Classifications

• • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/525—Tumour necrosis factor [TNF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • 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
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
  • A61P33/06—Antimalarials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • 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
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to glycosylated human tumor necrosis factor-alpha (TNF- ⁇ ), pharmaceutical composition containing it, and methods for its preparation and use.
• TNF- ⁇ glycosylated human tumor necrosis factor-alpha
• Cytokines are polypeptide mediators secreted by cells that affect the function of other cells and which play an important role in the interactions between cells in the immune system. Together with other substances, cytokines constitute the molecular language of inflammation and immunity and form a complex of interacting and overlapping networks of signals that orchestrate the body's defensive reactions.
• One such cytokine is tumor necrosis factor (TNF).
• TNF is one of a family of proteins that orchestrate the body's remarkably complex response to injury and infection.
• TNF is implicated in a wide variety of human diseases, may be effective against certain cancers, plays a role in septic shock and cachexia, is a mediator of inflammation in various immunological reactions, and also seems to be important in some pathological effects seen in malaria and menmgococcal septicemia.
• tumor necrosis factor As with many other factors produced by the body, there seems to be a fine line between benefit and harm: an agent that is helpful in the local control of injury and infection may be toxic when it is released in large amounts or in the wrong place. See Old, "Tumor Necrosis Factor", Scientific American. 258:59-60, 69-75 (May 1988). TNF exerts its effect by binding to specific receptors on cell membranes and causing clustering of the receptors.
• TNF receptors Two TNF receptors, with molecular weight of 55,000 Daltons and 75,000 Daltons, are known. After TNF binds to its receptor, the complex is internalized and degraded. Receptor clustering activates protein kinases and phospholipases, leading to production of signal mediators and secondary messengers, resulting in cell death.
• the TNF "family” includes two structurally and functionally related proteins, TNF-alpha and TNF-beta. The close relationship of these proteins was not known until 1984 when coding of the cDNAs for human TNF-alpha and TNF-beta, unexpectedly revealed that they are about 30% homologous at the amino acid level. Both TNF-alpha and TNF-beta bind to the same cell surface receptors, and they are very similar (though not identical) in the spectra of their activity. However, the regulation of expression of the TNF-alpha and the TNF-beta genes and the processing of the two corresponding proteins by the producing cells are completely different.
• TNF- ⁇ has previously been referred to as cachectin and is produced primarily by mononuclear phagocytes, such as macrophages and monocytes. It is also produced by astrocytes, endothelial cells and some transformed cells. Mature human TNF-alpha is a polypeptide of 157 amino acid residues. The apparent molecular mass of human TNF-alpha is 17,000 Daltons. In its native form, TNF-alpha is a trimer.
• TNF- ⁇ is known are melanoma, EL-4 lymphoma, Meth-A tumors, melanocytes, leukemia, carcinomas of the liver or lung, metastases or mammary carcinomas, cancers including breast, anal, colorectal, head, neck and other body organs.
• TNF has been found to have potential anti-microbial properties and is extremely potent against malarial and other parasites.
• TNF has been found to protect cells against some viral infections and prevent autoimmune diseases such as Type 1 diabetes.
• TNF inhibits lipid accumulation and alterations in adipose specific RNAs. TNF prevents the expression of genes responsible for producing enzymes important in storing fat.
• TNF exhibits a synergistic cytotoxic activity with interferon.
• TNF-beta also known as lymphotoxin and produced primarily by lymphocytes, contains 171 residues and has an apparent molecular mass of 25,000 Daltons.
• the difference in the size of the two molecules has been attributed to the absence of N-glycosylation in human TNF-alpha, as opposed to the presence of one N-glycosylation site in TNF-beta.
• murine TNF-alpha is a glycoprotein
• human TNF-alpha is glycosylated. Indeed it is described in the literature as being non-glycosylated (Markham et al, Eur. Cytokine Netw., 6: 49-54 (1995); Fiers, FEBS letters, 285: 199-212 (1991)).
• Human TNF-alpha can be isolated from human cells or prepared by recombinant techniques. While, theoretically, human TNF-alpha can be prepared by recombinant methods using any type of cells, conventionally TNF-alpha is prepared using transfected E. coli, cf. Wallace et al., U.S. Patent No. 4,879,226 and Aggarwal et al., EP 0168214. In fact, Aggarwal et al. specifically state that cultures of cells derived from multicellular organisms may be used as hosts, but that this is not preferred because of the excellent results obtained with TNF expressing microbes.
• TNF was ever separated from the medium in any way. Indeed, there is no disclosure that it was even subjected to SDS-PAGE chromatography. Marmenout states that the available data suggests that human TNF is not glycosylated. Because of the substantially greater expression achieved in E. coli than from the COS cells, the remaining work reported in the Marmenout publication was done with the E. coli expression product, which is necessarily non-glycosylated.
• Malik et al., in European Journal of Cancer. 26(1): 1031-1034 (1990) studied the action of TNF as a tumor-promoting agent by causing Chinese hamster ovary (CHO) cells to act as a tumor to demonstrate that TNF can stimulate tumor progression. In this case, the CHO cells transfected with the gene for human TNF were implanted directly into mice and the TNF produced by these cells was never isolated.
• TNF-alpha has now been produced in Chinese hamster ovary (CHO) cells and purified to homogeneity. This purified TNF-alpha has surprisingly been found to be glycosylated.
• the pure recombinant human TNF-alpha produced in CHO cells was partially characterized by a variety of methods including capillary electrophoresis, carbohydrate composition, carbohydrate mapping, isoelectric focusing, SDS-PAGE, and RP-HPLC. These analyses indicated that recombinant human TNF-alpha produced in CHO cells was different from recombinant human TNF-alpha produced in E. coli.
• human TNF-alpha is described in the literature as a non-glycosylated protein
• the recombinant human TNF-alpha obtained from CHO cells, according to the present invention was found to contain multiple charged species.
• an enzyme that cleaves sialic acid an enzyme that cleaves sialic acid
• CHO recombinant human TNF-alpha multiple charged species collapsed to one major isoform/charged form as determined by isoelectric focusing and capillary electrophoresis, indicating that the variability of isoforms/charged forms stems from sialic acid and, hence, the molecule is glycosylated.
• Carbohydrate composition and mapping demonstrated the existence of O-glycosylation.
• TNF-alpha co-migrated with E. coli recombinant human TNF-alpha on SDS-PAGE, isoelectric focusing gels, and co-eluted on the capillary electrophoresis and reverse phase-high performance liquid chromatography (RP-HPLC), indicating that both proteins are similar in their polypeptide core but differ in the presence of O-linked oligosaccharide in the CHO recombinant human TNF-alpha.
• RP-HPLC reverse phase-high performance liquid chromatography
• glycosylated human recombinant TNF prepared according to the present invention is particularly useful because, compared to non-glycosylated human TNF, the glycosylated TNF may have an increased half-life in body fluids, may improve binding to receptors, and may be better protected against the influence of proteases.
• Figures 1A and IB are oligomaps of CHO recombinant human
• FIG. 1A shows the "O" mode, in which O-linked glycoside linkages are preferentially released
• Figure IB shows the "N + 0" mode, in which both N-linked and O-linked glycosidic linkages are released.
• the oligomaps indicate only the presence of one main O-linked oligoform.
• Figures 2A-2E show capillary electrophoresis analysis of CHO recombinant human TNF-alpha.
• Figure 2A shows CHO recombinant h-TNF
• Figure 2B shows CHO recombinant h-TNF plus neuraminidase
• Figure 2C shows CHO recombinant h-TNF plus neuraminidase plus O-glycanase
• Figure 2D shows CHO recombinant h-TNF plus neuraminidase plus O-glycanase + N-glycanase.
• Figure 2E shows E. coli recombinant human TNF.
• tumor necrosis factor-alpha will be referred to as tumor necrosis factor or TNF and tumor necrosis factor-beta will be referred to as lymphotoxin.
• DNA which encodes tumor necrosis factor is ligated into a replicable (reproducible) vector, the vector used to transform host CHO cells, the CHO host cells are cultured, and TNF is recovered from the culture medium. This general process is used to construct glycosylated human TNF.
• the rumor necrosis factor species which are capable of synthesis herein include mature (valyl amino-terminal) tumor necrosis factor, pre-tumor necrosis factor ("pre-TNF", defined herein), and derivatives of TNF including fusion proteins wherein TNF (including mature tumor necrosis factor) is linked to other proteins or polypeptides by a peptide bond at the amino and/or carboxyl terminal amino acids to TNF.
• pre-TNF pre-tumor necrosis factor
• Human tumor necrosis factor synthesized in CHO recombinant culture is characterized by the presence of non-human cell components, including proteins, in amounts and of a character which are physiologically acceptable for administration to patients in concert with the rumor necrosis factor. These components are generally present in innocuous contaminant quantities, on the order of less than 1 percent by weight. Further, CHO recombinant cell culture enables the production of tumor necrosis factor absolutely free of homologous proteins.
• Homologous proteins are those which are normally associated with the tumor necrosis factor as it is found in nature, e.g., in cells, cell exudates, or body fluids.
• a homologous protein for human tumor necrosis factor is human serum albumin.
• Heterologous proteins are the converse, i.e., they are not naturally associated or found in combination with the tumor necrosis factor in question.
• “Substantially homogeneous" glycosylated TNF means glycosylated
• TNF which is substantially free of other proteins native to the source from which the glycosylated TNF was isolated.
• homogeneous glycosylated TNF is substantially free of blood plasma proteins such as albumin, fibrinogen, serine proteases, alpha- globulins, non-TNF cytotoxic polypeptides such as lymphotoxin or interferons, or other proteins of the cell or organism which serve as the synthetic origin of the glycosylated TNF, including whole cells and particulate cell debris.
• a mammalian cell is transformed with (a) a vector containing the entire tumor necrosis factor structural gene (including a 5' start codon), or (b) the gene for mature tumor, necrosis factor or a TNF derivative operably ligated to a secretory leader, which may also include the TNF secretory leader presequence, and the cell cultured, then mature TNF is recovered from the culture medium.
• a vector containing the entire tumor necrosis factor structural gene including a 5' start codon
• the gene for mature tumor, necrosis factor or a TNF derivative operably ligated to a secretory leader which may also include the TNF secretory leader presequence
• DNA which encodes TNF is operably ligated in a vector to a secretory leader which is properly processed by the host cell to be transformed, the host transformed with the vector and cultured then the tumor necrosis factor is synthesized without amino-terminal methionyl or blocked methionyl.
• Secretory leaders and host cells may be selected that also result in improper transport of mature protein into cell periplasm.
• tumor necrosis factor is prepared by cell bioprocess, it generally is purified by recovering the supernatant culture fluid, removing solids, absorbing TNF from the supernatant admixture (containing TNF and other proteins) onto a hydrophobic substance, eluting TNF from the substance, adsorbing TNF onto a tertiary amino anion exchange resin, eluting TNF from the resin, adsorbing TNF onto an anion exchange resin (preferably quaternary amino-substituted) having substantially uniform particle size, and eluting TNF from the resin.
• an anion exchange resin preferably quaternary amino-substituted
• the TNF composition can be optionally concentrated and purified by chromatofocusing at any point in the purification procedure, for example by isoelectric focusing or passage through a sieving gel such as SEPHADEX G-25.
• the tumor necrosis factor obtained according to the present invention from transfected CHO cells is always obtained as a glycoprotein.
• Purified glycosylated TNF from recombinant cell culture is combined for therapeutic use with pharmaceutically acceptable carriers and excipients, such as physiologically innocuous stabilizers and excipients, and prepared in dosage form as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
• pharmaceutically acceptable carriers and excipients such as physiologically innocuous stabilizers and excipients
• TNF may be incorporated into a polymer matrix for implantation into tumors or surgical sites from which tumors have been excised, thereby effecting a time-release of the tumor necrosis factor in a localized high gradient concentration.
• glycosylated TNF may be advantageously combined with predetermined amounts of other proteins such as lymphotoxin and/or interferon.
• Glycosylated tumor necrosis factor also includes multimeric forms. TNF spontaneously aggregates into multimers, usually dimers or higher multimers. Multimers are cytotoxic and accordingly are suitable for use in vivo therapy. While it is desirable to express and recover glycosylated TNF as a substantially homogeneous multimer or monomer, glycosylated TNF may be used therapeutically as a mixture of different multimers.
• Derivatives of glycosylated TNF are included within the scope of the present invention.
• Derivatives include covalent or aggregative conjugates with other chemical moieties.
• Covalent derivatives are prepared by linkage of functionalities to groups which are found in the glycosylated TNF amino acid side chains or at the N- or C- terminal, by means known in the art. These derivatives may, for example, include aliphatic esters or amides of the carboxy terminus or residues containing carboxyl side chains; O-acyl derivatives of hydroxyl group-containing residues; and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, such as lysine or arginine.
• the acyl group is selected from the group of alkyl moieties, including C 3 to C !0 normal alkyl, thereby forming alkanoyl species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species.
• the reactive groups preferably are difunctional compounds known for use in cross-linking proteins to insoluble matrices through reactive side groups. Preferred derivatization sites are at cysteine and histidine residues.
• Covalent or aggregative derivatives are useful as reagents in immunoassay or for affinity purification procedures.
• glycosylated TNF is insolubilized by covalent bonding to cyanogen bromide-activated Sepharose by conventional methods or adsorbed to polyolefin surfaces, with or without glutaraldehyde cross-linking for use in the assay or purification of anti-TNF antibodies, cell surface receptors, or soluble extracellular portions thereof.
• Glycosylated TNF may also be labeled with a detectable group, e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in diagnostic assays, especially for diagnosis of glycosylated TNF levels in biological samples by competitive-type immunoassays.
• a detectable group e.g., radioiodinated by the chloramine T procedure
• covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in diagnostic assays, especially for diagnosis of glycosylated TNF levels in biological samples by competitive-type immunoassays.
• Such derivatives need not exhibit cytotoxic activity, only cross-reactivity with anti-TNF.
• DNA which encodes glycosylated TNF can be obtained by chemical synthesis, by screening reverse transcripts of mRNA from peripheral blood lymphocytes or cell line cultures, or by screening genomic libraries from any cell.
• Glycosylated TNF is synthesized in host CHO (or other eukaryotic) cells transformed with vectors containing DNA encoding TNF.
• a vector is a replicable DNA construct. Vectors are used either to amplify DNA encoding TNF and/or to express DNA which encodes TNF.
• An expression vector is a replicable DNA construct in which a DNA sequence encoding TNF is operably linked to suitable control sequences capable of effecting the expression of TNF in CHO host cells. Such control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control termination of transcription and translation.
• Vectors comprise plasmids, viruses (including phage), and integrable DNA fragments (i.e., integratable into the CHO genome by recombination). Once it has transformed a CHO host, the vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself.
• vector is generic to "plasmid”. However, all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. Suitable vectors contain replicon and control sequences which are derived from species compatible with the CHO host cells.
• Transformed host cells are CHO or other eukaryotic cells which have been transformed or transfected with tumor necrosis factor vectors constructed using recombinant DNA techniques.
• Transformed CHO or other eukaryotic host cells ordinarily express TNF.
• DNA regions are operably linked when they are functionally related to each other.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide.
• a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
• operably linked means contiguous, and, in the case of secretory leaders, contiguous and in reading phase.
• Vectors must contain a promoter which is recognized by the host organism. This is generally a promoter homologous to the intended host.
• Expression vectors for CHO cells ordinarily include, if necessary, an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intr on-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.
• the transcriptional and translational control sequences in expression vectors used in transforming CHO cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40).
• the early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin or replication.
• Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the viral origin of replication is included. Further, it is also possible, and often desirable, to use human genomic promoter, control, and/or signal sequences normally associated with
• An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adenovirus, VSV, or BPV source), or may be provided by the exogenous origin.
• an exogenous origin such as may be derived from SV40 or other viral (e.g., Polyoma, Adenovirus, VSV, or BPV source), or may be provided by the
• CHO cell replication mechanism If the vector is integrated into the CHO host cell chromosome, the latter is often sufficient. While CHO cells are disclosed as the preferred host cell it is expected that TNF- ⁇ recombinantly produced in any eukoryotic host cell system will be glycosylated in the same manner as the TNF- ⁇ obtained from CHO cells. Thus, for providing glycosylated human TNF- ⁇ , any suitable eukaryotic host cell system may be used. Glycosylated TNF initially is recovered from cultures media.
• Transformed nonsecreting cells are lysed by sonication or other acceptable method and debris separated by centrifugation, while the supernatants from secreting cells (such as induced cell lines) are simply separated from the cells by centrifugation. Then, any one or more of the following steps may be used, or other methods entirely may be substituted. The following method was used to purify glycosylated TNF to a degree sufficient for sequencing. This is not necessarily coextensive with the purification required for a therapeutic product.
• glycosylated TNF is adsorbed onto a hydrophobic substance from the lysed culture of supernatant culture medium.
• the hydrophobic substance preferably is a nongelatinous hydrophobic surface such as a silicate or polyolefm, although alkyl Sepharose is also suitable.
• the preferred embodiment is controlled pore glass. A ratio of about one volume of controlled pore glass is mixed with 50 volumes of supernatant and the adsorption allowed to proceed at about 4°C without agitation over a period of about 30 minutes to two hours, preferably about one hour, under slightly alkaline conditions.
• the adsorbent generally should thereafter be washed with a suitable buffer to remove entrapped contaminant proteins.
• the adsorbed glycosylated TNF is eluted from the hydrophobic substance by altering the solvation properties of the surrounding medium.
• the elution can be accomplished by passing a solution buffered at approximately pH 7 to 8.5, preferably around 8, containing 1M salt and an effective amount of an aqueous solution of a water miscible organic polyol, such as, for example, ethylene glycol or glycerin, ordinarily ethylene glycol in the range of 10-30 percent v/v, preferably around 20 percent v/v.
• ethylene glycol or glycerin ordinarily ethylene glycol in the range of 10-30 percent v/v, preferably around 20 percent v/v.
• the glycosylated tumor necrosis factor- containing elution fractions are detected by any conventional assay.
• Further purification may be obtained by adsorption of TNF onto a tertiary or quaternary amino anion exchange resin.
• the preferred resins for this purpose are hydrophilic matrix resins such as cross-linked polystyrene, dextran or cellulose substituted with alkyl tertiary or quaternary amino groups.
• Glycosylated tumor necrosis factor prepared according to the present invention is prepared for administration by mixing the glycosylated TNF having the desired degree of purity with physiologically acceptable carriers, i.e., carriers which are nontoxic to recipients at the dosages and concentrations employed. Ordinarily, this will entail combining the glycosylated TNF with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose or dextrins, chelating agents such as EDTA, and other stabilizers and excipients.
• the carrier should be formulated to stabilize the glycosylated TNF as a dimer and/or preferably, a trimer.
• Glycosylated TNF to be used for therapeutic administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Glycosylated TNF ordinarily is stored in lyophilized form, or may simply be frozen.
• Human TNF is known to be useful in the treatment of many human diseases and conditions, either alone or with other active principles, such as interferon, or inactive carriers, diluents or excipients.
• the glycosylated human TNF of the present invention may be substituted for the non-glycosylated recombinantly-produced TNF in any of such applications and will provide increased half-life in body fluids, improved binding to receptors, and protection against the influence of proteases.
• glycosylated human TNF can be used to prevent or mitigate parasitic, bacterial, and viral infections, as well as prevention of autoimmune diseases.
• the glycosylated human TNF of the present invention may be formulated and administered as an injection, eye drop, nasal drop, inhalant, external preparation, oral preparation, rectal preparation, or vaginal preparation.
• the daily doses of the glycosylated recombinant human TNF may generally be in the range of from 50 to about 100,000,000 units. The daily dose may be increased or decreased according to the direction for use and symptoms of the recipient.
• glycosylated recombinant human tumor necrosis factor The amount of glycosylated recombinant human tumor necrosis factor that is administered depends, for example, upon the route of administration, the condition in question, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain optimal activity for the patient. Such modification can be effected by one skilled in the art without undue experimentation.
• glycosylated human recombinant TNF prepared according to the present invention is particularly useful because, compared to non-glycosylated human
• the glycosylated TNF may have an increased half-life in body fluids, improved binding to receptors, and may be better protected against the influence of proteases.
• glycosylated TNF- ⁇ of the present invention may be used for every indication in which prior art non-glycosylated TNF- ⁇ is currently known.
• known protcols for formulation, administration and dosage for non-glycosylated TNF- ⁇ may be initially used for the glycosylated TNF- ⁇ of the present invention subject to experimental modification in light of the expected increased half-life and improved efficiency of the glycosylated form.
• Glycosylated TNF can optionally be combined with other antineoplastic agents such as chemotherapeutic antibiotics such as actinomycin-D, adriamycin, aclacinomycin A, or with agents to augment or stimulate the immune response, such as immunoglobulins such as gamma globulin, including immunoglobulins having affinity for the cell surface antigens of neoplasms.
• chemotherapeutic antibiotics such as actinomycin-D, adriamycin, aclacinomycin A
• agents to augment or stimulate the immune response such as immunoglobulins such as gamma globulin, including immunoglobulins having affinity for the cell surface antigens of neoplasms.
• immunoglobulins such as gamma globulin, including immunoglobulins having affinity for the cell surface antigens of neoplasms.
• alpha, beta or gamma interferon is desirably combined with glycosylated
• a typical formulation comprises glycosylated TNF and gamma interferon in a unit activity proportion of from about 0.1 : 1 to 200: 1, ordinarily 10: 1, and may contain lymphotoxin in place of a portion of the glycosylated TNF. These proportions, of course, are subject to modification as required by therapeutic experience.
• Glycosylated TNF compositions may be administered to any tumor-bearing animals.
• the route of administration is in accord with known methods, e.g., intravenous, intraperitoneal, intramuscular, intralesional infusion or injection of sterile glycosylated TNF solutions, or by timed release systems as noted below.
• Glycosylated TNF may be administered intralesionally, i.e., by direct injection into solid tumor.
• administration is preferably intravenous or into the lymphatic system.
• Tumors of the abdominal organs such as ovarian cancer are advantageously treated by intraperitoneal infusion using peritoneal dialysis hardware and peritoneal-compatible solutions.
• glycosylated TNF is administered continuously by infusion, although bolus injection is acceptable.
• Glycosylated TNF can also be administered from an implantable timed-release article.
• suitable systems for proteins having the molecular weight of glycosylated TNF dimers or trimers include copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroethyl-methacrylate) or ethylene vinyl acetate.
• Such an article is implanted at surgical sites from which tumors have been excised.
• glycosylated TNF is encapsulated in semipermeable microcapsules or liposomes for injection into the tumor. This mode of administration is particularly useful for surgically inexcisable tumors such as brain tumors.
• glycosylated TNF The amount of glycosylated TNF that is administered will depend, for example, upon the route of administration, the tumor in question and the condition of the patient. Intralesional injections will require less glycosylated TNF on a body weight basis than will intravenous infusion, while some tumor types, e.g., solid tumors, appear to be more resistant to glycosylated TNF than other, e.g., leukemic.
• Example 1 Production of Recombinant Human TNF from Chinese Hamster
• the clone was propagated in Dulbecco Modified Eagle's Medium (DMEM) supplemented with proline, glutamine and 10% fetal bovine serum until production started. Production was carried out in a one-liter spinner with disc carriers.
• DMEM Dulbecco Modified Eagle's Medium
• the spinner was seeded with 2.5 x 10 cells, which were maintained in serum-supplemented medium for five days, after which the medium was changed to production medium (DMEM, ADC-1 supplement, Beit Haemck Industries, Israel), 0.2 ⁇ g per ml insulin and 0.5 ⁇ g protein per ml serum active fraction. Production was carried out in serum-free medium. Two harvests were collected every day and the spinner was kept running in production for three months. Crude concentrated recombinant human TNF was purified on an affinity column which contained recombinant human tumor necrosis binding protein coupled to Fractogel TSK-AF-CDI resin, a resin manufactured by Toso-Haas.
• glycosylated human TNF is purified using the following steps: 1. Clarification and concentration
• the chromatography procedure consisted of loading the crude onto the column which had been pre-equilibrated with PBS, washing the 30 bed volumes of HSPB and washing back to PBS (30 bed volumes) before the start of the elution.
• the elution was carried out with 100 mM NaCl in 50 mM citric acid (pH 2.5).
• the elution main peak was cut at 65% of its way down and collected into 2 M Tris (pH 8.5) for titration of the acidic pH (which is destructive to TNF activity) to pH 8.
• the titration was performed in a ratio of 400 microliters Tris per 5 ml of elution buffer.
• the column was regenerated with 10 bed volumes of 50 mM citric acid without NaCl and then regenerated with 20 bed volumes of HSPB.
• the column was washed with 20 bed volumes of 1 M NaCl and 0.05% NaN 3 and maintained in this buffer at 4°C.
• Table 1 shows a typical distribution of the recombinant human TNF through the columns fractions as measured by spectrophotometric measurement (A 280 /EC) and bioassay.
• the elution fractions from several chromatography cycles were pooled together and concentrated in a Filtron Minisette system with lOkDa cutoff membrane to a concentration of 1.65 mg/ml according to absorbance (A280 divided by the extinction coefficient, A280/EC).
• the concentrated pool was dialyzed against 20mM Tris-CHl; lOOmM NaCl (pH 8.0).
• Table 2 shows a typical yield of the CDI-TBP- 1 affinity column.
• Table 3 shows a typical distribution for the r-hTNF through the column's fractions in the reprocess chromatography, as measured by A280/EC and Bioassay.
• Oligosaccharide Mapping Chemical release of oligosaccharide was performed on a
• the GlycoPrep-1000 system from Oxford GlycoSystems.
• the GP-1000 is a bench-top chemical reactor which automatically cleaves the glycosidic bond with anhydrous hydrazine.
• the GP-1000 can operate in three modes: N, N+O and O. These modes give a preferential release of N-linked, O-linked or both, through lower recoverY
• Fig. 1A a second in the N+O mode
• Fig. IB a second in the N+O mode
• the separation of charged glycoforms was effected using 0.25 mm thick gel in a 13 x 26 cm. casting unit.
• the gel composition was 2.6% acrylamide, 4% ampholytes, and 10% glycerol. Five micrograms of each sample were loaded. The gel was run for 5000 Volt-hours, using 0.2 M NaOH as cathode solution and 0.2 M
• TNF samples were separated by capillary zone electrophoresis on an ABI Capillary Electrophoresis system (270A-HT, Applied Biosystems).
• the electrophoresis conditions were as follows: Buffer: 100 mM phosphate (pH 2.5)
• Capillary 72 mm length capillary
• the chromatographic conditions used in this test were based on those described in European Patent 168214.
• the chromatography was conducted at room temperature using a C4 RP-HPLC column, 25cm x 4.6 mm, and eluted with a gradient of 1-propanol in 0.1% trifluro acetic acid. Detection was at 280 nm on a UV detector.
• FIG. 1A In the GlycoPrep-1000 "O-mode" (Fig. 1A), only one peak of oligosaccharide was detected, eluting in the chromatogram at the same time as O-linked oligosaccharide standards run separately (data not shown).
• the released oligosaccharide in the "N+O" mode (Fig. IB) gave the same chromatogram with no additional peaks, indicating the absence of any known N-linked oligosaccharide. Other oligosaccharides may be present in minor amounts.
• TNF produced in CHO cells were sensitive to enzymatic digestion.
• Figure 2 represents the electropherograms obtained in capillary electrophoresis assays.
• Figure 2A shows the results of capillary electrophoresis of recombinant human TNF from CHO cells;
• 2B shows the results for recombinant human TNF from CHO cells + neuraminidase;
• 2C shows results from recombinant human TNF from CHO cells plus neuraminidase plus O-glycanase;
• 2D shows the results for recombinant human TNF from CHO cells plus neuraminidase plus
• O-glycanase plus N-glycanase shows the results of capillary electrophoresis analysis of recombinant human TNF produced in E. coli.
• the profile of TNF produced by CHO cells, Figure 2A exhibits a main peak, which is probably unglycosylated recombinant human TNF, as well as additional peaks not present in the E. coli recombinant human TNF, Figure 2E.
• the glycosylated peaks represent about 35% of the molecules.
• Figure 2B demonstrates that neuraminidase removed most of the charge, sialic acid, and that O-glycanase removed the oligosaccharide core, as shown in Figure 2C.
• Figure 2D shows that additional incubation with N-glycanase did not alter the electropherogram.
• recombinant human TNF prepared in CHO cells has an additional form not present on recombinant human TNF obtained from E. coli.
• the form from CHO cells was found to be O-glycosylated. All of the tests performed indicated that recombinant human TNF obtained from CHO cells is similar to recombinant human TNF from E. coli in the protein core, but differs in that the CHO TNF is glycosylated.
• the nature of the glycosylated form was determined by composition analysis, oligomap and capillary electrophoresis.
• samples applied on the gel were: (1) Sample buffer; (2) 10 micrograms TNF Bulk-2; (3) 20 micrograms TNF-Bulk 2; (4) 40 micrograms TNF Bulk-2; (5) 75 ng HSA; (6) 100 ng HSA; (7) 150 ng HSA; (8)LMW markers Qualitative purity were obtained by SDS-PAGE-silver stained. In obtaining the results, 9 and 18 micrograms of the final TNF Bulk were run on SDS-PAGE and stained by silver stain.
• the samples applied on the gel were: (1) Sample buffer; (2) 9 micrograms TNF Bulk-1; (3) 9 micrograms TNF Bulk-2; (4) 18 micrograms TNF Bulk-2; (5) LMW markers. No impurity was visible.
• Size exclusion chromatography was performed. Its purpose was to determine the purity and the molecular weight of the TNF form which is present in the final bulk.
• the SEC profile revealed one major peak with apparent MW by retention time of about 50 kD. It is known from the literature, and from our SDS-PAGE analysis, that the TNF molecular weight is 17 kD. The conclusion is that TNF exists in the bulk in the trimer form which is reported to be the bioactive form.
• Isoelectric focusing of r-hTNF Bulk samples was conducted on gel -5% total acrylamide 2.6% bis-acrylamide containing 2% ampholytes, 32% (v/v) glycerol, 10 mM GLU, 10 mM LYS, pH gradients 3-10, 15 cm wide (anode to cathode), run conditions: 15°C, 20,000 V/hr.
• Samples applied on the gel were: (1) Internal marker (cytochrome C); (2) pH markers; (3) 5 microliters 0.5 mg/ml TNF Bulk-1; (4) 5 microliters 0.5 mg/ml
• TNF Bulk-1 5 microliters 0.5 mg/ml TNF Bulk-2; (6) 0.5 mg/ml TNF Bulk-2; (7)
• the degree of amino acid sequence homology which brings a polypeptide within the scope of the definition of glycosylated tumor necrosis factor fragments or mutants herein will vary depending upon whether the homology between the candidate protein and glycosylated tumor necrosis factor falls within or without the tumor necrosis factor regions responsible for cytotoxic activity. Domains which are critical for cytotoxic activity should exhibit a high degree of homology in order to fall within the definition, while sequences not involved in maintaining glycosylated TNF conformation or in effecting receptor binding may show comparatively low homology.
• critical domains may exhibit cytolytic activity and yet remain homologous as defined herein if residues containing functionally similar amino acid side chains are substituted.
• Functionally similar refers to dominant characteristic of the side chains such as basic, neutral or acid, or the presence or absence of steroid bulk.
• tumor necrosis factor as defined herein specifically excludes lymphotoxin, or TNF-beta, as well as glycosylated TNF of other species such as murine TNF-alpha.
• glycosylated TNF A significant factor in establishing the identity of a polypeptide as glycosylated TNF is the ability of antisera which are capable of substantially neutralizing the cytolytic activity of mature glycosylated TNF to also substantially neutralize the cytolytic activity of the polypeptide in question.
• immunological identity and cytotoxic identity are not necessarily coextensive.
• Glycosylated tumor necrosis factor as defined herein includes, for example, TNF derivatives like insertion mutants, deletion mutants, or fusion proteins described above. These derivatives will bring glycosylated TNF outside of the molecular weight established for native human glycosylated TNF.
• glycosylated TNF may be engineered in order to reduce or eliminate susceptibility to hydrolysis by trypsin or other proteases.
• tumor necrosis factor includes polypeptides which can be converted, as by enzymatic hydrolysis, from an inactive state analogous to a zymogen to a polypeptide fragment which exhibits the desired biological activity.
• inactive precursors will be fusion proteins in which mature glycosylated TNF is linked by a peptide bond at its carbonyl terminus to a human protein or fragment thereof.
• the sequence at this peptide bond or nearby is selected so as to be susceptible to proteolytic hydrolysis to release glycosylated TNF, either in vivo or, as part of a manufacturing protocol, in vitro.
• the glycosylated TNF factor that is so generated then will exhibit the defmitionally-required cytotoxic activity.
• Mutant glycosylated TNF derivatives include the predetermined, i.e., site-specific, mutations of glycosylated TNF or its fragments.
• the objective of mutagenesis is to construct DNA that encodes glycosylated TNF as defined above, i.e., glycosylated TNF which exhibits cytotoxic activity towards tumor cells in vitro or acts like glycosylated TNF in vivo, and which retains residual homology with glycosylated TNF, but which also exhibits improved properties and activity.
• Mutant glycosylated tumor necrosis factor is defined as a polypeptide otherwise falling within the homology definition for glycosylated tumor necrosis factor set forth herein but which has an amino acid sequence different from that of glycosylated TNF whether by way of deletion, substitution, or insertion.
• the lysine or arginine residues of glycosylated TNF may be mutated to histidine or another amino acid residue which does not render the protein proteolytically labile.
• cysteine could be replaced by other residues and cross-linked chemically in order to confer oxidative stability.
• mutants meet the activity requirements for glycosylated TNF, for even biologically inactive mutants will be useful upon labeling or immobilization as reagents in immunoassays. However, in this case the mutants will retain at least one epitopic site which is cross-reactive with antibody to glycosylated TNF.
• DNA substitution mutations are those in which a different secretory leader or signal has been substituted for the native human secretory leader, either by deletions within the leader sequence or by substitutions, wherein most or all of the native leader is exchanged for a leader more likely to be recognized by the CHO cells.
• the secretory leader is "recognized" by the host CHO cells, the fusion protein consisting of glycosylated TNF and the leader ordinarily is cleaved at the leader-glycosylated TNF peptide bond in the events that lead to secretion of the glycosylated TNF.
• DNA mutants that are not expressed as glycosylated TNF derivatives are nucleotide substitutions made to enhance expression, primarily to avoid amino terminal loops in the transcribed mRNA or to provide codons that are more readily transcribed by the CHO, e.g., the well-known CHO preference codons for CHO expression.
• C CITY Ness-Ziona (E COUNTRY: Israel (F POSTAL CODE (ZIP) : 76110 (G TELEPHONE: 972-8-9382609 (H TELEFAX: 972-8-9407297
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO: 1 :

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