Title:  Chimeric proteins for use in transport of a selected substance into cells

United States Patent:  6,858,578

Issued:  February 22, 2005

Inventors:  Heartlein; Michael W. (Boxborough, MA); Lemontt; Jeffrey F. (West Newton, MA); Concino; Michael F. (Newton, MA)

Assignee:  Transkaryotic Therapies, Inc. (Cambridge, MA)

Appl. No.:  753385

Filed:  January 3, 2001

Abstract

Chimeric proteins useful in transporting a selected substance present in extracellular fluids, such as blood or lymph, into cells; quantitative assays for the selected substance using chimeric proteins; DNA encoding the chimeric proteins; plasmids which contain DNA encoding the chimeric proteins; mammalian cells, modified to contain DNA encoding the chimeric proteins, which express and, optionally, secrete the chimeric proteins; a method of producing the chimeric proteins; a method of isolating the chimeric proteins; a method of using the chimeric proteins to assay the selected substance; and a method of reducing extracellular levels of the selected substance through administration of the chimeric protein, which results in transport of the selected substance into cells.

Description of the Invention

BACKGROUND OF THE INVENTION

The transport of molecules across cell membranes is an important component of the physiologic mechanisms that mediate homeostasis at the levels of the cell and the organism as a whole. Molecules that are used directly or indirectly in the assembly of cellular components are transported from the extracellular fluid into the cell, usually by the action of specific cell-surface receptors which bind to the selected substance and mediate its uptake into specific cell types. Many hormones, enzymes, and drugs which influence cellular activity are also transported into cells by specific cell-surface receptors. Furthermore, toxic molecules which are either produced in the body (i.e. through normal or defective metabolic pathways) or introduced by ingestion or exposure can be taken up and sequestered or metabolized by certain cells.

Removal of substances, both endogenously-produced and foreign substances, from extracellular fluids, such as blood or lymph, is often physiologically appropriate. However, in many instances, removal is impaired or occurs to a lesser extent than desirable and disease occurs. An example concerns LDL cholesterol, a naturally-occurring substance which must be removed at a controlled rate if abnormally elevated levels and the accompanying adverse effects are to be avoided. Hypercholesterolemia in humans is a condition characterized by elevated levels of total serum cholesterol. It is usually caused by an excess of low density lipoprotein (LDL) cholesterol or a deficiency of high density lipoprotein (HDL) cholesterol and often leads to atherosclerosis and coronary artery disease. LDL is continually formed in the blood from apolipoproteins produced by the liver. In order to maintain a steady-state level, LDL is removed from the blood at a rate equal to its formation. If LDL removal is impaired, the blood level of LDL increases and atherosclerosis is a greater risk. Atherosclerosis is by far the leading cause of death in the United States, accounting for over one-half of all deaths. (Harrison's Principles of Internal Medicine, Ed. J. D. Wilson et al., 12th ed., p. 995, McGraw-Hill, New York, 1991).

LDL particles carry approximately 60-70% of total serum cholesterol. LDL is a large spherical particle with an oily core composed of approximately 1500 cholesterol molecules, each of which is linked to a long-chain fatty acid by an ester linkage. Surrounding the core is a layer of hospholipid and unesterified cholesterol molecules, arranged in such a manner that the hydrophilic heads of the phospholipids are on the outside, and thus making it possible for the LDL to be dissolved in blood or intercellular fluid. Each LDL particle contains one molecule of Apolipoprotein B-100 (ApoB-100), a large protein molecule which is embedded in the hydrophilic coat of LDL. ApoB-100 is recognized and bound by the LDL receptor, which is present on the surfaces of cells. LDL bound to a LDL receptor is carried into the cell, in which the two are separated. The LDL receptor is recycled to the cell surface and the LDL is delivered to a lysosome. In the lysosome, LDL is processed to liberate unesterified cholesterol. The liberated cholesterol is incorporated into newly synthesized cellular membranes in all cells and, in specialized cells, is used for other purposes (e.g., steroid hormone synthesis, bile acid production).

The steady-state level of serum LDL is determined to a large extent by the number of functional hepatic LDL receptors (LDLRS), which play a central role in the removal of circulating LDL. (Brown, M. S. and Goldstein, J. L., Science, 232:34-47 (1986)) Individuals with familial hypercholesterolemia (FH) may be either heterozygous or homozygous for mutations leading to defective LDLRs and, as a result, these individuals have excess serum LDL. Other individuals who have elevated serum LDL levels may carry leaky or previously uncharacterized LDLR mutations or might be producing too much LDL due to elevated intake of dietary fat. Both FH and non-FH patients have elevated cardiovascular risk and could benefit from a therapy based on increasing the catabolism of LDL as a result of increased cellular uptake.

Thus, there exists a need to develop methods for increasing the uptake of selected substances into cells. These substances may be destined for catabolism as discussed above, or they may be designed to influence intracellular processes and thus be considered regulatory agents. Thus, cellular activity may be altered by introducing new regulatory agents which can alter specific intracellular processes into recipient cells. For example, cellular patterns of protein phosphorylation, expression of specific cellular genes, and cell growth properties may be altered by introduction of an appropriate regulatory agent into a cell. These regulatory agents may be proteins which have enzymatic activity or they may be proteins that bind specific cellular targets, targets which may be comprised of nucleic acid, protein, carbohydrate, lipid, or glycolipid.

SUMMARY OF THE INVENTION

Cell surface receptors provide a route for introducing selected substances into cells. The natural ligand of the receptor may be a portion of a chimeric protein in which the ligand domain is functionally linked to a protein domain that exerts a desired effect within a cell and is therapeutic in vivo. Alternatively, the protein domain may be bound to a selected substance which is to be removed from extracellular fluids for catabolism or other metabolic processing.

The present invention relates to chimeric proteins useful in transporting a selected substance present in extracellular fluids, such as blood or lymph, into cells; quantitative assays for the selected substance using chimeric proteins; DNA encoding the chimeric proteins; plasmids which contain DNA encoding the chimeric proteins; mammalian cells, modified to contain DNA encoding the chimeric proteins, which express and, optionally, secrete the chimeric proteins; a method of producing the chimeric proteins; a method of isolating the chimeric proteins; a method of using the chimeric proteins to assay the selected substance; and a method of reducing extracellular levels of the selected substance through administration of the chimeric protein, which results in transport of the selected substance into cells. The present invention also relates to a method of gene therapy, in which mammalian cells expressing and secreting the chimeric protein are implanted into an individual, in whom the chimeric protein is expressed and secreted and binds the selected substance. The resulting selected substance-chimeric protein complex is taken up into somatic cells and, as a result, the extracellular levels of the selected substance are reduced.

The selected substance can be a normally-occurring (endogenously produced) constituent of the blood, such as a nutrient, metabolite, naturally-occurring hormone or lipoprotein, or a foreign constituent, such as a pathogen, toxin, environmental contaminant or drug or pharmacologic agent. In either case, the selected substance is removed from the extracellular fluid, such as blood or lymph, by means of a chimeric protein which selectively binds the selected substance and also binds a cell surface receptor present on one or more types of somatic cells, particularly human somatic cells. The resulting chimeric protein-selected substance complex binds to the cell surface receptor and is transported into the cell, where it is sequestered or metabolized, resulting in reduced extracellular levels of the selected substance.

Chimeric proteins of the present invention include at least two components: a functional domain and a carrier domain. The functional domain comprises an amino acid (polypeptide) sequence which binds the selected substance to be transported into cells or contains a sequence which will affect the target cell in a specific way. The carrier domain comprises an amino acid (polypeptide) sequence which binds a cell surface receptor present on one or more types of somatic cells. The amino acid sequence which is the functional domain can be a ligand binding domain of the selected substance; the amino acid sequence which is the carrier domain can bind to a cell-surface receptor and is thus a cell surface receptor ligand. Both the functional and carrier domains may be modified post-translationally, for example, by glycosylation at certain sites. In the case in which the selected substance is a normally-occurring constituent of the blood, lymph, or extracellular fluid, the ligand-binding domain which binds the selected substance is an amino acid sequence which normally binds the selected substance (i.e., binds the selected substance in humans), a modified form of such a sequence with altered binding properties, or an amino acid sequence which is not usually found in humans but has been produced by synthetic or genetic engineering methods and binds the selected substance. For example, the functional and/or carrier domains may be amino acid sequences selected from a combinatorial peptide library or phage display library. The functional and/or carrier domains may also comprise the antigen binding domain of an immunoglobulin or single-chain antibody, wherein the antigen binding domain of the immunoglobulin or single-chain antibody recognizes the desired selected substance or cell surface receptor. In the case in which the selected substance is a foreign constituent, the amino acid sequence which binds the selected substance is one selected from naturally-occurring ligand-binding domains which bind the foreign constituent or an amino acid sequence designed to bind the foreign constituent. The amino acid sequence which binds the cell surface receptor typically binds a cell surface receptor other than the receptor to which the selected substance normally binds. Thus, the method causes a selected substance to enter a cell by a route which is different from that which it normally takes in an organism.

The domains of the chimeric protein can be linked in a variety of configurations, as long as the resulting chimeric protein is able to bind both the selected substance and the cell surface receptor. Typically, the two domains are encoded by a single reading frame in a recombinant DNA molecule, and the two domains are linked by a peptide bond. The two domains may be separated by one or more amino acids also encoded by the open reading frame. Alternatively, the two domains may be expressed from separate DNA molecules and become linked in vitro or in vivo through either non-covalent (e.g., hydrophobic or ionic interaction) or covalent (e.g., disulfide) linkage.

Once the selected substance is bound to the ligand binding domain of the chimeric protein, the resulting complex is referred to as the selected substance-chimeric protein complex. The cell surface receptor ligand present in the selected substance-chimeric protein complex binds to its cell surface receptor and the complex is transported into the cell (e.g., by endocytosis), thus reducing circulating levels of the selected substance.

In one embodiment, the present invention relates to chimeric proteins useful in transporting LDL into cells; pharmaceutical compositions containing chimeric proteins; assays for LDL using the chimeric proteins; DNA encoding the chimeric proteins; plasmids which contain DNA encoding the chimeric proteins; mammalian cells, modified to contain DNA encoding the chimeric proteins, which express and, optionally, secrete the proteins (genetically modified cells); a method of producing the chimeric proteins; a method of isolating the chimeric proteins; a method of using the chimeric proteins to assay LDL and a method of reducing extracellular levels of LDL cholesterol (LDL) by administering a chimeric protein which transports LDL into cells.

In one embodiment of reducing extracellular LDL levels, a chimeric protein which binds LDL and a cell surface receptor other than LDL receptor (LDLR) is administered to a human patient in whom serum cholesterol level is to be reduced. In another embodiment of reducing extracellular LDL levels, cells modified to contain DNA which encodes the chimeric protein, are implanted into an individual. In the individual, the chimeric protein is expressed in and secreted by the genetically modified cells, binds LDL in the blood and forms LDL-chimeric protein complexes, which are transported into non-genetically modified cells, thus reducing serum LDL cholesterol levels.

Chimeric proteins of the present invention useful for LDL transport and for assaying LDL in a sample include at least two components: a functional domain, which comprises the amino acid sequence of the ligand-binding domain of the LDLR and a carrier domain, which comprises an amino acid sequence which binds a cell surface receptor other than the LDLR on one or more types of somatic cells, particularly human somatic cells. In one embodiment of the chimeric protein useful for increasing uptake of LDL into cells, an amino-terminal sequence comprising the ligand binding domain (i.e., the LDL binding domain) of the LDLR is joined to a C-terminal sequence comprising a cell surface receptor ligand. The carboxy-terminus of the LDL binding domain is joined to the amino-terminus of the cell surface receptor ligand domain.

In one embodiment of the chimeric protein, the two components are the ligand-binding domain of the human LDLR and human transferring the LDL binding domain of the LDLR is joined to the amino-terminus of the mature human transferrin polypeptide. This chimeric protein can bind both LDL and the transferrin receptor. Once bound to the transferrin receptor on the surface of a cell, such as a liver cell, the chimeric protein and the LDL bound to the chimeric protein is LDLR component are endocytosed by transferrin receptor-mediated endocytosis, with the result that LDL enters the cell and the extracellular LDL concentration is reduced. The transferrin receptor is present on a wide variety of mammalian cell types, allowing the chimeric protein to promote LDL uptake in a wide variety of mammalian cells.

In a second embodiment, the chimeric protein comprises a functional domain which is a ligand-binding domain of LDLR, and a carrier domain which is an amino acid sequence which binds a cell surface receptor other than the transferrin receptor, such as the serum albumin receptor, asialoglycoprotein receptor, an adenovirus receptor, a retrovirus receptor, CD4, lipoprotein (a), immunoglobulin Fc receptor, a-fetoprotein receptor, LDLR-like protein (LRP) receptor, acetylated LDL receptor, mannose receptor, or mannose-6-phosphate receptor. In general, an amino acid sequence that binds to any receptor which can bind and internalize bound ligand may be used. These chimeric proteins bind both LDL and a cell surface receptor and can be used to enhance the uptake of LDL into a wide variety of cells. Once bound to the receptor, the chimeric protein-LDL complex is endocytosed with the result that the LDL enters the cell and the extracellular LDL concentration is reduced.

A chimeric protein of the present invention is produced by an appropriate mammalian cell which contains DNA encoding the chimeric protein. Modified mammalian cells of the present invention (i.e., mammalian cells modified to contain nucleic acid encoding a chimeric protein of the present invention) include mammalian cells which are stably or transiently transfected or infected with a plasmid, nucleic acid fragment, or other vector, including a viral vector, comprising DNA or RNA encoding the chimeric protein or which are derived (directly or indirectly) from a progenitor modified mammalian cell which contains DNA or RNA (e.g., plasmid, nucleic acid fragment, or other vector comprising DNA or RNA) encoding the chimeric protein.

In one embodiment of the present method of producing chimeric proteins, the mammalian cell used is a cell line, such as a Chinese hamster ovary (CHO) cell line, which contains and expresses DNA which encodes the chimeric protein. Optionally, the chimeric protein is secreted into the medium in which the transfected CHO cells are cultured. In a second embodiment of the present method of producing chimeric proteins, the modified mammalian host cell is a primary or secondary cell, such as a primary or secondary human fibroblast, transfected or infected with DNA encoding the chimeric protein. The modified cell (e.g., a transfected or infected primary or secondary human fibroblast) expresses the encoded chimeric protein and, optionally, secretes it into the culture medium. Alternatively, the transfected or infected primary or secondary cells may be implanted into an individual, such as a human, in whom the chimeric protein is secreted for therapeutic purposes.

The chimeric protein produced by cultured modified cells may be isolated from cell lysates or the culture medium by any appropriate method. One such method, described herein, is based on affinity chromatography in which the chimeric protein is isolated by first binding to an antibody column prepared using an antibody directed against the cell surface receptor ligand, eluting, and subsequently by binding to a column bearing the selected substance to which the ligand binding domain of the chimeric protein binds. For example, the chimeric protein which binds LDL and the cell surface receptor for transferrin can be isolated by first being separated by binding to an anti-transferrin antibody column, and subsequently by binding to LDL bound to an anti-LDL antibody column. As a result of the isolation method, purified intact chimeric protein is obtained. Alternatively, in the first separation step, the chimeric protein is bound to a column bearing the selected substance to which the ligand binding domain of the chimeric protein binds and in the second separation step, is bound to a column bearing an antibody directed against the cell surface receptor ligand. As described herein, chimeric protein which includes domains from both LDLR and transferrin has been purified.

The chimeric protein of the present invention is useful to transport the selected substance, LDL, into cells, such as liver cells, thus reducing extracellular levels of the LDL. This has clinical or therapeutic applications, such as in controlling or lowering serum LDL levels in humans, such as hypercholesterolemic individuals. In one embodiment of the method of the present invention in which LDL is transported into cells through the use of the chimeric protein, cells expressing the chimeric protein are implanted in an individual in whom serum LDL levels are to be lowered. In the individual, the cells produce the chimeric protein, which enters the interstitial fluid. From the interstitium the chimeric protein can enter the lymphatics and ultimately, the bloodstream, where it binds LDL resulting in formation of a chimeric protein-LDL complex. The complex passes (e.g., via the bloodstream) to a cell which bears a transferrin receptor (e.g., a hepatocyte), and is bound to the cell as a result of the transferrin domain-transferrin receptor interaction. The chimeric protein-LDL complex is taken up by means of the transferrin receptor-mediated endocytosis pathway that normally functions to internalize transferrin. In another embodiment of the method, purified or partially purified chimeric protein is administered to an individual (particularly a human) in whom increased LDL transport into cells is desired.

The chimeric protein has a wide variety of other clinical or therapeutic applications, such as in reducing the circulating levels of normal or abnormal endogenously produced metabolites or nutrients (e.g. acetylated low density lipoprotein, apolipoprotein E4, tumor necrosis factor a, transforming growth factor .beta., a cytokine, an immunoglobulin, a hormone, glucose, a bile salt, a glycolipid [such as glucocerebroside which accumulates in patients with Gaucher disease or ceramidetrihexoside which accumulates in patients with Fabry disease], or a glycosaminoglycan [such as those that accumulate in patients with Hunter, Hurler, or Sly syndromes]) or of foreign substances (e.g., pathogens, environmental contaminants, or alcohol).

The chimeric protein of the present invention is also useful to assay, particularly to quantitatively assay, the selected substance to which the ligand binding domain binds. For example, it may be used in an assay to determine levels of LDL, immunoglobulins, growth hormone, and Apolipoprotein E in the blood. For example, the chimeric protein can be used as a component in known methods, such as an enzyme-linked assay, to assay a selected substance.

DETAILED DESCRIPTION OF THE INVENTION

The present invention exemplifies methods for introducing selected substances into cells for in vivo therapy. Further, the invention teaches the use of gene therapy to produce therapeutic proteins designed to transport selected substances into cells.

As described herein, Applicants have developed a new strategy for uptake of a selected substance into cells through the use of a chimeric protein and by means of a mechanism by which the selected substance is not normally taken up in cells. The chimeric protein binds the selected substance and also binds a cell surface receptor present on somatic cells, particularly on human somatic cells. Binding of the chimeric protein and the selected substance results in formation of a chimeric protein-selected substance complex, which is bound by the cell surface receptor and transported into the cell bearing the receptor. The selected substance can be a normally-occurring (endogenously produced) constituent of extracellular fluid, such as blood or lymph, or a foreign constituent such as a drug or a toxin. The chimeric protein of the present invention is itself delivered or administered to an individual or is provided to an individual by a gene therapy method in which cells which express and secrete the chimeric protein are introduced into an individual, in whom the chimeric protein is produced and secreted. The chimeric protein selectively binds the selected substance in the extracellular fluid (e.g., blood, lymph) of the individual, thus producing a selected substance-chimeric protein complex. The ligand-binding domain of the complex is bound by cell surface receptors on somatic cells in the individual. The complex is transported into the cell to which it is bound and the extracellular level of the selected substance is, thus, reduced.

Chimeric Proteins for Reducing Serum LDL Levels

In one embodiment, Applicants have developed a new strategy for uptake of human LDL which does not normally occur in humans, circumvents the defective LDLR or supplements the reduced level of LDLR known to occur in individuals with familial hypercholesterolemia, and augments or increases the ability of cells (those with a normal number of functional LDLRs and those with abnormal numbers of LDLRs) to take up LDL. This new method enhances LDL transport into liver cells, whether they have an abnormal number of functional LDLRs or a normal number of functional LDLRs. Applicants have determined that a receptor which is present on human cell surface membranes can be used as a means by which LDL can be transported into cells, such as liver cells. Cell surface receptors, such as the transferrin receptor, the serum albumin receptor, the asialoglycoprotein receptor, an adenovirus receptor, a retrovirus receptor, CD4, lipoprotein (a) receptor, immunoglobulin Fc receptor, a-fetoprotein receptor, LDLR-like protein (LRP) receptor, acetylated LDL receptor, mannose receptor, or mannose-6-phosphate receptor, can be used as a means by which LDL can be transported into cells. These receptors are present, respectively, on a wide variety of different cell types and allow uptake of LDL into a wide variety of cell types.

Applicants have produced chimeric proteins useful for transport of LDL into cells. These chimeric proteins are the subject of the present invention, as are nucleic acid sequences encoding the chimeric proteins; mammalian host cells containing DNA (or RNA) encoding the chimeric proteins, which is expressed in the cells; a method of producing the chimeric proteins; a method of isolating the chimeric proteins, a method in which the chimeric proteins are used to quantitatively assay for LDL, and a method of reducing extracellular LDL levels, including a therapeutic method of reducing serum LDL levels in an individual. In the therapeutic method, a chimeric protein which binds LDL and a human cell surface receptor other than the human LDLR is provided to an individual, either by administration of the chimerid protein itself or by administration (e.g., implantation) of cells which express and secrete the chimeric protein. In either case, the chimeric protein binds LDL and a human cell surface receptor and the complex is transported into the cell to which it is bound, reducing extracellular levels of LDL.

In one embodiment, the chimeric protein comprises a first domain, which is the ligand-binding domain of the LDLR and a second domain, which is transferrin (TF). The human transferrin receptor has a very high affinity for its ligand; the equilibrium dissociation constant (Kd) is 2-7 nM (Trowbridge, I. S. et al., Biochem. Pharmacol., 33:925-993 (1984)). Transferrin can be bound to its receptor even when the transferrin concentration is very low in comparison with total blood protein concentration. Similarly, the LDL receptor has a high affinity for its ligand (Kd=7.2 nM for liver receptors (Krampler, F. et al., J. Clin. Invest., 80:401-408 (1987) and 2.8 nM for fibroblast receptors (Innerarity, T. L. et al., Meth. Enzymol., 129:542-565 (1986)). Thus, the chimeric protein of this embodiment has a high affinity for the two components (human LDL and the human transferrin receptor) which must be brought together for LDL to be transported into cells by transferrin receptor-mediated endocytosis. A diagram of how an LDLR-transferrin (LDLR/TF) chimeric protein functions to promote LDL uptake via the transferrin receptor is shown in FIG. 1. LDL taken up by either pathway is metabolized to release cholesterol and free amino acids. In the LDLR-pathway, LDLR is recycled to the cell surface after LDL is released. In the TFR pathway, LDL is released and the TFR and LDLR/TF chimeric protein are recycled to the cell surface.

The entire human transferrin protein can be present in the chimeric protein; alternatively, only the portions of human transferrin necessary for binding to iron and the human transferrin receptor present on human cells are included in the chimeric protein. At neutral pH, each TFR binds two diferric (i.e., iron-saturated) TF molecules. The binding site is in the N-terminal domain of each TF. Monoferric TF binds TFR less readily, while apoTF (i.e., TF without bound iron) fails to bind TFR. In contrast, when iron is released in the acidic environment of the lysosome, ApoTF remains bound to TFR in order for recycling of the receptor and apoTF to the cell surface. As used herein, the term "human transferrin" refers to the entire human transferrin molecule or those segments of the protein necessary for binding to iron and to transferrin receptors on human cell surface membranes.

In one embodiment, the ligand-binding domain of the human LDL receptor is joined to human transferrin at the N-terminus of the mature TF molecule. The ligand-binding domain may include other LDL receptor regions which are present in the naturally-occurring receptor protein.

Production of Chimeric Proteins

The chimeric protein of the present invention, such as the chimeric protein for transporting LDL into cells, can be produced using host cells expressing a single nucleic acid encoding the entire chimeric protein or more than one nucleic acid sequence, each encoding a domain of the chimeric protein and, optionally, an amino acid or amino acids which will serve to link the domains. The chimeric proteins can also be produced by chemical synthesis.

A. Host Cells

Host cells used to produce chimeric proteins are bacterial, yeast, insect, non-mammalian vertebrate, or mammalian cells; the mammalian cells include, but are not limited to, hamster, monkey, chimpanzee, dog, cat, bovine, porcine, mouse, rat, rabbit, sheep and human cells. The host cells can be immortalized cells (a cell line) or non-immortalized (primary or secondary) cells and can be any of a wide variety of cell types, such as, but not limited to, fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), ovary cells (e.g., Chinese hamster ovary or CHO cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), muscle cells, hepatocytes and precursors of these somatic cell types.

Cells which contain and express DNA or RNA encoding the chimeric protein are referred to herein as genetically modified cells. Mammalian cells which contain and express DNA or RNA encoding the chimeric protein are referred to as genetically modified mammalian cells. Introduction of the DNA or RNA into cells is by a known transfection method, such as electroporation, microinjection, microprojectile bombardment, calcium phosphate precipitation, modified calcium phosphate precipitation, cationic lipid treatment, photoporation, fusion methodologies, receptor mediated transfer, or polybrene precipitation. Alternatively, the DNA or RNA can be introduced by infection with a viral vector. Methods of producing cells, including mammalian cells, which express DNA or RNA encoding a chimeric protein are described in co-pending patent applications U.S. Ser. No. 08/334,797, entitled "In Vivo Protein Production and Delivery System for Gene Therapy", by Richard F Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov. 4, 1994); U.S. Ser. No. 08/334,455, entitled "In Vivo Production and Delivery of Erythropoietin or Insulinotropin for Gene Therapy", by Richard F Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov. 4, 1994) and U.S. Ser. No. 08/231,439, entitled "Targeted Introduction of DNA Into Primary or Secondary Cells and Their Use for Gene Therapy", by Douglas A. Treco, Michael W. Heartlein and Richard F Selden (filed Apr. 20, 1994). The teachings of each of these applications are expressly incorporated herein by reference.

B. Nucleic Acid Constructs

A nucleic acid construct used to express the chimeric protein can be one which is expressed extrachromosomally (episomally) in the transfected mammalian cell or one which integrates, either randomly or at a pre-selected targeted site through homologous recombination, into the recipient cell's genome. A construct which is expressed extrachromosomally comprises, in addition to chimeric protein-encoding sequences, sequences sufficient for expression of the protein in the cells and, optionally, for replication of the construct. It typically includes a promoter, chimeric protein-encoding DNA and a polyadenylation site. The DNA encoding the chimeric protein is positioned in the construct in such a manner that its expression is under the control of the promoter. Optionally, the construct may contain additional components such as one or more of the following: a splice site, an enhancer sequence, a selectable marker gene under the control of an appropriate promoter, and an amplifiable marker gene under the control of an appropriate promoter.

In those embodiments in which the DNA construct integrates into the cell's genome, it need include only the chimeric protein-encoding nucleic acid sequences. Optionally, it can include a promoter and an enhancer sequence, a polyadenylation site or sites, a splice site or sites, nucleic acid sequences which encode a selectable marker or markers, nucleic acid sequences which encode an amplifiable marker and/or DNA homologous to genomic DNA in the recipient cell to target integration of the DNA to a selected site in the genome (targeting DNA or DNA sequences).

C. Cell Culture Methods

Mammalian cells containing the chimeric protein-encoding DNA or RNA are cultured under conditions appropriate for growth of the cells and expression of the DNA or RNA. Those cells which express the chimeric protein can be identified, using known methods and methods described herein, and the chimeric protein isolated and purified, using known methods and methods also described herein; either with or without amplification of chimeric protein production. Identification can be carried out, for example, through screening genetically modified mammalian cells displaying a phenotype indicative of the presence of DNA or RNA encoding the chimeric protein, such as PCR screening, screening by Southern blot analysis, or screening for the expression of the chimeric protein. Selection of cells having incorporated chimeric protein-encoding DNA may be accomplished by including a selectable marker in the DNA construct and culturing transfected or infected cells containing a selectable marker gene under conditions appropriate for survival of only those cells which express the selectable marker gene. Further amplification of the introduced DNA construct can be effected by culturing genetically modified mammalian cells under conditions appropriate for amplification (e.g., culturing genetically modified mammalian cells containing an amplifiable marker gene in the presence of a concentration of a drug at which only cells containing multiple copies of the amplifiable marker gene can survive).

Genetically modified mammalian cells expressing the chimeric protein can be identified, as described herein, by detection of the expression product. For example, mammalian cells expressing chimeric protein in which the second domain is transferrin can be identified by a sandwich enzyme immunoassay in which the chimeric protein is captured on a microtiter plate by binding to a monoclonal antibody specific for the LDL binding domain, and the bound chimeric protein is detected by binding to an anti-human TF monoclonal antibody specific for the human TF domain. Routine assay for the level of chimeric protein in most genetically modified mammalian cell types that otherwise do not synthesize human TF can be performed by an ELISA which detects human TF, as each chimeric protein expressed contains an assayable TF domain. Alternatively, mammalian cells expressing the chimeric protein can be identified by an LDL binding assay. Further, they can be identified using other methods known to one of ordinary skill in the art. (Sambrook, J. et al., Molecular Cloning; a Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F. A. et al., eds., Current Protocols in Molecular Biology, Wiley, N.Y. 1994.

D. Purification of Chimeric Proteins

The chimeric protein produced by genetically modified mammalian cells can be isolated from the cells and purified or partially purified using methods known to those of skill in the art as well as by methods described herein. As described in "the example of this patent", chimeric protein has been obtained, from cells in which it was expressed, through a method based on direct binding to both domains of the chimeric protein. The method includes two steps: one involving immunoaffinity binding to the transferrin epitope involved in transferrin receptor binding and another involving ligand (LDL) affinity binding to the LDLR domain. To purify LDLR ligand-binding domain transferrin chimeric protein, the method was carried out as follows: Medium in which cells expressing the chimeric protein were cultured is separated from the cells and, generally, concentrated by tangential flow ultrafiltration. The resulting culture medium, which is enriched or concentrated for the chimeric protein, is contacted with an anti-human transferrin monoclonal antibody (anti-TF MAb) bound to a column matrix, which results in binding of all proteins in the medium which contain a transferrin epitope. The bound protein is eluted from the immunoaffinity column and the resulting immunoaffinity purified chimeric protein is subsequently subjected to a ligand-affinity purification step, in which it is contacted with LDL indirectly bound to a column matrix through an anti-LDL antibody fixed to the column matrix, under conditions appropriate for LDL-LDLR binding. As a result, chimeric protein containing transferrin and an intact LDLR ligand binding domain is bound to LDL; the chimeric protein is eluted from the column to yield a highly purified preparation of the chimeric protein. As demonstrated in "the example of this patent", this process resulted in separation of intact chimeric protein which contains transferrin and binds LDL. The method can be modified to purify chimeric proteins in which the first domain is a ligand binding domain other than the LDLR ligand binding domain and the second domain is a cell surface receptor ligand other than transferrin.

In one embodiment of the present method of purifying chimeric protein comprising intact LDLR binding domain and transferrin, the purification is carried out as follows: Culture medium from host cells expressing the chimeric protein is separated from the host cells and concentrated by tangential-flow ultrafiltration using a membrane with a molecular weight cut off of 100,000 daltons. In one experiment, the culture medium is concentrated approximately 32-fold.

An anti-TF MAb is (e.g., HTF-14, Biodesign, Kennebunk, Me., isolated from ascites fluid) bound to a solid support, such as polystyrene beads (e.g., cyanogen-bromide [CNBr]--activated Sepharose 4B), to form an immunoaffinity column. The concentrated culture medium is contacted with the solid-support-bound anti-TF MAb, by loading the medium onto an HTF-14 immunoaffinity column, and maintained in contact with the anti-TF MAb under appropriate conditions and for sufficient time for the transferrin domain in the chimeric protein in the medium to bind to the anti-TF MAb (i.e., for the chimeric protein to bind to MTF-14 through an interaction with the chimeric protein's transferrin domain). As a result, chimeric protein containing transferrin is non-covalently bound to the solid support (e.g., to beads to which HTF-14 is bound). The solid support-bound chimeric protein is subjected to appropriate conditions (e.g., washing with 0.1 M glycine, pH 2.3) to elute the chimeric protein, which is collected.

In the embodiment described in "the example of this patent", the HTF-14 immunoaffinity column containing bound chimeric protein was washed with 0.1M Tris-HCl, 0.15M NaCl, pH 7.4 (TBS), and then eluted with 0.1 M glycine pH 2.3. Two milliliter fractions were collected into buffer of appropriate pH to neutralize the elution buffer and certain of the fractions were pooled. Analysis showed that this step resulted in an approximately 8,000-fold purification of chimeric protein from the concentrated culture media. The resulting immunoaffinity purified chimeric protein was a mixture which contained, as described in "the example of this patent", chimeric protein which includes transferrin and intact LDLR binding domains and chimeric protein which had been degraded by a serine protease and, thus, did not include intact LDLR binding domain. The second step in the purification process is a ligand-affinity (LDL-affinity) based step which results in separation of chimeric protein which comprises transferrin and intact LDLR binding domain from the chimeric protein which does not include intact LDLR binding domain. In this step, the immunoaffinity purified chimeric protein mixture was loaded onto a ligand affinity column containing CNBr activated Sepharose beads to which human LDL (hLDL) was bound (e.g., by means of polyclonal rabbit anti-human LDL antibody immobilized on the beads). The immunoaffinity purified chimeric protein was loaded onto the anti-LDL (LDL) ligand affinity column for sufficient time and under appropriate conditions for LDL on the beads and LDLR in the chimeric protein to bind, producing chimeric protein non-covalently bound to the column. The column was washed with TBS and eluted with 20 mM EDTA in TBS, pH 7.4. Fractions were collected and assayed, and fractions 2 and 3 were shown to contain the peak concentrations of the protein.

E. In Vitro Characterization of Chimeric Proteins

Analysis of chimeric protein produced as described herein showed that it binds LDL in a divalent cation-dependent manner, which is a well-established property of the LDL-LDLR binding interaction. Further analysis showed that binding of the chimeric protein to LDL did not occur in acidic buffer conditions and that EDTA and acidic buffer were each effective in dissociating chimeric protein bound to LDL. It is also a well-established property of the LDL-LDLR binding interaction that LDL is released from LDLR in vivo in the acidic endosomal compartment. This suggests that the chimeric protein may be able to act in a similar way in cells, which would result in release of the LDL from the chimeric protein in the cell. Taken together, these findings support the function of the chimeric protein with respect to binding to serum LDL and uptake into cells for further metabolism.

Additional analysis demonstrated that half-maximal binding occurs at an LDL concentration of approximately 3 nM, which is comparable to that of purified LDLR in a solid-phase binding assay (Innerarity, T. L. et al., Meth. Enzymol., 129:542-565 (1986)) as well as to the published K_d value for the dissociation of LDL from LDLR in human fibroblast cells. This supports the idea that the chimeric protein has a binding affinity for LDL which is comparable to the binding affinity of full-length, plasma membrane-bound LDLR for LDL.

The functional activity of chimeric protein in which the first domain is the ligand-binding domain of LDLR and the second domain is transferrin is assessed as described in "the examples in this patent". To determine whether chimeric protein can result in cellular uptake of LDL via the transferrin receptor, a hepatic cell line, such as HepG2, is used. As described in "the example of this patent", hepatic cells and LDL in vitro are exposed to chimeric protein and LDL. To determine whether chimeric protein can bind human LDL in culture medium and mediate uptake into hepatic cells via the transferrin receptor, the ability of unlabeled LDL or unlabeled transferrin to inhibit cellular uptake of labeled LDL (e.g., ¹²⁵ I-LDL) in the presence of chimeric protein is assessed, as described in "the example of this patent". Whether LDL taken up by the transferrin receptor is metabolized to a cholesterol pool can be assessed as described in "the example of this patent". For example, inhibition of cholesterol biosynthesis as a result of LDL uptake can be assessed by determining production of an enzyme, such as 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), which is down-regulated (inhibited) by an oversupply of cholesterol in a cell. Alternatively, determination of levels or activity of an enzyme, such as acyl-coenzyme A cholesterol acyltransferase (ACAT), which increases in response to increased cholesterol in cells, can be used to assess whether LDL is taken up by the transferrin receptor and metabolized to cholesterol. Increased ACAT activity is indicative of increased cholesterol levels in the cell.

F. In Vivo Characterization of Chimeric Proteins

The anti-hypercholesterolemic effect of the chimeric protein can be assessed, as described in "the example of this patent". Briefly, chimeric protein is administered in an animal model system for human familial hypercholesterolemia, such as the LDLR-knockout mouse or the Watanabe rabbit, and its effect on serum cholesterol levels is determined. A decrease in serum cholesterol levels after administration of an appropriate amount of chimeric protein to an LDLR-knockout mouse or a Watanabe rabbit is indicative of the ability of the chimeric protein to transport cholesterol into cells.

G. Therapeutic Use of Chimeric Proteins

Chimeric proteins of the present invention are useful to enhance LDL transport into cells, such as hepatic cells. Chimeric proteins, such as those with the LDLR-ligand-binding domain fused to a transferrin domain, can be administered to an individual in whom LDL metabolism is to be enhanced. Chimeric proteins are administered in an appropriate carrier, which can be physiologic saline or water or mixed with stabilizers or excipients, such as albumin or low molecular weight sugars. They are administered using known techniques and by a variety of routes, such as by intramuscular, intravenous, intraperitoneal injection. Alternatively, genetically modified mammalian cells expressing chimeric protein can be implanted in an individual. Non-immortalized cells (primary or secondary cells) and/or immortalized cells can be transfected. These include, but are not limited to fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), ovary cells (e.g., Chinese hamster ovary or CHO cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), muscle cells, hepatocytes and precursors of these somatic cell types. Immortalized cells can also be transfected by the present method and used for either protein production or gene therapy. Examples of immortalized human cell lines useful for protein production or gene therapy by the present method include, but are not limited to, HT1080, HeLa, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, 2780AD ovarian carcinoma cells, Raji (ATCC CCL 86) cells, Jurkat (ATCC TIB 152) cells, Namalwa (ATCC CRL 1432) cells, HL-60 (ATCC CCL 240) cells, Daudi (ATCC CCL 213) cells, RPMI 8226 (ATCC CCL 155) cells and MOLT-4 (ATCC CRL 1582) cells. In cases where genetically modified immortalized cells are used for gene therapy, the cells may be enclosed within a semi-permeable barrier device which allows for the diffusion of the chimeric protein out of the device.

Preferably, cells (e.g., fibroblasts) to be used in the gene therapy method of the present invention are obtained from the individual to be treated. The cells are modified by introduction of a DNA construct of the present invention, such as a DNA construct encoding a chimeric protein in which domain 1 is the ligand-binding domain of human LDLR and domain 2 is comprised of an amino acid sequence derived from transferrin. The resulting genetically modified cells are expanded in culture and introduced into the individual. Cells expressing a DNA construct encoding such a chimeric protein can be produced as described in co-pending U.S. patent application Ser. No. 07/787,840, entitled "In Vivo Protein Production and Delivery System for Gene Therapy", by Richard F Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov. 5, 1991), the teachings of which are incorporated herein by reference. The cells express and secrete the chimeric protein, which binds LDL and transports it to cells bearing transferrin receptors on their surfaces. Transferrin receptor-mediated endocytosis of the chimeric protein-LDL complex would result in introduction of LDL into cells and lowering of extracellular LDL levels. DNA encoding a chimeric protein in which the second domain is a cell surface receptor ligand other than transferrin, such as those listed above, can be introduced into mammalian cells. Genetically modified mammalian host cells which express and secrete the chimeric protein can be implanted in an individual, in whom the chimeric protein binds LDL. The resulting chimeric protein-LDL complex is bound to cells bearing the receptor for which the amino acid sequence of the second domain is a ligand and, through receptor-mediated endocytosis, enters the cells. Thus, circulating LDL levels would be reduced.

The number of genetically modified mammalian host cells implanted in an individual is determined by the amount of chimeric protein to be delivered and the level of its expression by the cells. The amount needed by an individual will depend on considerations such as age, body size, sex, serum LDL level, and serum half-life of the chimeric protein. The required dose can be determined empirically or calculated based on the pharmacodynamic properties established for the chimeric protein.

Other, related, chimeric proteins can be used to clear oxidized LDL, which represents a clinically significant fraction of the total LDL, from the blood. The early stages of atherosclerotic plaque formation are believed to occur when high levels of circulating LDL interact with endothelial cells in blood vessels, where oxidation of amine groups (e.g. on lysine and arginine residues ) on apoB-100 takes place by free radical attack. Oxidation (including acetylation) of apoB-100 in LDL particles is known to result in loss of affinity for the LDL receptor. Oxidized LDL is not cleared by the liver, but instead accumulates in the circulation until it can interact with the scavenger receptor (AcLDLR; previously known as the Acetyl LDL Receptor), which is predominantly found on macrophages. Macrophages taking up large quantities of oxidized LDL become bloated with lipid vacuoles and are known as "foam cells". Foam cells with cell-surface determinants for adhesion to endothelium are believed to play a role in the early development of atherosclerotic plaques and indeed are found in plaques at various stages.

A chimeric protein containing a scavenger receptor ligand-binding domain fused to human transferrin (AcLDLR/TF) is therapeutically useful by binding to and removing oxidized LDL via transferrin receptors in the liver. In addition, since the chimeric protein can be supplied at high concentration, it can function as a competitor of the macrophage AcLDLR, such that most oxidized LDL will be bound to AcLDLR/TF, rather than bound to AcLDLR on macrophages. In this way, oxidized LDL accumulation in macrophages can be reduced and thus the numbers of foam cells found in cardiovascular endothelium will also be reduced, thereby interfering with early-stage atherosclerotic plaque formation.

More generally, other chimeric proteins are useful therapeutically to reduce levels of other biochemical substances associated with certain disease states. The high-affinity ligand-binding domain of chimeric proteins need not be restricted to those of known receptor molecules (e.g. LDLR or AcLDLR), but may also include other types of proteins with high binding affinity for protein ligands or small molecules. Molecules with the antigen binding properties of antibodies (for example, single chain antibodies) or other proteins having reversible binding activities can be used to construct chimeric protein-encoding sequences with human transferrin or with ligands which bind to other cell receptor ligands.

In one embodiment the chimeric protein has high-affinity binding specificity for apolipoprotein E4 (apoE4), but not to apoE3 or apoE2. Approximately half of all cases of Alzheimer Disease are associated with specific allelic forms of apoE, and the E4 allelic form of apoE is found to accumulate in amyloid fibrils in the brain and is a risk factor for the disease. The apoE2 and apoE3 alleles are not associated with increased risk and might play a role in disease resistance. A chimeric protein able to reduce apoE4 levels may be used therapeutically to slow the development of Alzheimer Disease. By the methods described herein, chimeric proteins containing an apoE4 binding domain could be fused with transferrin to produce a molecule which could remove apoE4 from the circulation and ultimately reverse the accumulation that occurs in peripheral tissues.

Diagnostic Use of Chimeric Proteins

Chimeric proteins are also useful diagnostic reagents in biochemical assays. For example, chimeric proteins can be used to determine the quantity of a selected substance, such as LDL, in a biological sample (e.g., cell lysates, blood, lymph, urine, water or milk). The biological sample to be analyzed is processed, if needed, to render the selected substance available for binding to the first domain of an appropriate chimeric protein (e.g., for LDL, one in which the first domain is the ligand-binding domain of human LDLR) and contacted with the chimeric protein under conditions appropriate for binding of the first domain and the selected substance. The chimeric protein may be generally bound to a solid surface, such as a microtiter plate, polymeric beads or other surface in such a manner that it remains bound to the surface under conditions used for binding of the selected substance to the first domain of the chimeric protein. If the selected substance is present in the biological sample, it is bound to the chimeric protein and the resulting selected substance-chimeric protein complex is detected using known means (e.g., using an antibody which binds the selected substance and is covalently linked (conjugated) to an active enzyme or radioactive nuclide. The activity of the enzyme is monitored, for example, by measuring cleavage of a chromogenic or fluorogenic substance.) Chimeric proteins can detect a substance in a direct assay, with a high degree of specificity in a convenient format, such as in a microtiter plate format. In one example presented herein, LDL is quantified by binding to LDLR/TF chimeric protein bound to a microtiter plate. The resulting bound LDL is detected by reaction with an anti-LDL antibody.

In other embodiments, chimeric proteins can substitute for antibodies in ELISA assays. For example, LDL either directly or indirectly bound to a plate can capture a chimeric protein with an LDLR ligand-binding domain. This captured chimeric protein is then detected by reaction with an HRP-conjugated antibody directed against the second domain of the chimeric protein, for example, an anti-TF antibody can react with the TF domain when an LDLR/TF chimeric protein is used. As another example, chimeric proteins which bind to components of the human immunodeficiency virus (HIV) may be used to detect the presence of the virus in complex biological samples, such as blood or tissue specimens.

Claim 1 of 42 Claims

What is claimed is:

1. A method of lowering the amount of an endogenously produced substance in an extracellular fluid of a subject, comprising administering to the subject a chimeric protein comprising a functional domain and a carrier domain, wherein

the functional domain comprises a ligand-binding domain of a first receptor, wherein the ligand-binding domain binds an endogenously produced substance;

the carrier domain comprises an amino acid sequence which binds a mammalian cell surface receptor other than the first receptor, wherein (a) the amino acid sequence is from a protein other than the first receptor, and (b) the cell surface receptor is selected from the group consisting of low density lipoprotein receptor (LDLR), transferrin receptors, asialoglycoprotein receptors, adenovirus receptors, retrovirus receptors, lipoprotein (a) receptors, LDLR-like protein (LRP) receptors, acetylated LDLR, mannose receptors and mannose-6-phosphate receptors,

such that the chimeric protein binds to the endogenously produced substance in the extracellular fluid of the subject and to the cell surface receptor on the cell, whereupon the cell surface receptor on the cell transports the chimeric protein and the endogenously produced substance into the cell, thereby lowering the amount of the endogenously produced substance in the extracellular fluid of a subject.

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