, "LMB3,” that has a sequence (5'-CAGGAAACAGCTAGAC-3', SEQ ID NO: ) that is complementary to regions 5' ofthe sFv5AF coding region in pSyn), and "cys-long," a second oligonucleotide primer having the sequence:
- the latter primer is complementary to the last 4 codons of sFv5 AF, with the 5 ' end ofthe primer encoding the amino acid sequence GGGGSC in frame with sFv5AF, followed by a Notl restriction site.
- Amplification was performed using the Taq-plus precision polymerase (Sfratagene) according to the manufacturer's instructions.
- the PCR product was cleaved with Ncol and Notl, and then ligated into pSyn expression vector DNA that had been cleaved with Ncol and Notl.
- the resultant expression construct encodes sFv5AF-Cys, which has, from an amino- to carboxy-terminal direction, a pelb leader sequence (for secretion in E.
- sFv5AF-Cys i.e., a heavy chain variable region, a spacer sequence [GGGGS repeated three times, i.e., (6 4 8) 3 ], a light chain
- variable region another (0 4 8) 3 linker, a cysteine residue (emboldened “C") that has been
- sFv5A single chain antibody
- sFv5AF single chain antibody
- sFv5 AF-Cys single chain antibody
- sFv5A single chain antibody
- sFv5 AF-Cys single chain antibody
- sFv5A single chain antibody
- sFv5 AF-Cys single chain antibody
- sFv5A single chain antibody
- sFv5 AF-Cys a nucleotide sequence
- In vitro genetic manipulation is used to alter the amino acid sequence of sFv5A so as to favor the formation of dimers, trimers and other multimers; to add or enhance desirable attributes of sFv5A, and/or to reduce or remove undesirable attributes.
- the sFv single-chain molecule sFv5A was altered via PCR amplification in order to substitute the myc-6xHis-tags at the carboxy terminal region ( Figure 4) with a GGGG-Cys C-terminal tail.
- PCR amplification reactions were assembled using High Fidelity Platinum Taq (Life Technologies) according to manufacturer's instructions (IX High Fidelity PCR buffer, 0.2 mM each dNTP, 2 mM MgSO4, 0.2 ⁇ M of each primer, 2.5 units Platinum Taq High Fidelity, and template DNA as required), which allows for "hot start" PCR to minimize the generation of early stage nonspecific priming events.
- Amplification was carried out using a modified procedure adapted from Roux and Hecker (PCR Cloning Protocols, B.A. White, eds., Humana Press, 1997, pp. 39-45), where thermocycling reactions were ran using linked files in a PCR program as follows: 1) denaturation at 94°C for 10 minutes; 2) 30 cycles of denaturation for 1 minute at 94°C, primer annealing for 1 minute at 60°C, primer extension for 60 seconds at 72°C, and 3) a final 4°C chill step (until analyzed). The size ofthe PCR products were confirmed by agarose gel elecfrophoresis and then purified away from contaminating primers by spin column chromatography (Qiagen QIAquick purification kit).
- the template a pSyn expression vector encoding sFv5A, was amplified using a
- the latter primer is complementary to 7 codons near the C-terminus ofthe sFv5A coding region, with the 5' end ofthe primer encoding the Notl restriction site followed by the amino acid sequence GGGGC in frame with sFv5A, followed by a two (2) tandem TAG stop codons and an EcoRI restriction site.
- the PCR product was cleaved with BamHI and EcoRI, and then ligated into pSyn expression vector DNA that had been cleaved with BamHI and EcoRI.
- the resultant expression constract encodes sFv5 A-G Cys, which has, from an amino- to carboxy- terminal direction, a pelb leader sequence (for secretion in E.
- sFv5A-Cys i.e., a heavy chain variable region, a spacer sequence [GGGGS repeated three times, i.e., (G 4 S) ], a light chain variable region, another G 4 S linker, and a C- terminal cysteine residue that has been introduced into the sFv relative to sFv5A, replacing the c-myc epitope and 6xHis tags encoded by vector sequences shown in Figure 5.
- sFv single-chain molecules sFv5A and sFv5 AF were altered via PCR amplification in order to remove the Notl restriction site and substitute the myc-6xHis-tags at the carboxy terminal region with a GGGG-Cys C-terminal tail.
- PCR amplification reactions were assembled using High Fidelity Platinum Taq (Life Technologies) according to manufacturer's instructions as described above.
- the template a pSyn expression vector encoding sFv5A
- a "Forward" oligonucleotide primer, "Pelb-5 Forward,” (SEQ ID NO: ) that is complementary to the 5 '-portion of the pelb-coding sequence in the pSyn5A vector)
- the latter primer is complementary to the 7 C-terminal codons ofthe sFv5A coding region, followed by the amino acid sequence GGGGC in frame with sFv5A, followed by a two (2) tandem TAG stop codons and sequential Sail and EcoRI restriction sites.
- the PCR product was cleaved with BamHI and EcoRI, and then ligated into either the pSyn-5A or pSyn-5AF expression vector DNA that had been cleaved with BamHI and EcoRI.
- the resultant expression constructs encode sFv5A-(deltaN)Gly 4 -Cys or sFv5AF- (deltaN)Gly 4 -Cys, respectively.
- the amino acid sequence contains, from the amino- to carboxy-terminal direction, a pelb leader sequence (for secretion in E.
- sFv5A-Cys i.e., a heavy chain variable region, a spacer sequence [GGGGS repeated three times, i.e., (G 4 S) ], a light chain variable region, another G S linker, and a C-terminal cysteine residue that has been introduced into the sFv relative to sFv5A, replacing the Notl restriction site and the c-myc epitope and 6xHis tags encoded by vector sequences shown in Figure 4.
- sFv5A-Cys i.e., a heavy chain variable region, a spacer sequence [GGGGS repeated three times, i.e., (G 4 S) ], a light chain variable region, another G S linker, and a C-terminal cysteine residue that has been introduced into the sFv relative to sFv5A, replacing the Notl restriction site and the c-myc epitope and 6xHis tags encoded by vector sequences shown in Figure 4.
- V(H) and N(L) The two variable regions of a sFv that combine to form a ligand binding site are known as V(H) and N(L).
- V(H) and N(L) of each molecule are associated with each other.
- V(H) of one monomer [V(H)1] is associated with the V(L) of another monomer [V(L)2], and vice versa [i.e., V(H)2 is associated with V(L)1].
- the length and composition ofthe linker between the V(H) and V(L) regions in an sFv is one factor that influences the tendency of an sFv to form monomers or multimers (Todorovska et al., Design and application of diabodies, triabodies and tefrabodies for cancer targeting, J Immunol Methods 2001 Feb l;248(l-2):47-66; Amdt et al., "Factors Influencing the Dimer to Monomer Transition of an Antibody Single-Chain Fv Fragment" ,
- sFv molecule in which there is a relatively short linker between the V(H) and V(L) regions may be less likely to fold back upon itself and form a monomer.
- "short linker" sFv derivatives are often more likely to form dimers, as their V(H) and V(L) regions must pair with, respectively, the V(L) and V(H) regions of a second sFv molecule.
- sFv derivatives with relatively long linkers between the V(H) and V(L) regions may fold back upon themselves, and therefore may have a greater tendency to form monomers.
- sFv derivatives with long linkers between V(H) and V(L) may have some tendency to form multimers.
- the number of linkers between the V(H) and V(L) regions of sFv5A has been altered to produce a set of sFv derivatives that are screened and assayed for desirable attributes. That is, the sFv5A derivatives are assayed for their ability to form either monomers or multimers, and multimeric forms are analyzed to determine whether dimers, trimers, and the like, or mixtures thereof, are present.
- Assays including by way of non- limiting example those described herein, are performed on the derivatives in order to determine their paracellular transporting and transcytotic properties, pharmacokinetics, stability and the like, in absolute terms as well as compared to the unaltered sFv5A molecule.
- sequences that may serve as suitable spacers in the compounds ofthe invention (for a review, see Simons, Spacers, probability, and yields, Bioconjug Chem 1999 Jan-Feb;10(l):3-8).
- sequences that have been used in sFv's include include EGKSSGSGSESKEF, one or more copies of GGGGS [a.k.a. (G 4 S) X ] (Newton et al., Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains, Biochemistry 1996 Jan 16;35(2):545-53), GSGS [a.k.a. (GSGS) X ] and GSSG [a.k.a. (GSSG) X ].
Abstract
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Also Published As
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IL155204A0 (en) | 2003-11-23 |
US20060099150A1 (en) | 2006-05-11 |
AU2001296494A1 (en) | 2002-04-15 |
JP2005500002A (en) | 2005-01-06 |
WO2002028408A9 (en) | 2003-07-10 |
EP1324778A2 (en) | 2003-07-09 |
CA2424730A1 (en) | 2002-04-11 |
WO2002028408A3 (en) | 2003-03-20 |
US20030161809A1 (en) | 2003-08-28 |
MXPA03002918A (en) | 2004-04-20 |
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