Infectious Diseases – Steven Jacobs, Karyn O’neil, Janssen Biotech Inc
Abstract for “Fibronectin type 3 domain-based scaffold compositions and methods, as well as their uses”
A protein scaffold is a combination of fibronectin III (FN3) proteins. This includes isolated nucleic acid sequences that encode a protein structure, vectors, host cells and methods of making or using them. These applications can be used in diagnostic and/ortherapeutic compositions, methods, and devices. IgG binding protein scaffold molecules have been shown to be useful in diagnostic and/or therapeutic applications.
Background for “Fibronectin type 3 domain-based scaffold compositions and methods, as well as their uses”
Monoclonal antibodies are the most commonly used class of therapeutic protein when high affinity or specificity are required for a target molecule. Non-antibody proteins can also be engineered to bind these targets. This is why the biopharmaceutical sector is so interested in non-antibody protein. These “alternative scaffolds” are also available. These proteins could have advantages over traditional antibody because of their small size, absence of disulphide bonds and high stability. They can also be expressed in prokaryotic host cells. They are easy to conjugate drugs/toxins, penentrate easily into tissues, and can be readily formatted into multispecific binding compounds (Skerra 2000; Binz; Pluckthun 2005).
“The immunoglobulin fold (Ig) is an alternative scaffold. This fold can be found in both the variable regions of antibodies and thousands of non-antibody protein proteins. One such Ig protein, the tenth of fibronectin type III (FN3) repeats from human fibronectin can tolerate mutations in the surface exposed loops, while maintaining the overall Ig fold structure. These loops are enriched with a variety of amino acids and specific binding agents that can be used to target a variety of targets (Koide, et al. 1998; Karatan et al. 2004). These engineered FN3 domains were found to bind to targets of high affinity while retaining important biophysical property (Parker et. al. 2005).”
Potential alternative scaffold molecules should have high thermal stability and the ability to reverse thermal folding and unfolding. There have been many methods to improve the thermal stability of proteins or enzymes. These include rational design, comparison of highly similar sequences, design and stabilization of disulfide bridges and mutations to increase?helix propensity. Engineering of salt bridges and alteration of the protein’s surface charge. Consensus sequence composition has also been used (Lehmann & Wyss 2001). Such scaffolds have high thermal stability, which is desirable because it can increase yield, solubility, activity, immunogenicity, and reduce the need for a cold chain.
“The present invention is a protein scaffold that is based on fibronectin type III repeat proteins, encoding and complementary nucleic acid vectors, vectors host cells compositions, combinations formulations devices and methods of making them. The preferred embodiment of the protein scaffold is composed of a consensus sequence of multiple FN3domains from human Tenascin C (hereinafter?Tenascin’). A further preferred embodiment of the invention’s protein scaffold is a consensus sequence comprising 15 FN3 domains. The invention’s protein scaffolds can be configured to bind different molecules, such as a cellular target proteins.
“The invention’s protein scaffolds may contain additional molecules or moieties such as the Fc region or albumin binding domain or any other moiety that influences half-life. Further embodiments allow the protein scaffolds to be attached to a nucleic acids molecule which may encode the protein.
“The present invention also offers at least one method of expressing at minimum one protein scaffold in a host cells based on a consensus sequence FN3 domains.” This involves culturing host cells as described herein, under conditions in which at least one scaffold is detectable and/or recoverable.
“The present invention also includes at least one composition that comprises (a) a protein-stack based on a consensus sequence containing multiple FN3 domains or encoding nucleic acids as described herein; (b) a suitable carrier or diluent and/or pharmaceutically acceptable.”
The present invention also includes a method for generating libraries of a protein-stack based on a fibronectin III (FN3) repeat, preferably a consensus sequence with multiple FN3domains, and more preferably a consensus sequence with multiple FN3domains from human Tenascin. By altering the amino acids or number of amino acid in the molecules at particular locations in the scaffold (e.g. loop regions) and creating successive scaffolds, the library can be formed. You can alter the amino acid composition of one loop, or multiple loops simultaneously. You can alter the length or shortening of loops. These libraries can include all the possible amino acids at each position or a selected subset. These library members can be used to screen by display, such in vitro (DNA,RNA, ribosome, etc. phage, yeast, and bacterial display.
“The protein scaffolds of the present invention provides enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations; they may be expressed and folded in prokaryotic systems, such as E. coli, in eukaryotic systems, such as yeast, and in in vitro transcription/translation systems, such as the rabbit reticulocyte lysate system”
“In addition, the present invention provides a method for generating a scaffold molecular that binds with a target protein by scanning the scaffold library of invention with the target and detecting binding binders. The invention also includes screening methods that can be used to create affinity mature protein scaffolds. These may, for example, be able to bind to specific target proteins with a particular affinity. Iterative rounds can be used to mutagenesis and select using systems such as in vitro or phage display. This process can result in site-directed mutagenesis to specific scaffold residues or random mutagenesis due error-pronePCR, DNA shuffling and/or a combination thereof. Any invention described in the present invention is also included in the present invention.
“DESCRIPTION OFF THE FIGURES”
“FIG. 1. SDS-PAGE analysis was performed on purified Tencon using a NuPAGE 4-12% bis-Tris gel (Invitrogen). It was stained with coomassie bleu. N is for natural conditions, and R for reduced.
“FIG. 2. Tencon’s circular dichroism analysis in PBS
“FIG. 4. Phagemid plasmid design for pTencon pIX. The OmpA signal sequence drives expression and secretion.
“FIG. 5. Display of Myc-Tencon in M13 phage. ELISA results showed binding of phages to?-Myc, CNTO95, and uncoated wells.
“FIG. 6. Loop structure of the third FN3 domain for human Tenascin.”
“FIG. 7. Screening IgG output by ELISA. Each clone was tested for binding to biotinylated IgG and biotinylated HSA.
“DESCRIPTION THE INVENTION”
The present invention provides an isolated, recombinant, and/or synthetic protein-stack based upon a consensus sequence fibronectin III (FN3) repeat proteins, including mammalian-derived scaffolds, as well compositions and encoding DNA molecules comprising at minimum one polynucleotide encoding scaffold based the consensus FN3 sequence. This invention also includes methods for making and using these nucleic acids and protein-scaffolds, as well as diagnostic and therapeutic compositions and devices.
The present invention offers many advantages over traditional therapeutics. These include the ability to administer local, orally or through the blood-brain barrier, the ability to express in E. Coli, the ability to conjugate to drugs, polymers and probes, the ability to form high concentrations and the ability of such molecules penetrate cancerous tissues and tumors.
“Moreover, protein scaffolds have many of the same properties as antibodies due to the fold that mimics an antibody’s variable region. This orientation allows the FN3 loops (similar to CDRs) to be exposed. They must be able to bind with cellular targets. The loops can also be modified, such as affinity maturation, to enhance certain binding properties or other related properties.
Three of the six loops in the protein scaffold correspond topologically with complementarity determining areas (CDRs 1-3), which are antigen-binding region of an antibody. The remaining loops, however, are surface exposed in a similar manner to antibody CDRs. These loops are located at or near residues 13-16 and 22-28, 38-40, 51-54 and 60-64 and 75-81 of the SEQ ID No:16, as shown in Table 3 and FIG. 6. For binding specificity and affinity, it is preferred that the loop regions around residues 52-54, 51-54, 75-81, and 22-28 are modified. A few of these loop regions can be randomized with other loop areas and/or strands that maintain their sequence to populate a library. Potent binders can then be selected from the library that has high affinity for a specific protein target. A loop region can interact with a target protein in a similar way to an antibody CDR interaction.
“Scaffolds of the invention can contain other subunits, for example, through covalent interaction. To impart antibody-like properties such as complement activity (ADCC), half life, and so on, the scaffold may contain all or part of an antibody constant area. You can modify C1q binding or Fc?R binding to provide and/or control effector function, and thus change CDC activity and/or ADCC activation. ?Effector functions? They are responsible for activating/disrupting a biological activity (e.g. in a subject). Some examples of effector functions are: complement dependent cytotoxicity, CDC; Fc receptor binding, antibody-dependent cell mediated cytotoxicity, ADCC; phagocytosis, downregulation of cell surface receptors (e.g. B cell receptor; BCR), and others. These effector functions can require that the Fc region be combined with a binding area (e.g. protein scaffold loops). They can be evaluated using various assays (e.g. Fc binding assays; ADCC assays; CDC assays etc
For desired properties, “Additionally, a xin conjugate, albumin, albumin binders, or polyethylene glycol (PEG), molecules can be attached to the scaffold. These fusions can be made using standard techniques such as expression of the fusion protein by a recombinant gene sequence.
The scaffolds of the invention are monospecific in monomeric or bi- or multiple-specific (for different proteins targets or epitopes on a single protein target) in multimer forms. Attachments can be covalent or not. A dimeric bispecific scaffold, for example, has one subunit that is specific for a first target protein/epitope and another subunit that is specific for a second target proteins/epitope. The valency of antigen binding can be increased by joining subunits of the scaffold in different conformations.
“Antibody” is defined herein as: “Antibody” is any protein or peptide that contains at least one portion of an immunoglobulin molecular, including but not limited to: a complementarity determining area (CDR) of a heavy- or light-chain variable region, a light chain constant region, and a framework region or any portion thereof. Optionally, such antibody may also affect a specific ligand. This includes but is not limited to: modulating, decreasing, increasing, antagonizing, agonizing, mitigation, alleviates and/or interfering with at least one activity, binding, receptor activity, binding, in vitro and/or in situ, and/or in-vivo.
“Antibody” is a broad term. “Antibody” can also refer to antibodies, digestion fragments and specified portions, as well as variants and variants thereof. Functional fragments are antigen-binding pieces that bind to one target. Antibody fragments that are capable of binding to specific targets or portions thereof include, but are not limited to: Fab (e.g. by papain digestion), F(ab?) (e.g. by partial reduction and pepsin digestion) and F(ab?) )2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc? (e.g. by pepsin digest or plasmin digestion), facb (e.g. by plasmin digestion), pFc?
These fragments can be made using enzymatic or synthetic cleavage techniques as well as recombinant methods, as is known in the art. You can also produce antibodies in various shortened forms by using antibody genes that have one or more stop codons upstream of the natural stopping site. A combination gene that encodes a F(ab) could be an example. A combination gene encoding a F(ab)2 heavy-chain portion can include DNA sequences encoding both the CH1 domain or the hinge region of the heavy chains. You can either join the different parts of antibodies chemically using conventional techniques or make them contiguous by genetic engineering techniques.
“A scaffold protein according to the invention can be used for measuring or effect in a cell or tissue, organ, or animal (including mammals or humans) to diagnose, monitor and treat, modulate or help prevent the incidence or reduction of at least one disease or condition. This includes but is not limited to an immune disorder, disease, cardiovascular disorder, infectious, malignant, or neurologic disorder, or any other related condition.
“As such, a method may include administering an effective amount (or a pharmaceutical composition) to a patient, cell, tissue, organ or animal in need of modulation, treatment or alleviation. The effective amount may be between 0.001 and 500 mg/kg (e.g., single), multiple, or continuous administration. It can also include a serum concentration of 0.01 to 5000?g/ml per single, multiple, continuous administration or any other effective range or value as determined by known methods as described herein.
“Scaffold Protein: Production and Generation”
“A minimum of one scaffold protein can be optionally made by a cell-line, a mixed cell-line, an immortalized cell, or clonal population, of immortalized cells,” as is well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).”
“Amino acid from a scaffold protein may be modified, added or deleted to decrease immunogenicity or reduce or enhance or modify binding, affinity or on-rate.
“Optionally, scaffold protein can be engineered with high affinity for antigens and other beneficial biological properties. The process of analyzing the parental sequences and preparing scaffold proteins using various conceptual engineered products can be used to achieve this goal. Experts in the field are familiar with three-dimensional models. There are computer programs that can display and illustrate the three-dimensional conformational structure of selected candidate sequences. They also allow for measurement of immunogenicity (e.g., Immunofilter program by Xencor, Inc., Monrovia, Calif.). These displays allow for analysis of the role of potential residues in the functioning and function of the candidate sequence. This includes the analysis of residues that may affect the candidate scaffold protein’s ability to bind its antigen. This allows residues to be chosen and combined from parent and reference sequences in order to achieve the desired characteristic such as the affinity for the target antigen(s). Other engineering methods are also possible.
“Screening”
“Screening protein structures for specific binding to fragments or proteins can be done easily using nucleotide display (DNA orRNA display) or peptide library libraries, such as in vitro display. This involves screening large numbers of peptides to identify those that have the desired function or structure. Displayed nucleotide sequences or peptides may range in length from 3 to 5100 amino acids or more, often between 8 and 25 amino acids, depending on the peptide. There are many recombinant DNA methods that can be used to create peptide libraries in addition to chemical synthetic methods. One method involves the display of the peptide sequence on a cell or bacteriophage. The nucleotide sequence that encodes the displayed peptide sequence is contained in every bacteriophage and cell. These methods are described in PCT Patent Publication Nos. 91/17271; 91/18980; 91/19818 and 93/08278.
“Other systems to generate libraries of peptides include aspects of both in vitro and recombinant chemical synthesis. See, PCT Patent Publication Nos. 92/05258,92/14843,and 96/19256. U.S. Pat. Nos. 5,658,754 and 5,643,768. Commercially available peptidide display libraries, vector, or screening kits from suppliers such as Invitrogen (Carlsbad), Calif., and Cambridge Antibody Technologies, (Cambridgeshire), UK. See, e.g., U.S. Pat. Nos. Nos. Nos. Nos.
“The avidity or affinity of a protein scaffold to an antigen can all be determined experimentally by any suitable method. (See, for example, Berzofsky, et al., ?Antibody-Antigen Interactions,? Fundamental Immunology by Paul, W. E. Ed. Raven Press: New York (N.Y.) (1984); Kuby Janis Immunology, W. H. Freeman and Company New York, N.Y. (1992); and other methods described herein. If measured under different conditions (e.g. salt concentration or pH), the measured affinity of a specific protein scaffold-antigen interaction may vary. Therefore, it is best to measure affinity and other antigen binding parameters (e.g. KD, Kon and Koff) using standardized solutions of antigen and protein scaffold, as well as a standard buffer such the one described herein.
“Competitive assays are possible with the protein scaffold according to the invention to determine which proteins, antibodies, or other antagonists compete to bind to a target protein using the protein scaffold. These assays, which are well-known to those with ordinary skill in the arts, evaluate competition between antagonists and ligands for a restricted number of binding sites on proteins. Before or after competition, the protein and/or antibodies are immobilized/insolubilized and the bound sample to the target protein is separated. This can be done by either decanting (where it was preinsolubilized) and centrifuging where the protein/antibody has been precipitated following the competitive reaction. The competitive binding can also be affected by whether function is altered due to the binding or absence of binding of the protein scaffold molecules to the target protein. This could include, for example, whether the protein scaffold molecule inhibits, potentiates, or increases the enzyme activity of, say, a label. As is well-known in the art, ELISA and other functional tests can be used.
“Nucleic Acid Molecules”
The nucleic acid molecules of this invention that encode protein scaffolds can take the form of RNA (mRNA,hnRNA or any other form), or the form DNA (including, but not restricted to, cDNA, genomic DNA obtained by either cloning or synthetically), or any combination thereof. You can have DNA that is triple-stranded (double-stranded), single-stranded (or any combination thereof). Any portion of the DNA orRNA that is not at least one strand can be considered the coding or sense strand. It can also be called the non-coding or anti-sense or both.
“Isolated nucleic acids molecules of the invention can include nucleic acids molecules comprising an open-reading frame (ORF), optionally with one or more Introns; nucleic Acid molecules comprising the code sequence for a protein structure or loop region that binds the target protein; nucleic Acid molecules comprising the coding sequence of a protein scaffold; and nucleic Acid molecules which contain a nucleotide sequencing substantially different than those described above, but which, because of the degene, still encode the scaffold as described herein or as it as well as the gene. The genetic code is known to be well-known in the art. It would therefore be easy for a skilled person in the art to generate degenerate nucleic acids variants that code specifically for the protein scaffolds of this invention. Ausubel, above, shows that such nucleic acids variants are included within the present invention.
“As shown herein, nucleic acids molecules of the present invention that encode a nucleic Acid encoding a Protein Scaffold can include, without limitation, those encoding an amino acid sequence for a fragment of a protein-scaffold; the entire protein-scaffold or a portion thereof; and the coding sequencing for a fragment of a scaffold as well as additional sequences such as the coding of at least one signal lead or fusion peptide with or without the aforementioned non-coding sequences. 3, and 4? sequences such as the transcribed and non-translated sequences which play a role transcription, mRNA processing including splicing, polyadenylation signals and splicing (for example, stability and ribosome binding). An additional coding sequence codes for additional amino acid, such that they provide additional functionalities. The sequence that encodes a protein structure can be fused with a marker sequence. This could include a sequence that codes for a peptide to facilitate purification of the protein scaffold fragment or part.
“Polynucleotides Selectively hybridizing to a polynucleotide, as Described Herein
“The present invention allows for the isolation of nucleic acid that can hybridize to a polynucleotide under selective hybridization conditions. The polynucleotides in this embodiment can be used to detect, quantify, and/or isolate nucleic acid containing such polynucleotides. The polynucleotides can be used, for example, to identify, isolate or amplify partial or full-length copies in a deposited database. The polynucleotides may be genomic or cDNA sequences that are isolated from or complement a cDNA library of mammalian or human nucleic acids.
“Preferably the cDNA library contains at least 80% full length sequences. Preferably, at minimum 85% or 90% full length sequences. More preferably, at most 95% full-length sequencing. To increase the number of rare sequences, the cDNA libraries may be normalized. Low- or moderate-stringency hybridization conditions can be used with sequences that have a lower sequence identity than those of complementary sequences. Optionally, high and moderate stringency conditions may be used for sequences with greater identity. Low stringency conditions permit selective hybridization of sequences with about 70% sequence identity. This can be used to identify paralogous and orthologous sequences.
“Optionally, the polynucleotides encoding this invention will encode at most a portion of the protein scaffold encoded using the polynucleotides discussed herein. This invention includes nucleic acid sequences that are able to be used for selective hybridization to a particular polynucleotide that encodes a protein scaffold. See Colligan supra and Ausubel supra. Each is fully incorporated by reference in this document.
“Construction Nucleic Acids”
“The present invention allows for the isolation of nucleic acid using a variety of methods.
“The nucleic acid can include sequences in addition the polynucleotide according to the present invention. To aid in the isolation of the polynucleotide, one or more multi-cloning sites that contain endonuclease restrictions sites can be placed into the nucleic acids. Translatable sequences may also be used to aid in the isolation and translation of the polynucleotide according to the invention. A hexahistidine marker sequence, for example, is a simple way to purify the proteins according to the invention. The nucleic acids of the present invention, except the coding sequence are optionally a vector or adapter for cloning/or expression of a Polynucleotide.
“Additional sequences may be added to such expression and/or cloning sequences in order to optimize their function in expression and/or cloning, to aid in isolation or improve the introduction of polynucleotide to a cell. The art of using cloning vectors and expression vectors, adapters and linkers is well-known. (See, for example, Ausubel, supra, or Sambrook supra)
“Recombinant Methods to Construct Nucleic Acids”
“The isolated nucleic acids compositions of the invention, such RNA, cDNA and genomic DNA, can be obtained using any of the cloning methods known to those skilled in this art. Some embodiments use oligonucleotide probes to selectively hybridize to the polynucleotides in the present invention to identify the desired sequences in a cDNA library or genomic DNA library. Those with ordinary skill in the arts are familiar with methods for isolating RNA and building cDNA and genome libraries. (See, for example, Ausubel, supra, or Sambrook supra)
“Nucleic Acid Screening Methods and Isolation Methods”
A probe based on the sequence of a particular polynucleotide can be used to screen a cDNA or genome library. To isolate homologous genes from different organisms, probes can be used to cross-react with genomic DNA sequences or cDNA sequences. Experts in the art will be able to recognize that hybridization can take place in a variety of settings. The wash medium or hybridization can also be made stringent. To allow duplex formation, the hybridization conditions must be more stringent. Temperature, ionic strength and pH can all be adjusted to control the degree of stringency. You can change the polarity of the reactant solution to alter the stringency of hybridization, such as by manipulating the formamide concentration within the range of 0% – 50%. The stringency of hybridization medium or wash medium will determine the degree of complementarity required to detectable binding. The ideal complementarity level will be 100%, 70-100%, or any other value. It is possible to compensate for minor sequence differences in probes or primers by decreasing the stringency of hybridization and/or washing medium.
“Methods for amplifying RNA or DNA are well-known in the art. They can be used according the present invention without undue experimental, based upon the teachings and guidance provided herein.”
“Known methods for DNA or RNA amplification include but aren’t limited to polymerase chain reaction(PCR) and related processes (see, U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al. ; 4,683,195, 4,683,202, 4,800,159 and 4,921,794 respectively to Tabor, et. al.; U.S. Pat. No. No. No. Wilson, et. al. 5,122,464 ; U.S. Pat. No. No. No. 5,066,584 to Gyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas, U.S. Patent. No. No. No. No. (See, for example, Ausubel, supra, or Sambrook.)
“Polymerase chain reaction (PCR), for example, can be used to amplify the sequences polynucleotides according to the present invention and related genes from genomic DNA or other cDNA libraries. In vitro amplification techniques such as PCR can be used to create nucleic acids that can be used to detect the presence of desired mRNA, nucleic Acid sequencing, and for other purposes. Berger, Sambrook, supra and Ausubel, respectively, are examples of techniques that can be used to guide persons of skill through in vitro amplifying methods. Mullis, et. al., U.S. Patent. No. No. The art has many commercially available kits that allow for genomic PCR amplification. Look at Advantage-GC Genomic PCR Kit from Clontech. To increase the yield of long-term PCR products, you can also use, for example, the T4 gene 32 protein from Boehringer Mannheim.
“Synthetic Methods to Construct Nucleic Acids”
“The present invention also allows for the preparation of isolated nucleic acid by chemical synthesis using known methods (see Ausubel, supra). A single-stranded oligonucleotide is generally produced by chemical synthesis. This can be transformed into double-stranded DNA through hybridization with a complementary sequence or polymerization using a DNA polymerase that uses the single strand as its template. A skilled artist will know that chemical synthesis of DNA is limited to sequences up to 100 bases. However, it’s possible to obtain longer sequences by ligating shorter sequences.
“Recombinant Expression Cassettes”
“The present invention also provides recombinant transcription cassettes that contain a nucleic acids of the invention. The present invention also provides a nucleic acid sequence, such as a cDNA, or a genome encoding a protein-stack sequence, that can be used to create a recombinant transcription cassette that can be introduced into at most one host cell. The recombinant transcription cassette will usually contain a polynucleotide from the present invention that is linked to transcriptional initiating regulatory sequences. This will allow the polynucleotide to be directed into the desired host cell. To direct the expression of nucleic acid of the invention, heterologous as well as non-heterologous (i.e. endogenous) promoters are possible.
“In some embodiments, nucleic acid isolated for use as a promoter, enhancer or other element can be placed in the appropriate position (upstream or downstream) of a nonheterologous polynucleotide according to the present invention in order to regulate the expression of the polynucleotide. Endogenous promoters, for example, can be modified in vivo and in vitro through mutation, deletion, and/or substitution.
“Vectors & Host Cells”
The present invention also refers to vectors that contain isolated nucleic acids molecules of the invention, host cells genetically engineered using the recombinant Vectors, and the production at least one protein scaffold using recombinant techniques. This is well-known in the art. See, for example, Sambrook, Ausubel, and others supra. Each of these are fully incorporated by reference in this document.
“Polynucleotides may optionally be linked to a vector that contains a marker that can be used for propagation in a host. A plasmid is generally introduced in a precipitate such as a calcium-phosphate precipitate or in a complex containing a chargedlipid. The vector can be packaged in vitro with a suitable packaging cell line, and then transduced into host tissues if it is a virus.
“The DNA insert must be operationally linked to a suitable promoter. Sites for transcription initiation and termination will be included in expression constructs. There will also be a site for ribosome binding for translation. The coding portion of mature transcripts will include a translation starting at the beginning and terminating at the end. UAA and UAG are preferred for expression in mammalian and eukaryotic cells.
“Expression vectors should include at least one selectable marker, but this can be optional.” These markers include, but aren’t limited to, methotrexate, dihydrofolate reductase, U.S. Pat. Nos. Nos. Nos. Nos. The art identifies the appropriate culture media and conditions for the above-described host cell types. Skilled artisans will quickly identify suitable vectors. The skilled artisan can easily identify suitable vectors. These methods are described in art (e.g. Sambrook, supra Chapters 1-4, 16-18 and Ausubel supra Chapters 1, 9, 13, 15, 16).
“At minimum one protein scaffold according to the invention can be expressed in modified form such as a Fusion Protein. This modification can include secretion signals and additional heterologous functional areas. To improve stability and persistence in host cells, such as purification or storage, an area of additional amino acid, especially charged amino acids can be added to the Nterminus of a protein-stack. To facilitate purification, peptide moiety can be added to the protein scaffold of this invention. These regions can be removed before final preparation of a protein-stack or at least one portion thereof. These methods are covered in many laboratory manuals such as Sambrook (supra, Chapters 17.29-17.42, and 18.1-18.74) and Ausubel (supra, Chapters 16, 17, and 18).
“Those with ordinary skill in art know the many expression systems that allow for the expression of a nucleic acids encoding a protein according to the invention. Alternately, nucleic acid of the invention can be expressed in host cells by manipulating endogenous DNA to encode a protein scaffold. These methods are well-known in the art, e.g. as described in U.S. Pat. Nos. Nos. 5,580.734, 5,641,670 and 5,733,746, respectively, are all incorporated by reference.”
“An illustration of cell cultures that can be used to produce the protein scaffolds, specific portions or variants thereof,” are mammalian, yeast, and bacterial cells. Although mammalian cells are often monolayers, they can also be grown in bioreactors or cell suspensions. There are many host cell lines that can express intact glycosylated protein. These include the COS-1, ATCC CLRL 1650, COS-7, ATCC CLRL-1651, HEK293, CHO, ATCC RL-1610, HEK293, HEK293, HEK293, CHO, ATCC RL-1610, CHO, ATCC RL-1611, CHO, hep G2 cell lines, CHO, P3X63Ag8.653, 293 cells, 293, 293, and HeLa cells. Cells of lymphoid origin such as myeloma or lymphoma cells are preferred host cells. The P3X63Ag8.653 and SP2/0-Ag14 cell lines (ATCC Accession No CRL-1580) are preferred hosts. A P3X63Ab8.653 cell or an SP2/0-Ag14 cells are preferred in a particularly preferred embodiment.
“Expression vectors can be used to control the expression of these cells. They may include one or more of: an origin of replication; a enhancer (e.g. the late or early SV40 promors, or the CMV promoter (U.S. Patent. Nos. Nos. No. No. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. The American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org), and other commercial sources, also contain cells that can be used to produce the nucleic acid or protein of the invention.
Polyadenlyation sequences, also known as transcription terminator sequences, are commonly incorporated into vectors when eukaryotic hosts cells are used. A terminator sequence could be the polyadenlyation sequence of the bovine growthhormone gene. Sequences that allow for precise splicing can also be included. A good example of a splicing order is the VP1 Intron from SV40 (Sprague et al. J. Virol. 45:773-781 (1983)). Gene sequences that control replication in host cells can also be integrated into the vector as is known in the art.
“Purification of Protein Scaffold”
“A protein scaffold can also be extracted from recombinant cells by using well-known methods, including ammonium sulfate precipitation, acid extraction, anion exchange chromatography or ethanol precipitation, acid extract, phosphocellulose and hydrophobic interaction. Chromatography, affinity chromatography. Chromatography, hydroxylapatite.chromatography. High performance liquid chromatography (HPLC?) High performance liquid chromatography (?HPLC?) can be used for purification. You can also see Colligan, Current Protocols In Immunology or Current Protocols In Protein Science, John Wiley & Sons NY, NY (1997-2001).
The present invention includes protein scaffolds that are naturally purified, chemically synthesized products, and products made by recombinant methods from prokaryotic and eukaryotic hosts, such as E. Coli and yeast. The present invention’s protein scaffold can be either glycosylated, or non-glycosylated depending on the host used in the recombinant production process. These methods are described in many standard laboratory manuals such as Sambrook, supra Section 17.37-17.42 and Ausubel supra Chapters 10, 12, 13, 16 and 18 and 20, Colligan, Protein Science supra Chapters 12-14. All of these methods are fully incorporated herein by reference
“Amino Acid Codes”
The present invention uses abbreviated amino acids to describe the protein scaffolds. The designations of amino acids can be identified by either the single letter code, three-letter code, name, or three nucleotide codes. This is common practice in the art (see Alberts, B., et.al., Molecular Biology of The Cell, Third Edition, Garland Publishing, Inc., New York, 1994). The present invention allows for one or more amino acids to be added, deleted, or substituted, whether by natural mutations or human manipulation. The present invention allows for the identification of essential amino acids within a protein scaffold. These can be done using methods such as site-directed mutation or alanine scanning mutagenesis. This procedure creates single alanine mutants at each residue of the molecule. The mutant molecules that result are tested for biological activity. This includes, but is not limited to, at minimum one neutralizing activity. Structural analysis can be used to identify sites that are crucial for protein scaffold binding. This includes crystallization, nuclear magnet resonance, or photoaffinity labeling. Smith et al. Biol. Biol.
“The present invention contains at least one biologically activated protein scaffold, as those with skill will see. Biologically active protein-scaffolds must have a specific activity of at least 20%, 30% or 40%. Preferably, at minimum 50%, 60% or 70% and most preferably, at minimum 80%, 90% or 95%-1000% of that of the native (nonsynthetic), or endogenous, or related, protein scaffold. The art of quantifying substrate specificity and enzymatic activity is well-known to those skilled in the field.
“In another aspect of the invention, the invention refers to protein fragments and scaffolds as described herein. These are modified by covalent attachments of an organic moiety. This modification can result in a protein fragment with enhanced pharmacokinetic properties, such as an increase in vivo serum half life. An organic moiety may be a linear, branched, hydrophilic, fatty, or ester group. Particular embodiments can include a hydrophilic polymeric moiety with a molecular mass of 800 to approximately 120,000 Daltons. It can also be a polyalkane glycol (e.g. polypropylene glycol, polyethylene glycol, or PPG), carbohydrate polymer (e.g. amino acid polymer, polyvinylpyrolidone), and a fatty acid (or fatty acid ester) group that can contain between eight and forty carbon atoms.
“Modified protein scaffolds and fragments can contain one or more organic moieties, which are covalently bound, directly or indirectly to the antibody. Each organic moiety that is covalently bonded to a fragment or protein scaffold of the invention can be either a hydrophilic or fatty acid group. The term “fatty acid” is used herein. It includes mono-carboxylic and di-carboxylic acid. What is a ‘hydrophilic polymeric groups? The term “hydrophilic polymeric group” is used herein to refer to organic polymers that are more easily soluble in water than octane. Polylysine, for example, is more easily soluble in water than octane. The invention covers a protein scaffold that is modified by covalent attachment of polylysine. Hydrophilic polymers suitable for modifying protein scaffolds of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. The preferred hydrophilic polymer used to modify the protein scaffold according to the invention should have a molecular mass of between 800 and 150,000 Daltons. PEG5000 or PEG20,000 can be used, where the subscript indicates the average molecular mass of the polymer in Daltons. You can substitute the hydrophilic polymeric element with up to six alkyl, or fatty acids, or fatty acid esters groups. You can prepare hydrophilic polymers by substituting a fatty or fatty acids ester group with the appropriate methods. A polymer containing an amine group can be linked to a carboxylate or fatty acids ester of the fatty acids or fatty acids, or an activated carboxylate (e.g. activated with N. N-carbonyl dimazole on a fat acid or fatty oil ester) can be attached to a hydroxyl link on a polymer.
Fatty acids and fatty acids suitable for modifying protein scaffolds according to the invention can either be saturated or contain one or more units. Fatty acids that are suitable for modifying protein scaffolds of the invention include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-?9-octadecanoate (C18, oleate), all cis-?5,8,11,14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Mono-esters of dicarboxylic acid that contain a lower alkyl group, or have a branched lower one, are suitable fatty acids esters. One to twelve carbon atoms can be found in the lower alkyl group, but it is preferred to have six.
“Modified protein fragments and scaffolds can be prepared by using appropriate methods such as reaction with one or more modifiable agents. What is a?modifying agent? The term “modifying agent” is used herein to refer to any organic group (e.g. hydrophilic polymer or fatty acid, or fatty acid ester) that contains an activating group. An ?activating group? An?activating group? is a chemical moiety, or functional, that reacts with another chemical group under the right conditions. This forms a covalent bond between both the modifying agent (and the second chemical) group. For example, amine-reactive activating groups include electrophilic groups, such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NETS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. A functional aldehyde can be linked to amine- and hydrazide-containing molecules. An azide can react with a trivalent-phosphorous group to form either phosphoramidate, or phosphorimide links. There are many methods that can be used to insert activating groups into molecules. See Hermanson, G. T. Bioconjugate Techniques. Academic Press, San Diego, Calif. (1996). You can bond an activating group directly to the organic (e.g., hydrophilic fatty polymer, fatty acids, fatty acids, fatty ester) or via a linker moiety. For example, a divalent, C1-C12, where one or more carbon atoms are replaced by a heteroatom such as oxygen, nitrogen, or sulfur. Tetraethylene glycol is a good example of a suitable linker moiety. (CH2)3?, ?NH? (CH2)6?NH?, ?(CH2)2?NH? and ?CH2?O?CH2?CH2?O?CH2?CH2?O?CH?NH?. Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. Trifluoroacetic Acid (TFA), which can be used to remove the Boc protecting group, can expose a primary amino that can be linked to another carboxylate. Or, the product can be reacted to create an activated maleimido derivative by using maleic anhydride. ”
The invention allows for the production of modified protein scaffolds by reacting a fragment or protein scaffold with a modifying ingredient. The amine-reactive agent can be used to bond the organic moieties to the protein scaffold in an non-site-specific manner, such as an NHS ester or PEG. Modified protein scaffolds or fragments comprising an organ moiety that is bonded at specific sites on a protein scaffold can be prepared by suitable methods such as reverse proteolysis. Werlen et. al. Bioconjugate Chemistry, 3:47-153 (1992); Fisch et. al. Bioconjugate Chemical, 5:411-417 (1994); Kumaran and al. Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng. 56(4):456-463 (1997); and the methods described by Hermanson, G. T. Bioconjugate Techniques. Academic Press: San Diego. Calif. (1996).”
“Protein Scaffold Compositions Containing Additional Therapeutically Active Ingredients”
The invention may also contain an effective amount (or combination of compounds or proteins) of at least one drug. This can be an anti-infective, cardiovascular, or central nervous system drug, or an autonomic nervous systems (ANS) drug. It can also include a respiratory tract drug and a gastrointestinal drug. These drugs are well-known in the art, with formulations, indications, dosage and administration for each (e.g., Nursing 2001 Handbook of Drugs, Springhouse Corp., Springhouse, P.A. 2001; Health Professional’s Drug Guide 2001, ed. Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J., Wells et al., Appleton & Lange, Stamford, Conn., each completely e, a topical drug, otic or nasal drug, ophthalmic, otic, a nutritional drug, a topical drug, ophthalmic, e, e, e, e, e, Stamford, Stamford, Stamford, Stamford,
“The anti-infective drug may be one or more of the following: antiprotozoals, antimalarials and antituberculotics. You can choose from antiarrhythmics and antianginal s as well as antihypertensives and antilipemics. CNS drugs can include at least one of the following: antipyretics, nonsteroidal analgesics, antianginal s, antihypertensives, antipyretics and antidepressants. The ANS drug can be at least one selected from cholinergics (parasympathomimetics), anticholinergics, adrenergics (sympathomimetics), adrenergic blockers (sympatholytics), skeletal muscle relaxants, and neuromuscular blockers. At least one respiratory tract drug can be selected from antihistamines (bronchodilators), expectorants, or at most one antitussive. At least one GI tract drug can be selected from antacids, bronchodilators, expectorants, or at most one adsorbent, or at minimum one gallstone solubilizer or digestive enzyme, or antidiarrheals. At least one hormonal drug can be selected from corticosteroids or anabolic steroids, estrogen or gonadotropin or at most one glucagon. It may also include thyroid hormone, pituitaryhormone antagonist, thyroid hormone and thyroid hormone antagonist. Fluid and electrolyte balance drugs can include at least one of the following: diuretics, electrolytes, or at minimum one replacement solution, acidifier, or alkalinizer. At least one drug for hematology can be selected from anticoagulants, blood derivatives and thrombolytics. Antineoplastics may include antineoplastics such as antimetabolites and alkylating drugs. An immunomodulation drug may be any combination of immunosuppressants or vaccines, or at minimum one toxoid or antitoxin, or at most one antivenin or immune serum, or biological response modifier. You can choose from at least one of the following: otic, nasal, anti-infectives, anti-inflammatories or ophthalmic ophthalmics. You can choose from at least one of the following: local anti-infectives; scabicides; or at least one topical corticosteroid or pediculicide. A minimum of one nutritional drug can be chosen from vitamins, minerals, and calorics. You can see the contents of Nursing 2001 Drug Handbook, supra.
“The minimum one antiprotozoal or amebicide can be any one of the following: atovaquone; chloroquine hydrochloride; chloroquinephosphate; metronidazole; metronidazole hylloride and pentamidine isethionate. At least one antihelmintic can be mebendazole or pyrantel pamoate. At least one antifungal can include amphotericin, amphotericin cholesteryl sodium sulfate, amphotericin b lipid complex and amphotericin b liposomal. At least one antimalarial drug can be selected from chloroquine chloride, chloroquine-phosphate complex, doxycycline and hydroxychloroquine. At least one antituberculotic/antileprotic can be selected from cycloserine (doxycycline), doxycycline, chloroquine phosphate, mefloquine hydrochloride and ethambutol. At least one aminoglycoside may be selected from amikacin, gentamicin, neomycin, streptomycin, and tobramycin. The at least one penicillin can be at least one selected from amoxcillin/clavulanate potassium, amoxicillin trihydrate, ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillin sodium/sulbactam sodium, cloxacillin sodium, dicloxacillin sodium, mezlocillin sodium, nafcillin sodium, oxacillin sodium, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V potassium, piperacillin sodium, piperacillin sodium/tazobactam sodium, ticarcillin disodium, and ticarcillin disodium/clavulanate potassium. Cefaclor can be cefadroxil or cefazolin salt, cefdinir and cefepime hydrochloride. At least one tetracycline may be selected from demeclocycline, doxycycline sodium, cefazolin sodium, cefadroxil, cefazoline hyclate and doxycycline microchloride. At least one sulfonamide may be selected from co-trimoxazole or sulfadiazine. At least one fluoroquinolone may be selected from alatrofloxacin ciprofloxacin enoxacin levofloxacin hydrochloride or nalidixic acids, norfloxacin and ofloxacin. At least one fluoroquinolone may be selected from alatrofloxacin ciprofloxacin enoxacin levofloxacin lomefloxacin hyloride, trovafloxacin and sparfloxacin. The at least one antiviral can be at least one selected from abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and zidovudine. At least one macroline antiviral can be selected from azithromycin and clarithromycin. The at least one miscellaneous anti-infective can be at least one selected from aztreonam, bacitracin, chloramphenicol sodium sucinate, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, imipenem and cilastatin sodium, meropenem, nitrofurantoin macrocrystals, nitrofurantoin microcrystal s, quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim, and vancomycin hydrochloride. (See, e.g., pp. 24214 of Nursing 2001 Drug Handbook.
“The least one inotropic may be any one of the following: amrinone, digoxin, or milrinone. At least one antiarrhythmic can include adenosine or amiodarone hydrochloride. At least one antianginal can include amlodipine besylate (amyl nitrate), diltiazem hyloride. isosorbide mononitrate. nadolol, procainamide hydrochloride. propranolol hydrchloride. propranolol hydrochloride. verapamil. At least one antihypertensive may be selected from acebutolol Hydrochloride and amlodipine sylate. At least one antilipemic can include atorvastatin calcium (micronized), colestipol sodium, chlorestyramines, cholestyramines, colestipol hydrchloride and fenofibrate mesylate, fluvastatin salt, gemfibrozil. At least one miscellaneous CV medication can be abciximab or alprostadil. (See, e.g., pp. Nursing 2001 Drug Handbook, pp. 215-336.
“The minimum one non-narcotic anti-inflammatory drug or analgesic can be any of the following: acetaminophen; aspirin; choline magnesium trisalicylate; diflunisal; and acetaminophen. Celecoxib and diclofenac potassium and diclofenac salt can each be used as at least one nonsteroidal analgesic drug. At least one nonsteroidal opiod analgesic or narcotic can be selected from alfentanil, buprenorphine sodium, diclofenac potassium, indomethacin sodium trihydrate, ketoprofen, ketorolac tromethamine, ketoprofen, ketoprofen, ketorolac tromethamine, ketoprofen, ketoprofen, ketorolax tromethamine, pentazocine, propoxyphene napsylate and tramadolhydrol citrate, tramadol hydrochloride At least one sedative-hypnotic may be selected from chloralhydrate, estazolam and flurazepam Hydrochloride, pentobarbital salt, pentobarbital sodium phenobarbital potassium, secobarbital salt, temazepam or zaleplon. At least one anticonvulsant may be selected from acetazolamide, carbamazepine and clorazepate sodium. At least one antidepressant may be selected from amitriptyline, amitriptyline sulfate and amoxapine. At least one antianxiety drug may be selected from alprazolam and buspirone hydrochloride. At least one antipsychotic drug may be selected from clozapine hydrochloride and fluphenazine. At least one central nervous system stimulant may be amphetamine, caffeine, dextroamphetamine, doxapram, methamphetamine, methylphenidate, modafinil and pemoline. At least one antiparkinsonian may be selected from amantadine sulfate, benztropine sulfate, biperiden hyloride and biperiden lactate. At least one miscellaneous central nerve system drug can be bupropion hydrochloride or donepezil hloride. (See, e.g., pp. 337-530 Nursing 2001 Drug Handbook.
“The minimum one cholinergic (e.g. parasymathomimetic), can be any of the following: bethanechol chloride; edrophonium chloride; neostigmine bromide; neostigmine-methyl sulfate; physostigmine salticylate; pyridostigmine molemide. Atropine sulfate (dicyclomine hydrochloride), glycopyrrolate (hyoscyamine), hyoscyamine-sulfate (hyoscyamine) and propantheline bromide (scopolamine, Scopolamine butylbromide and scopolamine highlbromide can all be the at least one anticholinergic. Sympathomimetics can contain at least one adrenergic. This includes dobutamine hydrochloride and metaraminol bitartrates, dopamine sulfate, norepinephrinebitartrates, phenylephrine hylloride, pseudoephedrine chloride, and pseudoephedrine sodium sulfate. Sympatholytics can contain at least one adrenergic blocking agent (sympatholytic). This could be any one of the following: dihydroergotamine, ergotamine tartrate or methysergide manate. At least one skeletal muscle relaxant may be selected from baclofen (carisoprodol), chlorzoxazone (cyclobenzaprine Hydrochloride), cyclobenzaprine sodium, methocarbamol and tizanidine hyroide. At least one neuromuscular inhibitor can be selected from atracurium, cisatracurium, chlorzoxazone, doxacurium, mivacurium, pancuronium, pipecuronium, rapacuronium, rocuronium, succinylcholine, tubocurarine, and vecuronium. (See, e.g., pp. “Nursing 2001 Drug Handbook, 531-84.
The at least one antihistamine may be any of the following: brompheniramine, chlorpheniramine, chlorpheniramine malate, clemastine fumarate, clemastine hydrochloride and diphenhydramine hyride. At least one bronchodilator may be selected from albuterol or albuterol Sulfate. At least one antitussive or expectorant can be selected from benzonatate and codeine phosphate. At least one miscellaneous drug can be selected from acetylcysteine and beclomethasone dimethionate, beractants, budesonide and guaifenesin. (See, e.g., pp. “Nursing 2001 Drug Handbook, 585-642.
“The minimum one antiflatulent, adsorbent or antacid can be any one of the following: aluminum carbonate; aluminum hydroxide; calcium carbonate; magaldrate; magnesium hydroxide; magnesium oxide, simethicone and sodium bicarbonate. At least one of the following digestive enzymes or gallstone solubilizers can be used: pancreatin; pancrelipase; and ursodiol. Attapulgite can be selected from attapulgite and bismuth subsalicylate as well as calcium polycarbophil and diphenoxylate sulfate. Other options include loperamide and octreotide-acetate. At least one laxative may be selected from bisocodyl and calcium polycarbophil as well as cascara-sagrada aromatic fluidextract and cascara-sagrada fluidextract. At least one antiemetic may be selected from chlorpromazine, dimenhydrinate and dolasetron mesylate. At least one antiulcer drug may be selected from cimetidine and cimetidine hydrochloride. (See, e.g., pp. 643-95 Nursing 2001 Drug Handbook.
The minimum number of corticosteroids that can be used is one from betamethasone, betamethasone or betamethasone salt phosphate. Betamethasone sodium-phosphate, betamethasone sodium citrate, betamethasone sodium hydrophosphate, betamethasone sodium citrate, hydrocortisone sodium succinate. Hydrocortisone, hydrocortisone hydrocortisone hydrocortisone hydrocortisone hydrocortisone hydrocortisone to, prednisolone and prednisolone. At least one anabolic steroid or androgen can be selected from danazol and fluoxymesterone. The at least one estrogen or progestin can be at least one selected from esterified estrogens, estradiol, estradiol cypionate, estradiol/norethindrone acetate transdermal system, estradiol valerate, estrogens (conjugated), estropipate, ethinyl estradiol, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and levonorgestrel, ethinyl estradiol and norethindrone, ethinyl estradiol and norethindrone acetate, ethinyl estradiol and norgestimate, ethinyl estradiol and norgestrel, ethinyl estradiol and norethindrone and acetate and ferrous fumarate, levonorgestrel, medroxyprogesterone acetate, mestranol and norethindron, norethindrone, norethindrone acetate, norgestrel, and progesterone. At least one gonadroptropin may be selected from ganirelix, gonadoreline, histrelin, or progesterone. At least one antidiabetic/glucaon can be selected from acarbose or chlorpropamide, glimepiride and glipizide. At least one of the thyroid hormones can be selected from levothyroxine, liothyronine, liotrix and thyroid. At least one thyroid hormone antagonist may be methimazole or potassium iodide. At least one pituitary hormone may be corticotropin or cosyntropin. At least one parathyroid-like medication can be selected from calcifediol (human), calcium (salmon), and calcitriol. (See, e.g., pp. “Nursing 2001 Drug Handbook, pp. 696-796.
“The minimum one diuretic may be any of the following: acetazolamide, amiloride sodium, bumetanide and chlorthalidone; acetazolamide sodium; acetazolamide sodium; mannitol; metolazone; spironolactone; torsemide or triamterene. The at least one electrolyte or replacement solution can be at least one selected from calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, calcium lactate, calcium phosphate (dibasic), calcium phosphate (tribasic), dextran (high-molecular-weight), dextran (low-molecular-weight), hetastarch, magnesium chloride, magnesium sulfate, potassium acetate, potassium bicarbonate, potassium chloride, potassium gluconate, Ringer’s injection, Ringer’s injection (lactated), and sodium chloride. At least one acidifier/alkalinizer can be selected from sodium bicarbonate or sodium lactate. (See, e.g., pp. Nursing 2001 Drug Handbook, pp. 797-833.
“The minimum one hematinic may be any of the following: ferrous fumarate or ferrous glutarate, ferrous sulfurate, ferrous ferric gluconate, iron sorbil, ferrous iron gluconate (dried), ferrous ferroussulfate, iron sulfate(dried), ferrous ferric gluconate complex, and ferrous sulfate. At least one anticoagulant may be selected from ardeparin salt, dalteparin potassium, ferrous sulfate, ferrous sulfate (dried), iron dextran, iron sorbil, polysaccharide-iron complex, and sodium ferric gluconate complex. At least one blood derivative can include albumin 5% or 25%, antihemophilic factors, anti-inhibitor complex, antithrombin IX (human), factor IIX complex and plasma protein fractions. At least one thrombolytic agent can be selected from anistreplase or reteplase recombinant, streptokinase and urokinase. (See, e.g., pp. Nursing 2001 Drug Handbook, pp.834-66.
“Alkylating drugs can include busulfan (carboplatin), carmustine, chlorambucil and cisplatin), ifosfamides, lomustines, mechlorethamine Hydrochloride (melphalan), melphalan hydrochloride, streptozocin, or temozolomide. At least one antimetabolite may be capecitabine or cladribine, floxuridine and fludarabine. At least one antibiotic antineoplastic may be selected from bleomycin sulfate (dactinomycin), dactinomycin (dunorubicin citrate liquid liposomal), daunorubicin phosphate, doxorubicin hydrchloride liposomal), doxorubicin hyrosomal doxorubicin hydrchloride liposomal), epirubicin hydrochloride plicamycin, valrubicin, idamycin, velamycin, clon, tin, dis, dis, dis, dizin, dis, dis, dis, damycin, dis, dis, dis, dis, dis, dis, dis, izin, idis, izin, izin, izin, prakil, iz idis, izin, idis, idis, idis, idis, idamycin, idis, thiomycin, idamycin, cin, cin, cin, cin, cin, cin cin, cin cin cin cin cin cin cin cin cin cin cya, cin cin cin cin cin cin cin cin cin ida, cin cin cin cin cin cin cin cin cin, idarubicin cin cin cin cin cin cin cin cin cin, cin, cin, cin, cin,,,,,,,,,,,,,,,, methotrex,,,,,,,,,,, Anastrozole can be selected from bicalutamide and estramustinephosphate sodium. Asparaginase (BCG), (live intravesical), dacarbazine (BCG), (live intravesical), docetaxel (etoposide phosphate), gemcitabine hydrochloride), gemcitabine hydrochloride), mitotane (mitoxantrone phosphate), gemcitabine hydrochloride), mitotane (mitoxantrone hyoxantrone hydrchloride), mitotitan, vinblastine stine stine stine stine stine stine stine stine stine stine sine stine stine stine stine stine stine stine stine stine e stine stine stine stine stine stine stine stine stine striestine stine stine stine stine stine stine stine sulfate, vincristine stine stine sulfate and vinblastine stine stine sulfate and vincristine sulfate and vincristine sulfate and vincristine sulfate-sulfate and vinblastine sulfate and vincrisulfate (See, e.g., pp. “867-963 Nursing 2001 Drug Handbook.
At least one immunosuppressant may be any of the following: azathioprine (or basiliximab), cyclosporine (or daclizumab), lymphocyte immune globulin; muromonab CD3; mycophenolate Mofetil, mycophenolate hydrochloride; sirolimus. At least one vaccine or toxoid may be selected from BCG vaccine and cholera vaccine. At least one antitoxin/antivenin can be selected from Crotalidae antivenin (polyvalent), Diphtheria antivenom (equine), and amd Micrurus Fulvius antivenin. At least one immune serum may be selected from cytomegalovirus immuno globulin, intraveneous, hepatitis B immunity globulin, immuneglobulin intratoxin, immuneglobulin intermuscular, immuneglobulin intravenous, rabies vaccine, respiratory syncytial viral immuneglobulin, intravenous (human), Rh0 (D) immuneglobulin human), varicella-zoster immuneglobulin (human), or a) and varicella virus (human). At least one biological response modifier may be selected from aldesleukin (epoetin), filgrastim and glatiramer-acetate for injection. (See, e.g., pp. 964-1040 Nursing 2001 Drug Handbook.
Summary for “Fibronectin type 3 domain-based scaffold compositions and methods, as well as their uses”
Monoclonal antibodies are the most commonly used class of therapeutic protein when high affinity or specificity are required for a target molecule. Non-antibody proteins can also be engineered to bind these targets. This is why the biopharmaceutical sector is so interested in non-antibody protein. These “alternative scaffolds” are also available. These proteins could have advantages over traditional antibody because of their small size, absence of disulphide bonds and high stability. They can also be expressed in prokaryotic host cells. They are easy to conjugate drugs/toxins, penentrate easily into tissues, and can be readily formatted into multispecific binding compounds (Skerra 2000; Binz; Pluckthun 2005).
“The immunoglobulin fold (Ig) is an alternative scaffold. This fold can be found in both the variable regions of antibodies and thousands of non-antibody protein proteins. One such Ig protein, the tenth of fibronectin type III (FN3) repeats from human fibronectin can tolerate mutations in the surface exposed loops, while maintaining the overall Ig fold structure. These loops are enriched with a variety of amino acids and specific binding agents that can be used to target a variety of targets (Koide, et al. 1998; Karatan et al. 2004). These engineered FN3 domains were found to bind to targets of high affinity while retaining important biophysical property (Parker et. al. 2005).”
Potential alternative scaffold molecules should have high thermal stability and the ability to reverse thermal folding and unfolding. There have been many methods to improve the thermal stability of proteins or enzymes. These include rational design, comparison of highly similar sequences, design and stabilization of disulfide bridges and mutations to increase?helix propensity. Engineering of salt bridges and alteration of the protein’s surface charge. Consensus sequence composition has also been used (Lehmann & Wyss 2001). Such scaffolds have high thermal stability, which is desirable because it can increase yield, solubility, activity, immunogenicity, and reduce the need for a cold chain.
“The present invention is a protein scaffold that is based on fibronectin type III repeat proteins, encoding and complementary nucleic acid vectors, vectors host cells compositions, combinations formulations devices and methods of making them. The preferred embodiment of the protein scaffold is composed of a consensus sequence of multiple FN3domains from human Tenascin C (hereinafter?Tenascin’). A further preferred embodiment of the invention’s protein scaffold is a consensus sequence comprising 15 FN3 domains. The invention’s protein scaffolds can be configured to bind different molecules, such as a cellular target proteins.
“The invention’s protein scaffolds may contain additional molecules or moieties such as the Fc region or albumin binding domain or any other moiety that influences half-life. Further embodiments allow the protein scaffolds to be attached to a nucleic acids molecule which may encode the protein.
“The present invention also offers at least one method of expressing at minimum one protein scaffold in a host cells based on a consensus sequence FN3 domains.” This involves culturing host cells as described herein, under conditions in which at least one scaffold is detectable and/or recoverable.
“The present invention also includes at least one composition that comprises (a) a protein-stack based on a consensus sequence containing multiple FN3 domains or encoding nucleic acids as described herein; (b) a suitable carrier or diluent and/or pharmaceutically acceptable.”
The present invention also includes a method for generating libraries of a protein-stack based on a fibronectin III (FN3) repeat, preferably a consensus sequence with multiple FN3domains, and more preferably a consensus sequence with multiple FN3domains from human Tenascin. By altering the amino acids or number of amino acid in the molecules at particular locations in the scaffold (e.g. loop regions) and creating successive scaffolds, the library can be formed. You can alter the amino acid composition of one loop, or multiple loops simultaneously. You can alter the length or shortening of loops. These libraries can include all the possible amino acids at each position or a selected subset. These library members can be used to screen by display, such in vitro (DNA,RNA, ribosome, etc. phage, yeast, and bacterial display.
“The protein scaffolds of the present invention provides enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations; they may be expressed and folded in prokaryotic systems, such as E. coli, in eukaryotic systems, such as yeast, and in in vitro transcription/translation systems, such as the rabbit reticulocyte lysate system”
“In addition, the present invention provides a method for generating a scaffold molecular that binds with a target protein by scanning the scaffold library of invention with the target and detecting binding binders. The invention also includes screening methods that can be used to create affinity mature protein scaffolds. These may, for example, be able to bind to specific target proteins with a particular affinity. Iterative rounds can be used to mutagenesis and select using systems such as in vitro or phage display. This process can result in site-directed mutagenesis to specific scaffold residues or random mutagenesis due error-pronePCR, DNA shuffling and/or a combination thereof. Any invention described in the present invention is also included in the present invention.
“DESCRIPTION OFF THE FIGURES”
“FIG. 1. SDS-PAGE analysis was performed on purified Tencon using a NuPAGE 4-12% bis-Tris gel (Invitrogen). It was stained with coomassie bleu. N is for natural conditions, and R for reduced.
“FIG. 2. Tencon’s circular dichroism analysis in PBS
“FIG. 4. Phagemid plasmid design for pTencon pIX. The OmpA signal sequence drives expression and secretion.
“FIG. 5. Display of Myc-Tencon in M13 phage. ELISA results showed binding of phages to?-Myc, CNTO95, and uncoated wells.
“FIG. 6. Loop structure of the third FN3 domain for human Tenascin.”
“FIG. 7. Screening IgG output by ELISA. Each clone was tested for binding to biotinylated IgG and biotinylated HSA.
“DESCRIPTION THE INVENTION”
The present invention provides an isolated, recombinant, and/or synthetic protein-stack based upon a consensus sequence fibronectin III (FN3) repeat proteins, including mammalian-derived scaffolds, as well compositions and encoding DNA molecules comprising at minimum one polynucleotide encoding scaffold based the consensus FN3 sequence. This invention also includes methods for making and using these nucleic acids and protein-scaffolds, as well as diagnostic and therapeutic compositions and devices.
The present invention offers many advantages over traditional therapeutics. These include the ability to administer local, orally or through the blood-brain barrier, the ability to express in E. Coli, the ability to conjugate to drugs, polymers and probes, the ability to form high concentrations and the ability of such molecules penetrate cancerous tissues and tumors.
“Moreover, protein scaffolds have many of the same properties as antibodies due to the fold that mimics an antibody’s variable region. This orientation allows the FN3 loops (similar to CDRs) to be exposed. They must be able to bind with cellular targets. The loops can also be modified, such as affinity maturation, to enhance certain binding properties or other related properties.
Three of the six loops in the protein scaffold correspond topologically with complementarity determining areas (CDRs 1-3), which are antigen-binding region of an antibody. The remaining loops, however, are surface exposed in a similar manner to antibody CDRs. These loops are located at or near residues 13-16 and 22-28, 38-40, 51-54 and 60-64 and 75-81 of the SEQ ID No:16, as shown in Table 3 and FIG. 6. For binding specificity and affinity, it is preferred that the loop regions around residues 52-54, 51-54, 75-81, and 22-28 are modified. A few of these loop regions can be randomized with other loop areas and/or strands that maintain their sequence to populate a library. Potent binders can then be selected from the library that has high affinity for a specific protein target. A loop region can interact with a target protein in a similar way to an antibody CDR interaction.
“Scaffolds of the invention can contain other subunits, for example, through covalent interaction. To impart antibody-like properties such as complement activity (ADCC), half life, and so on, the scaffold may contain all or part of an antibody constant area. You can modify C1q binding or Fc?R binding to provide and/or control effector function, and thus change CDC activity and/or ADCC activation. ?Effector functions? They are responsible for activating/disrupting a biological activity (e.g. in a subject). Some examples of effector functions are: complement dependent cytotoxicity, CDC; Fc receptor binding, antibody-dependent cell mediated cytotoxicity, ADCC; phagocytosis, downregulation of cell surface receptors (e.g. B cell receptor; BCR), and others. These effector functions can require that the Fc region be combined with a binding area (e.g. protein scaffold loops). They can be evaluated using various assays (e.g. Fc binding assays; ADCC assays; CDC assays etc
For desired properties, “Additionally, a xin conjugate, albumin, albumin binders, or polyethylene glycol (PEG), molecules can be attached to the scaffold. These fusions can be made using standard techniques such as expression of the fusion protein by a recombinant gene sequence.
The scaffolds of the invention are monospecific in monomeric or bi- or multiple-specific (for different proteins targets or epitopes on a single protein target) in multimer forms. Attachments can be covalent or not. A dimeric bispecific scaffold, for example, has one subunit that is specific for a first target protein/epitope and another subunit that is specific for a second target proteins/epitope. The valency of antigen binding can be increased by joining subunits of the scaffold in different conformations.
“Antibody” is defined herein as: “Antibody” is any protein or peptide that contains at least one portion of an immunoglobulin molecular, including but not limited to: a complementarity determining area (CDR) of a heavy- or light-chain variable region, a light chain constant region, and a framework region or any portion thereof. Optionally, such antibody may also affect a specific ligand. This includes but is not limited to: modulating, decreasing, increasing, antagonizing, agonizing, mitigation, alleviates and/or interfering with at least one activity, binding, receptor activity, binding, in vitro and/or in situ, and/or in-vivo.
“Antibody” is a broad term. “Antibody” can also refer to antibodies, digestion fragments and specified portions, as well as variants and variants thereof. Functional fragments are antigen-binding pieces that bind to one target. Antibody fragments that are capable of binding to specific targets or portions thereof include, but are not limited to: Fab (e.g. by papain digestion), F(ab?) (e.g. by partial reduction and pepsin digestion) and F(ab?) )2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc? (e.g. by pepsin digest or plasmin digestion), facb (e.g. by plasmin digestion), pFc?
These fragments can be made using enzymatic or synthetic cleavage techniques as well as recombinant methods, as is known in the art. You can also produce antibodies in various shortened forms by using antibody genes that have one or more stop codons upstream of the natural stopping site. A combination gene that encodes a F(ab) could be an example. A combination gene encoding a F(ab)2 heavy-chain portion can include DNA sequences encoding both the CH1 domain or the hinge region of the heavy chains. You can either join the different parts of antibodies chemically using conventional techniques or make them contiguous by genetic engineering techniques.
“A scaffold protein according to the invention can be used for measuring or effect in a cell or tissue, organ, or animal (including mammals or humans) to diagnose, monitor and treat, modulate or help prevent the incidence or reduction of at least one disease or condition. This includes but is not limited to an immune disorder, disease, cardiovascular disorder, infectious, malignant, or neurologic disorder, or any other related condition.
“As such, a method may include administering an effective amount (or a pharmaceutical composition) to a patient, cell, tissue, organ or animal in need of modulation, treatment or alleviation. The effective amount may be between 0.001 and 500 mg/kg (e.g., single), multiple, or continuous administration. It can also include a serum concentration of 0.01 to 5000?g/ml per single, multiple, continuous administration or any other effective range or value as determined by known methods as described herein.
“Scaffold Protein: Production and Generation”
“A minimum of one scaffold protein can be optionally made by a cell-line, a mixed cell-line, an immortalized cell, or clonal population, of immortalized cells,” as is well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).”
“Amino acid from a scaffold protein may be modified, added or deleted to decrease immunogenicity or reduce or enhance or modify binding, affinity or on-rate.
“Optionally, scaffold protein can be engineered with high affinity for antigens and other beneficial biological properties. The process of analyzing the parental sequences and preparing scaffold proteins using various conceptual engineered products can be used to achieve this goal. Experts in the field are familiar with three-dimensional models. There are computer programs that can display and illustrate the three-dimensional conformational structure of selected candidate sequences. They also allow for measurement of immunogenicity (e.g., Immunofilter program by Xencor, Inc., Monrovia, Calif.). These displays allow for analysis of the role of potential residues in the functioning and function of the candidate sequence. This includes the analysis of residues that may affect the candidate scaffold protein’s ability to bind its antigen. This allows residues to be chosen and combined from parent and reference sequences in order to achieve the desired characteristic such as the affinity for the target antigen(s). Other engineering methods are also possible.
“Screening”
“Screening protein structures for specific binding to fragments or proteins can be done easily using nucleotide display (DNA orRNA display) or peptide library libraries, such as in vitro display. This involves screening large numbers of peptides to identify those that have the desired function or structure. Displayed nucleotide sequences or peptides may range in length from 3 to 5100 amino acids or more, often between 8 and 25 amino acids, depending on the peptide. There are many recombinant DNA methods that can be used to create peptide libraries in addition to chemical synthetic methods. One method involves the display of the peptide sequence on a cell or bacteriophage. The nucleotide sequence that encodes the displayed peptide sequence is contained in every bacteriophage and cell. These methods are described in PCT Patent Publication Nos. 91/17271; 91/18980; 91/19818 and 93/08278.
“Other systems to generate libraries of peptides include aspects of both in vitro and recombinant chemical synthesis. See, PCT Patent Publication Nos. 92/05258,92/14843,and 96/19256. U.S. Pat. Nos. 5,658,754 and 5,643,768. Commercially available peptidide display libraries, vector, or screening kits from suppliers such as Invitrogen (Carlsbad), Calif., and Cambridge Antibody Technologies, (Cambridgeshire), UK. See, e.g., U.S. Pat. Nos. Nos. Nos. Nos.
“The avidity or affinity of a protein scaffold to an antigen can all be determined experimentally by any suitable method. (See, for example, Berzofsky, et al., ?Antibody-Antigen Interactions,? Fundamental Immunology by Paul, W. E. Ed. Raven Press: New York (N.Y.) (1984); Kuby Janis Immunology, W. H. Freeman and Company New York, N.Y. (1992); and other methods described herein. If measured under different conditions (e.g. salt concentration or pH), the measured affinity of a specific protein scaffold-antigen interaction may vary. Therefore, it is best to measure affinity and other antigen binding parameters (e.g. KD, Kon and Koff) using standardized solutions of antigen and protein scaffold, as well as a standard buffer such the one described herein.
“Competitive assays are possible with the protein scaffold according to the invention to determine which proteins, antibodies, or other antagonists compete to bind to a target protein using the protein scaffold. These assays, which are well-known to those with ordinary skill in the arts, evaluate competition between antagonists and ligands for a restricted number of binding sites on proteins. Before or after competition, the protein and/or antibodies are immobilized/insolubilized and the bound sample to the target protein is separated. This can be done by either decanting (where it was preinsolubilized) and centrifuging where the protein/antibody has been precipitated following the competitive reaction. The competitive binding can also be affected by whether function is altered due to the binding or absence of binding of the protein scaffold molecules to the target protein. This could include, for example, whether the protein scaffold molecule inhibits, potentiates, or increases the enzyme activity of, say, a label. As is well-known in the art, ELISA and other functional tests can be used.
“Nucleic Acid Molecules”
The nucleic acid molecules of this invention that encode protein scaffolds can take the form of RNA (mRNA,hnRNA or any other form), or the form DNA (including, but not restricted to, cDNA, genomic DNA obtained by either cloning or synthetically), or any combination thereof. You can have DNA that is triple-stranded (double-stranded), single-stranded (or any combination thereof). Any portion of the DNA orRNA that is not at least one strand can be considered the coding or sense strand. It can also be called the non-coding or anti-sense or both.
“Isolated nucleic acids molecules of the invention can include nucleic acids molecules comprising an open-reading frame (ORF), optionally with one or more Introns; nucleic Acid molecules comprising the code sequence for a protein structure or loop region that binds the target protein; nucleic Acid molecules comprising the coding sequence of a protein scaffold; and nucleic Acid molecules which contain a nucleotide sequencing substantially different than those described above, but which, because of the degene, still encode the scaffold as described herein or as it as well as the gene. The genetic code is known to be well-known in the art. It would therefore be easy for a skilled person in the art to generate degenerate nucleic acids variants that code specifically for the protein scaffolds of this invention. Ausubel, above, shows that such nucleic acids variants are included within the present invention.
“As shown herein, nucleic acids molecules of the present invention that encode a nucleic Acid encoding a Protein Scaffold can include, without limitation, those encoding an amino acid sequence for a fragment of a protein-scaffold; the entire protein-scaffold or a portion thereof; and the coding sequencing for a fragment of a scaffold as well as additional sequences such as the coding of at least one signal lead or fusion peptide with or without the aforementioned non-coding sequences. 3, and 4? sequences such as the transcribed and non-translated sequences which play a role transcription, mRNA processing including splicing, polyadenylation signals and splicing (for example, stability and ribosome binding). An additional coding sequence codes for additional amino acid, such that they provide additional functionalities. The sequence that encodes a protein structure can be fused with a marker sequence. This could include a sequence that codes for a peptide to facilitate purification of the protein scaffold fragment or part.
“Polynucleotides Selectively hybridizing to a polynucleotide, as Described Herein
“The present invention allows for the isolation of nucleic acid that can hybridize to a polynucleotide under selective hybridization conditions. The polynucleotides in this embodiment can be used to detect, quantify, and/or isolate nucleic acid containing such polynucleotides. The polynucleotides can be used, for example, to identify, isolate or amplify partial or full-length copies in a deposited database. The polynucleotides may be genomic or cDNA sequences that are isolated from or complement a cDNA library of mammalian or human nucleic acids.
“Preferably the cDNA library contains at least 80% full length sequences. Preferably, at minimum 85% or 90% full length sequences. More preferably, at most 95% full-length sequencing. To increase the number of rare sequences, the cDNA libraries may be normalized. Low- or moderate-stringency hybridization conditions can be used with sequences that have a lower sequence identity than those of complementary sequences. Optionally, high and moderate stringency conditions may be used for sequences with greater identity. Low stringency conditions permit selective hybridization of sequences with about 70% sequence identity. This can be used to identify paralogous and orthologous sequences.
“Optionally, the polynucleotides encoding this invention will encode at most a portion of the protein scaffold encoded using the polynucleotides discussed herein. This invention includes nucleic acid sequences that are able to be used for selective hybridization to a particular polynucleotide that encodes a protein scaffold. See Colligan supra and Ausubel supra. Each is fully incorporated by reference in this document.
“Construction Nucleic Acids”
“The present invention allows for the isolation of nucleic acid using a variety of methods.
“The nucleic acid can include sequences in addition the polynucleotide according to the present invention. To aid in the isolation of the polynucleotide, one or more multi-cloning sites that contain endonuclease restrictions sites can be placed into the nucleic acids. Translatable sequences may also be used to aid in the isolation and translation of the polynucleotide according to the invention. A hexahistidine marker sequence, for example, is a simple way to purify the proteins according to the invention. The nucleic acids of the present invention, except the coding sequence are optionally a vector or adapter for cloning/or expression of a Polynucleotide.
“Additional sequences may be added to such expression and/or cloning sequences in order to optimize their function in expression and/or cloning, to aid in isolation or improve the introduction of polynucleotide to a cell. The art of using cloning vectors and expression vectors, adapters and linkers is well-known. (See, for example, Ausubel, supra, or Sambrook supra)
“Recombinant Methods to Construct Nucleic Acids”
“The isolated nucleic acids compositions of the invention, such RNA, cDNA and genomic DNA, can be obtained using any of the cloning methods known to those skilled in this art. Some embodiments use oligonucleotide probes to selectively hybridize to the polynucleotides in the present invention to identify the desired sequences in a cDNA library or genomic DNA library. Those with ordinary skill in the arts are familiar with methods for isolating RNA and building cDNA and genome libraries. (See, for example, Ausubel, supra, or Sambrook supra)
“Nucleic Acid Screening Methods and Isolation Methods”
A probe based on the sequence of a particular polynucleotide can be used to screen a cDNA or genome library. To isolate homologous genes from different organisms, probes can be used to cross-react with genomic DNA sequences or cDNA sequences. Experts in the art will be able to recognize that hybridization can take place in a variety of settings. The wash medium or hybridization can also be made stringent. To allow duplex formation, the hybridization conditions must be more stringent. Temperature, ionic strength and pH can all be adjusted to control the degree of stringency. You can change the polarity of the reactant solution to alter the stringency of hybridization, such as by manipulating the formamide concentration within the range of 0% – 50%. The stringency of hybridization medium or wash medium will determine the degree of complementarity required to detectable binding. The ideal complementarity level will be 100%, 70-100%, or any other value. It is possible to compensate for minor sequence differences in probes or primers by decreasing the stringency of hybridization and/or washing medium.
“Methods for amplifying RNA or DNA are well-known in the art. They can be used according the present invention without undue experimental, based upon the teachings and guidance provided herein.”
“Known methods for DNA or RNA amplification include but aren’t limited to polymerase chain reaction(PCR) and related processes (see, U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al. ; 4,683,195, 4,683,202, 4,800,159 and 4,921,794 respectively to Tabor, et. al.; U.S. Pat. No. No. No. Wilson, et. al. 5,122,464 ; U.S. Pat. No. No. No. 5,066,584 to Gyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas, U.S. Patent. No. No. No. No. (See, for example, Ausubel, supra, or Sambrook.)
“Polymerase chain reaction (PCR), for example, can be used to amplify the sequences polynucleotides according to the present invention and related genes from genomic DNA or other cDNA libraries. In vitro amplification techniques such as PCR can be used to create nucleic acids that can be used to detect the presence of desired mRNA, nucleic Acid sequencing, and for other purposes. Berger, Sambrook, supra and Ausubel, respectively, are examples of techniques that can be used to guide persons of skill through in vitro amplifying methods. Mullis, et. al., U.S. Patent. No. No. The art has many commercially available kits that allow for genomic PCR amplification. Look at Advantage-GC Genomic PCR Kit from Clontech. To increase the yield of long-term PCR products, you can also use, for example, the T4 gene 32 protein from Boehringer Mannheim.
“Synthetic Methods to Construct Nucleic Acids”
“The present invention also allows for the preparation of isolated nucleic acid by chemical synthesis using known methods (see Ausubel, supra). A single-stranded oligonucleotide is generally produced by chemical synthesis. This can be transformed into double-stranded DNA through hybridization with a complementary sequence or polymerization using a DNA polymerase that uses the single strand as its template. A skilled artist will know that chemical synthesis of DNA is limited to sequences up to 100 bases. However, it’s possible to obtain longer sequences by ligating shorter sequences.
“Recombinant Expression Cassettes”
“The present invention also provides recombinant transcription cassettes that contain a nucleic acids of the invention. The present invention also provides a nucleic acid sequence, such as a cDNA, or a genome encoding a protein-stack sequence, that can be used to create a recombinant transcription cassette that can be introduced into at most one host cell. The recombinant transcription cassette will usually contain a polynucleotide from the present invention that is linked to transcriptional initiating regulatory sequences. This will allow the polynucleotide to be directed into the desired host cell. To direct the expression of nucleic acid of the invention, heterologous as well as non-heterologous (i.e. endogenous) promoters are possible.
“In some embodiments, nucleic acid isolated for use as a promoter, enhancer or other element can be placed in the appropriate position (upstream or downstream) of a nonheterologous polynucleotide according to the present invention in order to regulate the expression of the polynucleotide. Endogenous promoters, for example, can be modified in vivo and in vitro through mutation, deletion, and/or substitution.
“Vectors & Host Cells”
The present invention also refers to vectors that contain isolated nucleic acids molecules of the invention, host cells genetically engineered using the recombinant Vectors, and the production at least one protein scaffold using recombinant techniques. This is well-known in the art. See, for example, Sambrook, Ausubel, and others supra. Each of these are fully incorporated by reference in this document.
“Polynucleotides may optionally be linked to a vector that contains a marker that can be used for propagation in a host. A plasmid is generally introduced in a precipitate such as a calcium-phosphate precipitate or in a complex containing a chargedlipid. The vector can be packaged in vitro with a suitable packaging cell line, and then transduced into host tissues if it is a virus.
“The DNA insert must be operationally linked to a suitable promoter. Sites for transcription initiation and termination will be included in expression constructs. There will also be a site for ribosome binding for translation. The coding portion of mature transcripts will include a translation starting at the beginning and terminating at the end. UAA and UAG are preferred for expression in mammalian and eukaryotic cells.
“Expression vectors should include at least one selectable marker, but this can be optional.” These markers include, but aren’t limited to, methotrexate, dihydrofolate reductase, U.S. Pat. Nos. Nos. Nos. Nos. The art identifies the appropriate culture media and conditions for the above-described host cell types. Skilled artisans will quickly identify suitable vectors. The skilled artisan can easily identify suitable vectors. These methods are described in art (e.g. Sambrook, supra Chapters 1-4, 16-18 and Ausubel supra Chapters 1, 9, 13, 15, 16).
“At minimum one protein scaffold according to the invention can be expressed in modified form such as a Fusion Protein. This modification can include secretion signals and additional heterologous functional areas. To improve stability and persistence in host cells, such as purification or storage, an area of additional amino acid, especially charged amino acids can be added to the Nterminus of a protein-stack. To facilitate purification, peptide moiety can be added to the protein scaffold of this invention. These regions can be removed before final preparation of a protein-stack or at least one portion thereof. These methods are covered in many laboratory manuals such as Sambrook (supra, Chapters 17.29-17.42, and 18.1-18.74) and Ausubel (supra, Chapters 16, 17, and 18).
“Those with ordinary skill in art know the many expression systems that allow for the expression of a nucleic acids encoding a protein according to the invention. Alternately, nucleic acid of the invention can be expressed in host cells by manipulating endogenous DNA to encode a protein scaffold. These methods are well-known in the art, e.g. as described in U.S. Pat. Nos. Nos. 5,580.734, 5,641,670 and 5,733,746, respectively, are all incorporated by reference.”
“An illustration of cell cultures that can be used to produce the protein scaffolds, specific portions or variants thereof,” are mammalian, yeast, and bacterial cells. Although mammalian cells are often monolayers, they can also be grown in bioreactors or cell suspensions. There are many host cell lines that can express intact glycosylated protein. These include the COS-1, ATCC CLRL 1650, COS-7, ATCC CLRL-1651, HEK293, CHO, ATCC RL-1610, HEK293, HEK293, HEK293, CHO, ATCC RL-1610, CHO, ATCC RL-1611, CHO, hep G2 cell lines, CHO, P3X63Ag8.653, 293 cells, 293, 293, and HeLa cells. Cells of lymphoid origin such as myeloma or lymphoma cells are preferred host cells. The P3X63Ag8.653 and SP2/0-Ag14 cell lines (ATCC Accession No CRL-1580) are preferred hosts. A P3X63Ab8.653 cell or an SP2/0-Ag14 cells are preferred in a particularly preferred embodiment.
“Expression vectors can be used to control the expression of these cells. They may include one or more of: an origin of replication; a enhancer (e.g. the late or early SV40 promors, or the CMV promoter (U.S. Patent. Nos. Nos. No. No. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. The American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org), and other commercial sources, also contain cells that can be used to produce the nucleic acid or protein of the invention.
Polyadenlyation sequences, also known as transcription terminator sequences, are commonly incorporated into vectors when eukaryotic hosts cells are used. A terminator sequence could be the polyadenlyation sequence of the bovine growthhormone gene. Sequences that allow for precise splicing can also be included. A good example of a splicing order is the VP1 Intron from SV40 (Sprague et al. J. Virol. 45:773-781 (1983)). Gene sequences that control replication in host cells can also be integrated into the vector as is known in the art.
“Purification of Protein Scaffold”
“A protein scaffold can also be extracted from recombinant cells by using well-known methods, including ammonium sulfate precipitation, acid extraction, anion exchange chromatography or ethanol precipitation, acid extract, phosphocellulose and hydrophobic interaction. Chromatography, affinity chromatography. Chromatography, hydroxylapatite.chromatography. High performance liquid chromatography (HPLC?) High performance liquid chromatography (?HPLC?) can be used for purification. You can also see Colligan, Current Protocols In Immunology or Current Protocols In Protein Science, John Wiley & Sons NY, NY (1997-2001).
The present invention includes protein scaffolds that are naturally purified, chemically synthesized products, and products made by recombinant methods from prokaryotic and eukaryotic hosts, such as E. Coli and yeast. The present invention’s protein scaffold can be either glycosylated, or non-glycosylated depending on the host used in the recombinant production process. These methods are described in many standard laboratory manuals such as Sambrook, supra Section 17.37-17.42 and Ausubel supra Chapters 10, 12, 13, 16 and 18 and 20, Colligan, Protein Science supra Chapters 12-14. All of these methods are fully incorporated herein by reference
“Amino Acid Codes”
The present invention uses abbreviated amino acids to describe the protein scaffolds. The designations of amino acids can be identified by either the single letter code, three-letter code, name, or three nucleotide codes. This is common practice in the art (see Alberts, B., et.al., Molecular Biology of The Cell, Third Edition, Garland Publishing, Inc., New York, 1994). The present invention allows for one or more amino acids to be added, deleted, or substituted, whether by natural mutations or human manipulation. The present invention allows for the identification of essential amino acids within a protein scaffold. These can be done using methods such as site-directed mutation or alanine scanning mutagenesis. This procedure creates single alanine mutants at each residue of the molecule. The mutant molecules that result are tested for biological activity. This includes, but is not limited to, at minimum one neutralizing activity. Structural analysis can be used to identify sites that are crucial for protein scaffold binding. This includes crystallization, nuclear magnet resonance, or photoaffinity labeling. Smith et al. Biol. Biol.
“The present invention contains at least one biologically activated protein scaffold, as those with skill will see. Biologically active protein-scaffolds must have a specific activity of at least 20%, 30% or 40%. Preferably, at minimum 50%, 60% or 70% and most preferably, at minimum 80%, 90% or 95%-1000% of that of the native (nonsynthetic), or endogenous, or related, protein scaffold. The art of quantifying substrate specificity and enzymatic activity is well-known to those skilled in the field.
“In another aspect of the invention, the invention refers to protein fragments and scaffolds as described herein. These are modified by covalent attachments of an organic moiety. This modification can result in a protein fragment with enhanced pharmacokinetic properties, such as an increase in vivo serum half life. An organic moiety may be a linear, branched, hydrophilic, fatty, or ester group. Particular embodiments can include a hydrophilic polymeric moiety with a molecular mass of 800 to approximately 120,000 Daltons. It can also be a polyalkane glycol (e.g. polypropylene glycol, polyethylene glycol, or PPG), carbohydrate polymer (e.g. amino acid polymer, polyvinylpyrolidone), and a fatty acid (or fatty acid ester) group that can contain between eight and forty carbon atoms.
“Modified protein scaffolds and fragments can contain one or more organic moieties, which are covalently bound, directly or indirectly to the antibody. Each organic moiety that is covalently bonded to a fragment or protein scaffold of the invention can be either a hydrophilic or fatty acid group. The term “fatty acid” is used herein. It includes mono-carboxylic and di-carboxylic acid. What is a ‘hydrophilic polymeric groups? The term “hydrophilic polymeric group” is used herein to refer to organic polymers that are more easily soluble in water than octane. Polylysine, for example, is more easily soluble in water than octane. The invention covers a protein scaffold that is modified by covalent attachment of polylysine. Hydrophilic polymers suitable for modifying protein scaffolds of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. The preferred hydrophilic polymer used to modify the protein scaffold according to the invention should have a molecular mass of between 800 and 150,000 Daltons. PEG5000 or PEG20,000 can be used, where the subscript indicates the average molecular mass of the polymer in Daltons. You can substitute the hydrophilic polymeric element with up to six alkyl, or fatty acids, or fatty acid esters groups. You can prepare hydrophilic polymers by substituting a fatty or fatty acids ester group with the appropriate methods. A polymer containing an amine group can be linked to a carboxylate or fatty acids ester of the fatty acids or fatty acids, or an activated carboxylate (e.g. activated with N. N-carbonyl dimazole on a fat acid or fatty oil ester) can be attached to a hydroxyl link on a polymer.
Fatty acids and fatty acids suitable for modifying protein scaffolds according to the invention can either be saturated or contain one or more units. Fatty acids that are suitable for modifying protein scaffolds of the invention include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-?9-octadecanoate (C18, oleate), all cis-?5,8,11,14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Mono-esters of dicarboxylic acid that contain a lower alkyl group, or have a branched lower one, are suitable fatty acids esters. One to twelve carbon atoms can be found in the lower alkyl group, but it is preferred to have six.
“Modified protein fragments and scaffolds can be prepared by using appropriate methods such as reaction with one or more modifiable agents. What is a?modifying agent? The term “modifying agent” is used herein to refer to any organic group (e.g. hydrophilic polymer or fatty acid, or fatty acid ester) that contains an activating group. An ?activating group? An?activating group? is a chemical moiety, or functional, that reacts with another chemical group under the right conditions. This forms a covalent bond between both the modifying agent (and the second chemical) group. For example, amine-reactive activating groups include electrophilic groups, such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NETS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. A functional aldehyde can be linked to amine- and hydrazide-containing molecules. An azide can react with a trivalent-phosphorous group to form either phosphoramidate, or phosphorimide links. There are many methods that can be used to insert activating groups into molecules. See Hermanson, G. T. Bioconjugate Techniques. Academic Press, San Diego, Calif. (1996). You can bond an activating group directly to the organic (e.g., hydrophilic fatty polymer, fatty acids, fatty acids, fatty ester) or via a linker moiety. For example, a divalent, C1-C12, where one or more carbon atoms are replaced by a heteroatom such as oxygen, nitrogen, or sulfur. Tetraethylene glycol is a good example of a suitable linker moiety. (CH2)3?, ?NH? (CH2)6?NH?, ?(CH2)2?NH? and ?CH2?O?CH2?CH2?O?CH2?CH2?O?CH?NH?. Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. Trifluoroacetic Acid (TFA), which can be used to remove the Boc protecting group, can expose a primary amino that can be linked to another carboxylate. Or, the product can be reacted to create an activated maleimido derivative by using maleic anhydride. ”
The invention allows for the production of modified protein scaffolds by reacting a fragment or protein scaffold with a modifying ingredient. The amine-reactive agent can be used to bond the organic moieties to the protein scaffold in an non-site-specific manner, such as an NHS ester or PEG. Modified protein scaffolds or fragments comprising an organ moiety that is bonded at specific sites on a protein scaffold can be prepared by suitable methods such as reverse proteolysis. Werlen et. al. Bioconjugate Chemistry, 3:47-153 (1992); Fisch et. al. Bioconjugate Chemical, 5:411-417 (1994); Kumaran and al. Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng. 56(4):456-463 (1997); and the methods described by Hermanson, G. T. Bioconjugate Techniques. Academic Press: San Diego. Calif. (1996).”
“Protein Scaffold Compositions Containing Additional Therapeutically Active Ingredients”
The invention may also contain an effective amount (or combination of compounds or proteins) of at least one drug. This can be an anti-infective, cardiovascular, or central nervous system drug, or an autonomic nervous systems (ANS) drug. It can also include a respiratory tract drug and a gastrointestinal drug. These drugs are well-known in the art, with formulations, indications, dosage and administration for each (e.g., Nursing 2001 Handbook of Drugs, Springhouse Corp., Springhouse, P.A. 2001; Health Professional’s Drug Guide 2001, ed. Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J., Wells et al., Appleton & Lange, Stamford, Conn., each completely e, a topical drug, otic or nasal drug, ophthalmic, otic, a nutritional drug, a topical drug, ophthalmic, e, e, e, e, e, Stamford, Stamford, Stamford, Stamford,
“The anti-infective drug may be one or more of the following: antiprotozoals, antimalarials and antituberculotics. You can choose from antiarrhythmics and antianginal s as well as antihypertensives and antilipemics. CNS drugs can include at least one of the following: antipyretics, nonsteroidal analgesics, antianginal s, antihypertensives, antipyretics and antidepressants. The ANS drug can be at least one selected from cholinergics (parasympathomimetics), anticholinergics, adrenergics (sympathomimetics), adrenergic blockers (sympatholytics), skeletal muscle relaxants, and neuromuscular blockers. At least one respiratory tract drug can be selected from antihistamines (bronchodilators), expectorants, or at most one antitussive. At least one GI tract drug can be selected from antacids, bronchodilators, expectorants, or at most one adsorbent, or at minimum one gallstone solubilizer or digestive enzyme, or antidiarrheals. At least one hormonal drug can be selected from corticosteroids or anabolic steroids, estrogen or gonadotropin or at most one glucagon. It may also include thyroid hormone, pituitaryhormone antagonist, thyroid hormone and thyroid hormone antagonist. Fluid and electrolyte balance drugs can include at least one of the following: diuretics, electrolytes, or at minimum one replacement solution, acidifier, or alkalinizer. At least one drug for hematology can be selected from anticoagulants, blood derivatives and thrombolytics. Antineoplastics may include antineoplastics such as antimetabolites and alkylating drugs. An immunomodulation drug may be any combination of immunosuppressants or vaccines, or at minimum one toxoid or antitoxin, or at most one antivenin or immune serum, or biological response modifier. You can choose from at least one of the following: otic, nasal, anti-infectives, anti-inflammatories or ophthalmic ophthalmics. You can choose from at least one of the following: local anti-infectives; scabicides; or at least one topical corticosteroid or pediculicide. A minimum of one nutritional drug can be chosen from vitamins, minerals, and calorics. You can see the contents of Nursing 2001 Drug Handbook, supra.
“The minimum one antiprotozoal or amebicide can be any one of the following: atovaquone; chloroquine hydrochloride; chloroquinephosphate; metronidazole; metronidazole hylloride and pentamidine isethionate. At least one antihelmintic can be mebendazole or pyrantel pamoate. At least one antifungal can include amphotericin, amphotericin cholesteryl sodium sulfate, amphotericin b lipid complex and amphotericin b liposomal. At least one antimalarial drug can be selected from chloroquine chloride, chloroquine-phosphate complex, doxycycline and hydroxychloroquine. At least one antituberculotic/antileprotic can be selected from cycloserine (doxycycline), doxycycline, chloroquine phosphate, mefloquine hydrochloride and ethambutol. At least one aminoglycoside may be selected from amikacin, gentamicin, neomycin, streptomycin, and tobramycin. The at least one penicillin can be at least one selected from amoxcillin/clavulanate potassium, amoxicillin trihydrate, ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillin sodium/sulbactam sodium, cloxacillin sodium, dicloxacillin sodium, mezlocillin sodium, nafcillin sodium, oxacillin sodium, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V potassium, piperacillin sodium, piperacillin sodium/tazobactam sodium, ticarcillin disodium, and ticarcillin disodium/clavulanate potassium. Cefaclor can be cefadroxil or cefazolin salt, cefdinir and cefepime hydrochloride. At least one tetracycline may be selected from demeclocycline, doxycycline sodium, cefazolin sodium, cefadroxil, cefazoline hyclate and doxycycline microchloride. At least one sulfonamide may be selected from co-trimoxazole or sulfadiazine. At least one fluoroquinolone may be selected from alatrofloxacin ciprofloxacin enoxacin levofloxacin hydrochloride or nalidixic acids, norfloxacin and ofloxacin. At least one fluoroquinolone may be selected from alatrofloxacin ciprofloxacin enoxacin levofloxacin lomefloxacin hyloride, trovafloxacin and sparfloxacin. The at least one antiviral can be at least one selected from abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and zidovudine. At least one macroline antiviral can be selected from azithromycin and clarithromycin. The at least one miscellaneous anti-infective can be at least one selected from aztreonam, bacitracin, chloramphenicol sodium sucinate, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, imipenem and cilastatin sodium, meropenem, nitrofurantoin macrocrystals, nitrofurantoin microcrystal s, quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim, and vancomycin hydrochloride. (See, e.g., pp. 24214 of Nursing 2001 Drug Handbook.
“The least one inotropic may be any one of the following: amrinone, digoxin, or milrinone. At least one antiarrhythmic can include adenosine or amiodarone hydrochloride. At least one antianginal can include amlodipine besylate (amyl nitrate), diltiazem hyloride. isosorbide mononitrate. nadolol, procainamide hydrochloride. propranolol hydrchloride. propranolol hydrochloride. verapamil. At least one antihypertensive may be selected from acebutolol Hydrochloride and amlodipine sylate. At least one antilipemic can include atorvastatin calcium (micronized), colestipol sodium, chlorestyramines, cholestyramines, colestipol hydrchloride and fenofibrate mesylate, fluvastatin salt, gemfibrozil. At least one miscellaneous CV medication can be abciximab or alprostadil. (See, e.g., pp. Nursing 2001 Drug Handbook, pp. 215-336.
“The minimum one non-narcotic anti-inflammatory drug or analgesic can be any of the following: acetaminophen; aspirin; choline magnesium trisalicylate; diflunisal; and acetaminophen. Celecoxib and diclofenac potassium and diclofenac salt can each be used as at least one nonsteroidal analgesic drug. At least one nonsteroidal opiod analgesic or narcotic can be selected from alfentanil, buprenorphine sodium, diclofenac potassium, indomethacin sodium trihydrate, ketoprofen, ketorolac tromethamine, ketoprofen, ketoprofen, ketorolac tromethamine, ketoprofen, ketoprofen, ketorolax tromethamine, pentazocine, propoxyphene napsylate and tramadolhydrol citrate, tramadol hydrochloride At least one sedative-hypnotic may be selected from chloralhydrate, estazolam and flurazepam Hydrochloride, pentobarbital salt, pentobarbital sodium phenobarbital potassium, secobarbital salt, temazepam or zaleplon. At least one anticonvulsant may be selected from acetazolamide, carbamazepine and clorazepate sodium. At least one antidepressant may be selected from amitriptyline, amitriptyline sulfate and amoxapine. At least one antianxiety drug may be selected from alprazolam and buspirone hydrochloride. At least one antipsychotic drug may be selected from clozapine hydrochloride and fluphenazine. At least one central nervous system stimulant may be amphetamine, caffeine, dextroamphetamine, doxapram, methamphetamine, methylphenidate, modafinil and pemoline. At least one antiparkinsonian may be selected from amantadine sulfate, benztropine sulfate, biperiden hyloride and biperiden lactate. At least one miscellaneous central nerve system drug can be bupropion hydrochloride or donepezil hloride. (See, e.g., pp. 337-530 Nursing 2001 Drug Handbook.
“The minimum one cholinergic (e.g. parasymathomimetic), can be any of the following: bethanechol chloride; edrophonium chloride; neostigmine bromide; neostigmine-methyl sulfate; physostigmine salticylate; pyridostigmine molemide. Atropine sulfate (dicyclomine hydrochloride), glycopyrrolate (hyoscyamine), hyoscyamine-sulfate (hyoscyamine) and propantheline bromide (scopolamine, Scopolamine butylbromide and scopolamine highlbromide can all be the at least one anticholinergic. Sympathomimetics can contain at least one adrenergic. This includes dobutamine hydrochloride and metaraminol bitartrates, dopamine sulfate, norepinephrinebitartrates, phenylephrine hylloride, pseudoephedrine chloride, and pseudoephedrine sodium sulfate. Sympatholytics can contain at least one adrenergic blocking agent (sympatholytic). This could be any one of the following: dihydroergotamine, ergotamine tartrate or methysergide manate. At least one skeletal muscle relaxant may be selected from baclofen (carisoprodol), chlorzoxazone (cyclobenzaprine Hydrochloride), cyclobenzaprine sodium, methocarbamol and tizanidine hyroide. At least one neuromuscular inhibitor can be selected from atracurium, cisatracurium, chlorzoxazone, doxacurium, mivacurium, pancuronium, pipecuronium, rapacuronium, rocuronium, succinylcholine, tubocurarine, and vecuronium. (See, e.g., pp. “Nursing 2001 Drug Handbook, 531-84.
The at least one antihistamine may be any of the following: brompheniramine, chlorpheniramine, chlorpheniramine malate, clemastine fumarate, clemastine hydrochloride and diphenhydramine hyride. At least one bronchodilator may be selected from albuterol or albuterol Sulfate. At least one antitussive or expectorant can be selected from benzonatate and codeine phosphate. At least one miscellaneous drug can be selected from acetylcysteine and beclomethasone dimethionate, beractants, budesonide and guaifenesin. (See, e.g., pp. “Nursing 2001 Drug Handbook, 585-642.
“The minimum one antiflatulent, adsorbent or antacid can be any one of the following: aluminum carbonate; aluminum hydroxide; calcium carbonate; magaldrate; magnesium hydroxide; magnesium oxide, simethicone and sodium bicarbonate. At least one of the following digestive enzymes or gallstone solubilizers can be used: pancreatin; pancrelipase; and ursodiol. Attapulgite can be selected from attapulgite and bismuth subsalicylate as well as calcium polycarbophil and diphenoxylate sulfate. Other options include loperamide and octreotide-acetate. At least one laxative may be selected from bisocodyl and calcium polycarbophil as well as cascara-sagrada aromatic fluidextract and cascara-sagrada fluidextract. At least one antiemetic may be selected from chlorpromazine, dimenhydrinate and dolasetron mesylate. At least one antiulcer drug may be selected from cimetidine and cimetidine hydrochloride. (See, e.g., pp. 643-95 Nursing 2001 Drug Handbook.
The minimum number of corticosteroids that can be used is one from betamethasone, betamethasone or betamethasone salt phosphate. Betamethasone sodium-phosphate, betamethasone sodium citrate, betamethasone sodium hydrophosphate, betamethasone sodium citrate, hydrocortisone sodium succinate. Hydrocortisone, hydrocortisone hydrocortisone hydrocortisone hydrocortisone hydrocortisone hydrocortisone to, prednisolone and prednisolone. At least one anabolic steroid or androgen can be selected from danazol and fluoxymesterone. The at least one estrogen or progestin can be at least one selected from esterified estrogens, estradiol, estradiol cypionate, estradiol/norethindrone acetate transdermal system, estradiol valerate, estrogens (conjugated), estropipate, ethinyl estradiol, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and levonorgestrel, ethinyl estradiol and norethindrone, ethinyl estradiol and norethindrone acetate, ethinyl estradiol and norgestimate, ethinyl estradiol and norgestrel, ethinyl estradiol and norethindrone and acetate and ferrous fumarate, levonorgestrel, medroxyprogesterone acetate, mestranol and norethindron, norethindrone, norethindrone acetate, norgestrel, and progesterone. At least one gonadroptropin may be selected from ganirelix, gonadoreline, histrelin, or progesterone. At least one antidiabetic/glucaon can be selected from acarbose or chlorpropamide, glimepiride and glipizide. At least one of the thyroid hormones can be selected from levothyroxine, liothyronine, liotrix and thyroid. At least one thyroid hormone antagonist may be methimazole or potassium iodide. At least one pituitary hormone may be corticotropin or cosyntropin. At least one parathyroid-like medication can be selected from calcifediol (human), calcium (salmon), and calcitriol. (See, e.g., pp. “Nursing 2001 Drug Handbook, pp. 696-796.
“The minimum one diuretic may be any of the following: acetazolamide, amiloride sodium, bumetanide and chlorthalidone; acetazolamide sodium; acetazolamide sodium; mannitol; metolazone; spironolactone; torsemide or triamterene. The at least one electrolyte or replacement solution can be at least one selected from calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, calcium lactate, calcium phosphate (dibasic), calcium phosphate (tribasic), dextran (high-molecular-weight), dextran (low-molecular-weight), hetastarch, magnesium chloride, magnesium sulfate, potassium acetate, potassium bicarbonate, potassium chloride, potassium gluconate, Ringer’s injection, Ringer’s injection (lactated), and sodium chloride. At least one acidifier/alkalinizer can be selected from sodium bicarbonate or sodium lactate. (See, e.g., pp. Nursing 2001 Drug Handbook, pp. 797-833.
“The minimum one hematinic may be any of the following: ferrous fumarate or ferrous glutarate, ferrous sulfurate, ferrous ferric gluconate, iron sorbil, ferrous iron gluconate (dried), ferrous ferroussulfate, iron sulfate(dried), ferrous ferric gluconate complex, and ferrous sulfate. At least one anticoagulant may be selected from ardeparin salt, dalteparin potassium, ferrous sulfate, ferrous sulfate (dried), iron dextran, iron sorbil, polysaccharide-iron complex, and sodium ferric gluconate complex. At least one blood derivative can include albumin 5% or 25%, antihemophilic factors, anti-inhibitor complex, antithrombin IX (human), factor IIX complex and plasma protein fractions. At least one thrombolytic agent can be selected from anistreplase or reteplase recombinant, streptokinase and urokinase. (See, e.g., pp. Nursing 2001 Drug Handbook, pp.834-66.
“Alkylating drugs can include busulfan (carboplatin), carmustine, chlorambucil and cisplatin), ifosfamides, lomustines, mechlorethamine Hydrochloride (melphalan), melphalan hydrochloride, streptozocin, or temozolomide. At least one antimetabolite may be capecitabine or cladribine, floxuridine and fludarabine. At least one antibiotic antineoplastic may be selected from bleomycin sulfate (dactinomycin), dactinomycin (dunorubicin citrate liquid liposomal), daunorubicin phosphate, doxorubicin hydrchloride liposomal), doxorubicin hyrosomal doxorubicin hydrchloride liposomal), epirubicin hydrochloride plicamycin, valrubicin, idamycin, velamycin, clon, tin, dis, dis, dis, dizin, dis, dis, dis, damycin, dis, dis, dis, dis, dis, dis, dis, izin, idis, izin, izin, izin, prakil, iz idis, izin, idis, idis, idis, idis, idamycin, idis, thiomycin, idamycin, cin, cin, cin, cin, cin, cin cin, cin cin cin cin cin cin cin cin cin cin cya, cin cin cin cin cin cin cin cin cin ida, cin cin cin cin cin cin cin cin cin, idarubicin cin cin cin cin cin cin cin cin cin, cin, cin, cin, cin,,,,,,,,,,,,,,,, methotrex,,,,,,,,,,, Anastrozole can be selected from bicalutamide and estramustinephosphate sodium. Asparaginase (BCG), (live intravesical), dacarbazine (BCG), (live intravesical), docetaxel (etoposide phosphate), gemcitabine hydrochloride), gemcitabine hydrochloride), mitotane (mitoxantrone phosphate), gemcitabine hydrochloride), mitotane (mitoxantrone hyoxantrone hydrchloride), mitotitan, vinblastine stine stine stine stine stine stine stine stine stine stine sine stine stine stine stine stine stine stine stine stine e stine stine stine stine stine stine stine stine stine striestine stine stine stine stine stine stine stine sulfate, vincristine stine stine sulfate and vinblastine stine stine sulfate and vincristine sulfate and vincristine sulfate and vincristine sulfate-sulfate and vinblastine sulfate and vincrisulfate (See, e.g., pp. “867-963 Nursing 2001 Drug Handbook.
At least one immunosuppressant may be any of the following: azathioprine (or basiliximab), cyclosporine (or daclizumab), lymphocyte immune globulin; muromonab CD3; mycophenolate Mofetil, mycophenolate hydrochloride; sirolimus. At least one vaccine or toxoid may be selected from BCG vaccine and cholera vaccine. At least one antitoxin/antivenin can be selected from Crotalidae antivenin (polyvalent), Diphtheria antivenom (equine), and amd Micrurus Fulvius antivenin. At least one immune serum may be selected from cytomegalovirus immuno globulin, intraveneous, hepatitis B immunity globulin, immuneglobulin intratoxin, immuneglobulin intermuscular, immuneglobulin intravenous, rabies vaccine, respiratory syncytial viral immuneglobulin, intravenous (human), Rh0 (D) immuneglobulin human), varicella-zoster immuneglobulin (human), or a) and varicella virus (human). At least one biological response modifier may be selected from aldesleukin (epoetin), filgrastim and glatiramer-acetate for injection. (See, e.g., pp. 964-1040 Nursing 2001 Drug Handbook.
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