Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• the present invention generally relates to ricin toxin.
• the present invention relates to ricin vaccines as well as methods of making and using thereof.
• Ricin is a very toxic protein obtained from the castor bean, Ricinus communis,
• Ricin is a heterodimer comprising an A chain and a B chain joined by a disulfide bond.
• Ricin A chain is an N-glycosidase enzyme that irreversibly damages a specific adenine base from 28S rRNA. Once the rRNA has been damaged, the cell cannot make protein and will inevitably die (cytotoxicity). As RTA exhibits this type of destructive catalytic activity, RTA is commonly referred to as a type II ribosome inactivating protein (RIP). See Lord, et al. (1991) Semin. Cell Biol. 2(l):15-22. RTA has been coupled with a targeting moiety to selectively destroy target cells such as tumor cells. See U.S. Pat.
• RTA Ricin B chain
• RTA binds the toxin to cell surface receptors and then RTA is transferred inside the cell where inhibition of ribosome activity occurs.
• the human lethal dose of ricin toxin is about 1 ⁇ g/kg.
• highly purified ricin is commercially available, the use of ricin toxin in biological warfare and terrorism is highly possible and probable.
• Ricin vaccines have been prepared by isolating the natural toxin from castor beans, and treating the toxin with harsh chemicals, such as typically formaldehyde, to reduce the toxic activity. See Hewetson, et al. (1993) Vaccine ll(7):743-746; Griffiths, et al. (1995) Hum. Exp. Toxicol. 14(2):155-164; Griffiths, et al. (1999) Vaccine 17(20- 21):2562-2568; and Yan, et al. (1996) Vaccine 14(11):1031-1038. These first generation vaccines are called "toxoid" vaccines as they are made directly from natural toxin itself.
• the current toxoid vaccine suffers from several important limitations that include: (1) the presence of both RTA and RTB; (2) the presence of trace amounts of active RTA and RTB; (3) the possibility that denatured toxoid could refold and, thereby, revert to the active form; (4) side-effects that arise from the presence of the harsh chemicals used in the vaccine formulation; (5) heterogeneity in the final vaccine product arising from the process of formaldehyde treatment; and (6) heterogeneity in the final vaccine resulting from the natural structural variations of ricin protein found in castor beans. See Despeyroux, et al. (2000) Anal. Biochem. 279(l):23-36.
• the second generation ricin vaccines comprise wild-type (wt) RTA, but not RTB.
• dgRTA deglycosylated RTA
• rRTA wt recombinant RTA
• the third generation ricin vaccines are based on the active site of RTA that comprises the amino acid residues that interact directly with, or are within about five angstroms from, the bound ribosomal RNA substrate.
• mutant substitution RTA vaccines are based on recombinant DNA technology and substitute amino acids of wt RTA in order to reduce N-glycosidase-rRNA activity. See Ready, et al. (1991) Proteins 10(3):270-278; Kim, et al. (1992) Biochem. 31:3294-3296; Roberts, et al. (1992) Targeted Diag. Ther. 7:81-97; Frankel, et al. (1989) Mol. Cell. Biol. 9(2):415- 420; and Gould, et al. (1991) Mol. Gen. Genet. 230(l-2):81-90.
• RTA El 77 A The x-ray crystallographic structure of RTA El 77 A demonstrates a remarkable rescue of electrostatic balance in the active site, achieved by the rotation of a proximal glutamic acid into the vacated space. See Kim, et al. (1992) supra. Despite the non-conservative substitution, the free energy of denaturation for RTA Bill A was anticipated from modeling studies to be two-fold more favorable than the conservative replacement of mutant substitution RTA E177Q See Olson (2001) supra, h terms of expression levels, RTA E177Q is far less well behaved than wt RTA. See Ready, et al. (1991) supra.
• RTA El 77 A and RTA E177Q remain active enzymes, thereby indicating plasticity in obtaining the catalytic transition-state. See Schlossman, et al. (1989) Mol. Cell. Biol. 9(11):5012-5021; and Ready, et al. (1991) supra..
• the present invention generally relates to polypeptides related to ricin toxin A chain.
• the present invention relates to an isolated polypeptide or variant thereof comprising a polypeptide sequence having substantial identity to a wild type ricin A chain first globular domain sequence and which lacks detectable N- glycosidase-rRNA activity or exhibits reduced N-glycosidase-rRNA activity as compared to a control.
• the polypeptide may retain the functional integrity of the neutralizing immunological epitope of wild type ricin A chain, h some preferred embodiments, the polypeptide has a solubility that is greater than the solubility of wild type ricin A chain in aqueous solutions of physiological pH and ionic strength.
• the wild type ricin A chain first globular domain sequence may be SEQ ID NO:2 or a variant thereof and the polypeptide of the present invention may comprise SEQ ID NO:3, SEQ ID NO:4, or variants thereof.
• the polypeptide of the present invention may be substantially identical to SEQ ID NO:3 or SEQ ID NO:4.
• the polypeptide of the present invention lacks a hydrophobic loop that corresponds to the hydrophobic loop of wild type ricin A chain.
• the polypeptide of the present invention may comprise at least one amino acid mutation, substitution, deletion, or a combination thereof.
• polypeptides of the present invention may be made by recombinant DNA techniques or by proteo lyrically cleaving the first globular domain and the second globular domain of ricin A chain and then purifying the first globular domain.
• the present invention relates to isolated polynucleotides that encode the polypeptides or variants of the present invention.
• the present invention relates to antibodies raised against the polypeptides or variants of the present invention.
• the antibody is a neutralizing antibody that is capable of neutralizing ricin, ricin A chain, or both.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least one polypeptide or variant of the present invention, or at least one polynucleotide of the present invention or at least one antibody of the present invention a pharmaceutically acceptable vehicle.
• the pharmaceutical composition may further comprise an adjuvant.
• the pharmaceutical composition may be capable of eliciting an immune response when administered to a subject.
• the immune response may be a protective immune response against ricin intoxication.
• the present invention also relates to vaccines comprising an immunogenic amount of at least one polypeptide or variant of the present invention.
• the present invention relates to a method of inducing an immune response in a subject which comprises administering to the subject at least one immunogenic amount of the polypeptide or variant of the present invention.
• the subject is mammalian, more preferably, the subject is human.
• the method may further comprise administering to the subject at least one booster dose.
• the present invention relates to a method of providing passive immunity against ricin intoxication in a subject comprising administering to the subject a therapeutically effective amount of at least one antibody raised against the polypeptides or variants of the present invention.
• the present invention relates to a method of treating or preventing ricin intoxication in a subject comprising administering to the subject an immunogenic amount of the polypeptide or variant of the present invention, or administering to the subject a therapeutically effective amount of an antibody raised against the polypeptide or variant of the present invention.
• kits comprising the polypeptides or variants of the present invention, or antibodies raised against the polypeptides or variants of the present invention, or polynucleotides of the present invention packaged together with instructions for use.
• the kits may further comprise diagnostic reagents such as labeling compounds for detecting the presence of ricin toxin or for diagnosing exposure of a subject to ricin.
• the kits may comprise drug delivery devices for administering the compositions of the present invention to a subject.
• Figure 1 is a ribbon diagram of wt RTA.
• Figure 2 is an SDS-PAGE that compares the heterogeneity of dgRTA, wt RTA, and RTA198 and RTA 1-33/44-198.
• Figure 3 is an SDS-PAGE that exhibits the consistency and reproducibility of making the polypeptides of the present invention as exemplified with RTA1-33/44-198.
• the present invention provides polypeptides derived from native or wt RTA amino acid sequences, such as the amino acid sequence set forth in SEQ ED NO: 1 (SwissProt accession no. P02879), for use in compositions and methods for treating or preventing ricin intoxication in a subject.
• the structure of RTA comprises two globular domains according to a widely accepted method of evaluating protein secondary structure elements and packing called PDBSum, a web-based database of summaries and analyses of all PDB structures. See Laskowski R A, et al. (1997) Trends Biochem. Sci. 22:488-490.
• the first globular domain of RTA comprises amino acid residues from about 1 to about 179 (counting from the amino terminus) of native RTA which is set forth in SEQ ID NO:2.
• the second globular domain of RTA comprises amino acid residues from about position 180 to about 267 of native RTA.
• the second globular domain of RTA is the part that mainly interfaces with RTB.
• the neutralizing immunogenic epitope, section 2 is believed to be the amino acid residues from about position 95 to about 110 of native RTA. See Aboud-Pirak, et al. (1993) Med. Def. Biosci. Rev., Baltimore, MD, U.S. Army Medical Research and Materiel Command, which is herein incorporated by reference. Based on structural analysis and protein chemistry, the first globular domain was determined to relate to immunogenicity and the second globular domain was determined to include the hydrophobic interfacial region that interacts directly with RTB.
• the aqueous solubility of native RTA was determined to be limited by the absence of RTB. Moreover, major structural changes were identified that could be made in native RTA to overcome limited solubility by optimizing solute-solvent interactions.
• the significant changes described herein were surprisingly successful as significant changes to a polypeptide sequence usually results in an unfolded protein and, therefore, loss of utility as a biological therapeutic.
• the engineered polypeptides do not retain any natural catalytic activity of RTA. Additionally, the engineered polypeptides retained the favorable thermodynamic equilibrium between folded and unfolded RTA. Most surprisingly, the functional integrity of the neutralizing immuno logical epitope of RTA was retained in the engineered polypeptides.
• the present invention provides polypeptides that: (1) do not retain any natural catalytic activity of RTA; (2) retain the favorable thermodynamic equilibrium between folded and unfolded RTA; (3) retain the functional integrity of the neutralizing immunological epitope of RTA; or (4) a combination thereof.
• RTA provided polypeptides having reduced surface hydrophobicity (increased solubility in aqueous solutions of physiological pH and ionic strength) or a reduced ability to form aggregates as well as reduced N-glycosidase-rRNA activity, if any, as compared to wt RTA.
• the engineered polypeptides that lack most of the second globular domain of RTA. Specifically, the second globular domain was deleted at position 198 of RTA since the second residue following the cleavage site is a proline (residue 200), and this residue was thought to be restrictive in the torsional backbone of the deleted domain.
• polypeptides of the present invention comprise at least one amino acid mutation, substitution, deletion, or a combination thereof, in the linker region of RTA.
• the polypeptides of the present invention comprise an amino acid sequence that has substantial identity to amino acid residues from about position 1 to about 210, preferably about 1 to about 202 of wt RTA, more preferably about 1 to about 198.
• the polypeptides of the present invention comprise an amino acid sequence that has a substantial identity to amino acid residues from position 1 to 198 of wt RTA.
• amino acid residues in the first globular domain that do not change its overall stability and do not change the neutralizing immunogenic epitope of wt RTA may be removed. Specifically, deletion of amino acid residues between positions 33 and 44 of wt RTA still provided a stable polypeptide that retained immunogenicity.
• polypeptides of the present invention comprise at least one amino acid mutation, substitution, deletion, or a combination thereof, in the linker region and in the first globular domain of wt RTA.
• the polypeptides of the present invention comprise an amino acid sequence that has substantial identity to amino acid residues from position 1 to about 210, preferably about 1 to about 200, more preferably about 1 to about 198 of wt RTA and include at least one amino acid mutation, substitution, deletion, or a combination thereof, in the first globular domain, preferably in the loop region, more preferably from about position 33 to about 44 of wt RTA.
• the amino acid mutation, substitution, deletion, or a combination thereof is a deletion of amino acid residues from about position 34 to about 43 of wt RTA.
• the polypeptides of the present invention are stable, non-toxic, and capable of eliciting an immune response in a subject.
• the polypeptides of the present invention may be used to prevent or treat systemic side effects of locally administered ricin toxin.
• the polypeptides of the present invention are capable providing a protective immune response in a subject.
• the subject is mammalian, more preferably, the subject is human.
• an "immune response" refers to a humoral or cellular response caused by exposure to an antigenic substance.
• an immune response against ricin or "ricin immune response” refers to a humoral or cellular response in a subject that is caused by exposing the subject to an antigenic substance such as polypeptides of the present invention.
• a "protective immune response” against ricin refers to humoral immune responses, cellular immune responses, or both, that are sufficient to inhibit or prevent ricin intoxication in a subject.
• polypeptides of the present invention comprise at least one amino acid mutation, substitution, deletion, or a combination thereof, in the linker region of the second globular domain of LTA such as the following:
• RTA198 (designated from the protein amino terminus to the protein carboxyl terminus):
• RTA1-33/44-198 (designated from the protein amino terminus to the protein carboxyl-terminus)
• the bold type face of SEQ ID NO:4 indicates the position where the
• polypeptides of the present invention need not be identical to those exemplified herein so long as the subject polypeptides are able to induce an immune response against RTA, ricin, or both.
• variants of the polypeptides of the present invention refer to polypeptides having insignificant changes such as a methionine as the first amino acid residue at the amino terminus, conservative amino acid substitutions, deletion of or insertion of up to about 10 amino acid residues in the linker or loop region of wt RTA, and co-translational or post-translational surface modifications such as the addition of covalently attached sugars or lipids.
• polypeptides of the present invention may also be modified to provide a variety of desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the immunological activity of the RTA.
• desired attributes e.g., improved pharmacological characteristics
• polypeptides having mutated linker regions By using conventional methods in the art, one of ordinary skill will be readily able to make a variety of polypeptides having mutated linker regions and then screen the polypeptides for stability, toxicity, and immunogenicity according to the present invention.
• amino acid substitutions can be used to determine which residues are relatively insensitive to modification.
• Amino acid substitutions are preferably made between relatively neutral moieties, such as alanine, glycine, proline, and the like.
• Substitutions with different amino acids, of either D or L isomeric forms, or amino acid mimetics can be made.
• the number and types of substitutions, deletions, and insertions depend on the functional attributes that are sought such as hydrophobicity, immunogenicity, three-dimensional structure, and the like.
• amino acid mimetic refers to a moiety other than a naturally occurring amino acid residue that conformationally and functionally serves as a suitable substitute for an amino acid residue in a polypeptide of the present invention.
• a moiety is a suitable substitute for an amino acid residue if it does not interfere with the ability of the peptide to elicit an immune response against ricin.
• amino acid mimetics include cyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine, adamantylacetic acid, and the like. See e.g. Morgan and Gainor, (1989) Ann. Repts. Med. Chem. 24:243-252.
• a peptide bond mimetic include peptide backbone modifications of the amide nitrogen, the ⁇ -carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See e.g. Spatola (1983) CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPT ⁇ DES AND PROTEINS, Vol. N ⁇ , Weinstein ed.
• the polypeptides of the present invention may include an additional methionine as the first amino acid residue on the protein amino terminus.
• the polypeptides may be truncated by up to about ten (10) amino acid residues from the carboxyl terminus of RTA198, RTA1-33/44-198, or the like. Similarly, up to about ten (10) amino acid residues from wt RTA bordering the hydrophobic loop, amino acid residues at about position 34 to about 43 may be deleted. Additionally, co-translational or post-translational surface modifications, such as the addition of covalently attached sugars or lipids, may be made to the polypeptides of the present invention.
• sequence identity means that two sequences are identical over a window of comparison. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the structural models and conclusions deduced for RTA may be applied to the solved three-dimensional structure of related proteins, such as other RTPs. This could be done by a structure-structure alignment that minimizes the root-mean-square-deviation of atomic coordinates among the aligned structure (for example, optimal superposition of the position of all atoms, or the protein backbone atoms or the alpha-carbon atoms).
• a structure-structure alignment that minimizes the root-mean-square-deviation of atomic coordinates among the aligned structure (for example, optimal superposition of the position of all atoms, or the protein backbone atoms or the alpha-carbon atoms).
• Such methods are now largely automated, see for example, the homology module of Insight ⁇ (Accelrys, e, San Diego, CA); the Combinatorial Extension (CE) of the optimal path method (Shindyalov and Bourne (1998) Protein Engineering 11.
• the amino acid residues in corresponding positions among aligned structures are identical or differ only by "conservative amino acid substitutions".
• a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
• Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
• basic side chains
• the polypeptides of the present invention may be made by conventional methods known in the art.
• the polypeptides of the present invention may be manually or synthetically synthesized using conventional methods and devices known in the art. See e.g., Stewart and Young (1984) SOLID PHASE PEPTIDE SYNTHESIS, 2 ed. Pierce, Rockford, EL, which is herein incorporated by reference.
• Ricin or RTA may be obtained by conventional methods. See U.S. Patent No. 5,547,867, which is herein incorporated by reference.
• the ricin or RTA may then be exposed to proteolytic enzymes that cleave the amino acid residues to obtain polypeptides of the present invention.
• Ricin and RTA may be purified from natural sources using conventional protein purification techniques such as reverse phase high-performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis.
• HPLC reverse phase high-performance liquid chromatography
• ion-exchange or immunoaffinity chromatography filtration or size exclusion
• electrophoresis electrophoresis. See Olsnes, S. and A. Pihl (1973) Biochem. 12(16):3121-3126; and see e.g., Scopes (1982) PROTEIN PURIFICATION, Springer- Verlag, NY, which are herein incorporated by reference.
• the polypeptides of the present invention are substantially purified.
• a “substantially purified” compound refers to a compound that is removed from its natural environment and is at least about 60% free, preferably about 75% free, and most preferably about 90% free from other macromolecular components with which the compound is naturally associated.
• the polypeptides of the present invention may be made by conventional recombinant DNA techniques such as those disclosed in the Examples below.
• the present invention provides polynucleotides that encode the polypeptides of the present invention.
• the polynucleotides are isolated.
• isolated polynucleotides refers to polynucleotides that are in an environment different from that in which the polynucleotide naturally occurs.
• SEQ ED NO:2 or SEQ ED NO:3 can be made by introducing one or more nucleotide substitutions, insertions, or deletions into the nucleotide sequence that encodes SEQ ED NO:l, SEQ ED NO:2, or SEQ ED NO:3 such that one or more amino acid substitutions, insertions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• a polynucleotide encoding a polypeptide of the present invention is then inserted in to a vector such as a cloning vector or an expression vector.
• An expression vector allows the polypeptide to be expressed when present in a host.
• Either the expression vector or the host may comprise the regulatory sequences necessary for expression of the polypeptide.
• the regulatory sequences are within the expression vector, the regulatory sequences are operatively linked to the sequence encoding the polypeptide.
• "operably linked” means that the nucleotide sequence of interest is linked to at least one regulatory sequence in a manner that allows the polypeptide to be expressed in an in vitro transcription/translation system or in a host cell.
• Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). See e.g., Goeddel (1990) GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY, Academic Press, San Diego, CA, which is herein incorporated by reference.
• the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the desired expression levels of the polypeptide, the compatibility of the host cell and the expressed polypeptide, and the like.
• the vectors can be designed for expressing the polypeptides of the present invention of in prokaryotic or eukaryotic host cells such as bacterial cells, insect cells, plant cells, yeast cells, or mammalian cells.
• the host cells are bacterial cells. Suitable host cells are discussed further in Goeddel supra; Baldari, et al. (1987) EMBO J. 6:229-234; Kurjan and Herskowitz (1982) Cell 30:933-943; Schultz, et al. (1987) Gene 54:113-123; Smith, et al. (1983) Mol. Cell Biol.
• the present invention also provides host cells comprising polynucleotides that encode the polypeptides of the present invention.
• Host cells include the progeny or potential progeny of the primary cell in which the polynucleotide was introduced. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope and meaning of host cell.
• a polypeptide of the present invention may be used to prepare antibodies against ricin by immunizing a suitable subject, e.g., rabbit, goat, mouse or other mammal with the polypeptide by conventional methods known in the art. Large quantities of neutralizing antibodies could be generated and then used as an antidote for ricin intoxication. See Lemley, et al. (1994) Hybridoma 13(5):417-427 and U.S. Patent No. 5,626,844, which are herein incorporated by reference.
• the antibodies raised against the polypeptides of the present invention may be used to prevent or treat systemic side effects of locally administered ricin toxin.
• the present invention also provides antibodies that are raised against or derived from the polypeptides of the present invention, and methods of using thereof.
• Antibodies of the present invention may be produced by conventional methods known in the art. See e.g., Coligan (1991) CURRENT PROTOCOLS IN IMMUNOLOGY. Wiley/Greene, NY; and Harlow and Lane (1989) ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press, NY; Stites, et al. (1986) BASIC AND CLINICAL IMMUNOLOGY. 4th ed. Lange Medical Publications, Los Altos, CA; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE. 2d ed.
• Therapeutic antibodies may be produced specifically for clinical use in humans by conventional methods known in the art. See Chadd, H.E. and S.M. Chamow (2001) Curr. Opin. Biotechnol. 12:188-194 and references therein, all of which are herein incorporated by reference.
• the present invention has the advantage of allowing safe exposure of subjects, such as humans, to the RTA neutralizing epitope. Thus, the present invention allows for the safe in vivo production of RTA antibodies directly subjects.
• antibody refers to immunoglobulin molecules and immunologically active portions that comprise an antigen binding site which specifically binds an antigen, such as ricin.
• immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which may be generated by treating the antibody with an enzyme such as pepsin.
• Polyclonal and monoclonal antibodies against the polypeptides of the present invention may be made by conventional methods known in the art.
• the polypeptides, polynucleotides, or antibodies of the present invention may be administered, preferably in the form of pharmaceutical compositions, to a subject.
• the subject is mammalian, more preferably, the subject is human.
• Preferred pharmaceutical compositions are those comprising at least one immunogenic composition against ricin, RTA, or both, in an immunogenic amount or a therapeutically effective amount, and a pharmaceutically acceptable vehicle.
• the immunogenic composition may be an active immunizing agent, such as a polypeptide of the present invention, or a passive immunizing agent, such as an antibody raised against the polypeptide of the present invention.
• the immunogenic composition may elicit an immune response that need not be protective or the immunogenic composition may provide passive immunity.
• a vaccine elicits a local or systemic immune response that is protective against subsequent challenge by the immunizing agent such as the polypeptides of the present invention, or an immunologically cross-reactive agent, such as ricin.
• the immunizing agent such as the polypeptides of the present invention, or an immunologically cross-reactive agent, such as ricin.
• an "immunogenic composition” can refer to vaccines as well as antibodies.
• a protective immune response may be complete or partial, i.e. a reduction in symptoms as compared with an unvaccinated mammal.
• the present invention provides immunogenic compositions comprising the polypeptides or the antibodies of the present invention that may be used to immunize a subject against ricin by methods known in the art. See U.S. Patent No. 5,453,271, which is herein incorporated by reference.
• an "immunogenic amount” is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the polypeptide, the manner of administration, the general state of health of the subject, and the like.
• the typical immunogenic amounts for initial and boosting immunization for therapeutic or prophylactic administration ranges from about 0.01 mg to about 0.1 mg per about 65-70 kg body weight of a subject.
• the typical immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject ranges from about 0.01 mg to about 0.1 mg.
• suitable immunization protocols include initial immunization injections at time 0 and 4 or initial immunization injections at 0, 4, and 8 weeks, which initial immunization injections may be followed by further booster injections at 1 or 2 years.
• a "therapeutically effective amount” refers to an amount of a polypeptide, a polynucleotide, or an antibody that may be used to treat, prevent, or inhibit ricin intoxication in a subject as compared to a control. Again, the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including the severity of ricin exposure, previous treatments, the general health and age of the subject, and the like. A therapeutically effective amount may be readily determined by conventional methods known in the art.
• treatment of a subject with a therapeutically effective amount of a polypeptide, a polynucleotide, or an antibody of the present invention can include a single treatment or, preferably, can include a series of treatments.
• the pharmaceutical compositions may include an adjuvant. As used herein, an
• adjuvant refers to any substance which, when administered with or before the polypeptide, polynucleotide, or antibody of the present invention, aids the polypeptide, polynucleotide, or antibody in its mechanism of action.
• an adjuvant in a vaccine is a substance that aids the immunogenic composition in eliciting an immune response.
• Suitable adjuvants include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N-acetylmuramyl- Lalanyl-D-isoglutaminyl-L-alanine-2-( -2'-dipa-lmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (CGP 19835 A, MTP-PE), and REBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
• compositions of the present invention may be administered to a subject by any suitable route including oral, transdermal, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical fonnulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular polypeptide, polynucleotide, or antibody used.
• a "pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.
• Pharmaceutically acceptable vehicles include those known in the art. See e.g. REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY. 20 th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is herein incorporated by reference.
• compositions of the present invention may be provided in dosage unit forms.
• dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, one may determine the lethal dose of toxin, LCt 5 o (the dose expressed as concentration of toxin x exposure time that is lethal to 50% of the population) or the LD 5 o (the dose lethal to 50% of the population), and the ED 5 o (the dose therapeutically effective in 50% of the population) by conventional methods in the art.
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
• Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 5 o with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 5 o (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
• IC 5 o i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
• levels in plasma may be measured, for example, by high performance liquid chromatography.
• kits comprising a pharmaceutical composition may include the pharmaceutical composition as a single dose or multiple doses.
• the kit may include a device for delivering the pharmaceutical composition.
• the device may be a multi-chambered syringe for intramuscular delivery, a microneedle or set of microneedle arrays for transdermal delivery, a small balloon for intranasal delivery, or a small aerosol generating device for delivery by inhalation.
• Ricin exposure is presently detected by medical history and symptoms, and is confirmed by antibody- or activity-based measurements of ricin in bodily fluids.
• Ricin detection or medical diagnosis of ricin exposure may based upon immunoassays utilizing the antibodies or polypeptides of the present invention or combinations thereof. Since a subject may be safely exposed to the polypeptides of the present invention, exposure to ricin may be determined by detecting RTA antibodies in a subject in vivo. Additionally, as the polypeptides of the present invention are relatively non-toxic and safe, immunoassays utilizing the polypeptides of the present invention for detecting antibodies against ricin would be also be safe. Thus, the present invention provides diagnostic assays for detecting ricin toxin, exposure to ricin, or both.
• the diagnostic assays may comprise the polypeptides of the present invention, antibodies of the present invention, or a combination thereof.
• the diagnostic assays may be provided in the form of kits that may be used outside of a laboratory setting, such as in the field.
• the polypeptides of the present invention are fundamentally superior as immunogenic compositions such as ricin vaccines as compared ricin toxoid, wt RTAs, dgRTAs, and mutant substitution RTAs as the polypeptides do not include RTB, and lack detectable N-glycosidase-rRNA activity or exhibit reduced N-glycosidase- rRNA activity as compared to controls.
• detectable N-glycosidase- rRNA activity refers to N-glycosidase-rRNA activity that may be detected by assays conventional in the art. See Hale (2001) Pharmacol. & Toxicol.
• polypeptides of the present invention that lack detectable N-glycosidase-rRNA activity refers to polypeptides that do not exhibit ribosome inactivating activity at concentrations of less than about 0.5 nM, preferably, less than about 0.1 nM.
• the polypeptides of the present invention have an aqueous solubility that is greater than the aqueous solubility of wt RTA as evidenced by high expression yields in the soluble fraction and by the absence of protein aggregation or precipitation upon storage in physiological saline solutions.
• a direct comparison of dgRTA and polypeptides of the present invention was made using standard methods of SDS-PAGE and isoelectric focusing. Each method showed evidence of higher molecular weight species, indicative of aggregation, in the dgRTA samples that were absent from samples of the polypeptides of this invention.
• polypeptides of the present invention are more homogenous than dgRTA as the polypeptides may be consistently expressed and substantially purified as explained in Examples 1 and 2 and evidenced by the SDS-PAGEs in Figures 2 and 3. [76] The following Examples are intended to illustrate, but not to limit the present invention.
• a stop codon and a Sal I restriction endonuclease cleavage site were introduced by use of the following primer: 5' GTC GAC CTA GGA TCT ACG GTT GTA TCT AAT TC 3 ' (SEQ ED NO: 13).
• the reaction mixture comprising RTA DNA template, PCR primer pairs, pfu DNA polymerase and dNTP, was subjected to PCR as outlined in the commercial kit (Stratagene, La JoUa, CA). Template DNA is then eliminated by incubating at 37 °C for 1 hour with Dpn I restriction endonuclease. A 1 ⁇ l sample of the PCR mixture containing the mutated polynucleotide was used to transform E.
• coli XLl-Blue competent E. coli cells (Stratagene, La Jolla, CA). Mutagenesis was conducted under contract by Clinical Research Management (Frederick, MD). The resultant mutated polynucleotide was purified using a commercial Miniprep kit (Qiagen, Valencia, CA) and sequenced using conventional methods to confirm that only the desired changes were made. [79] The DNA products were purified, ligated to a commercial pET-24 expression vector based upon the T7 promoter system of Studier, et al. (1990) Methods Enzymol. 185:60-89, which is herein incorporated by reference, and then used to transform competent E. coli BL21 DE3 cells (Invitrogen, CA). Plasmid DNA was purified by conventional methods in the art.
• the transformed E. coli BL 21 cells were cultured in Terrific Broth (TB; a standard bacterial cell culture medium comprising 12 g/L tryptone, 24 g/L yeast extract, 0.4% (v/v) glycerol, 2.31 g/L KH 2 PO 4 , and 12.54 g/L K 2 HPO 4 ) containing 50 ⁇ g/ml of kanamycin until a cell density of 0.4 to 0.6 OD 6 oo was reached.
• Expression of the RTA198 polynucleotide was induced by adding 1 mM isopropyl- ⁇ -D- thiogalactopyranoside (EPTG) at 25°C, for about 18 to about 20 hours.
• the cells were centrifuged in a Sorvall GS3 rotor at 6,000 rpm for 15 minutes at 4 °C. The cell media was decanted and discarded. The pellet containing E. coli cells were bathed in TB medium with kanamycin and EPTG to create a cell paste that was frozen at -20 °C until further use. Generally, the frozen cell paste was stored for at least about 24 hours prior to protein purification.
• E. coli cells were analyzed using polyclonal antiserum specific for RTA and standard methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.
• the antiserum was affinity purified previously using dgRTA by conventional methods in the art. Briefly, wet E. coli cells were solubilized in 50 mM sodium phosphate buffer, pH 7.3, and centrifuged to give a soluble fraction (supernatant) and a pellet. The pellet was then solubilized by boiling in 6M urea, pH 7. RTA198 was detected by Western blot analysis in both the phosphate buffer soluble fraction, and in the phosphate buffer insoluble fraction.
• FIG. 2 is an SDS-PAGE result wherein lane 1 shows the molecular weight standards.
• BCA bicinchoninic acid assay
• the gel is 10% (w/v) total acrylamide stained with Coomassie blue.
• Lane 2 is dgRTA
• lane 3 is purified recombinant wt RTA
• lane 4 is RTA198. As shown, the RTA198 produced a clear dark band thereby indicating a high degree of homogeneity.
• the purified RTA198 did not possess detectable levels of N-glycosidase activity as evidenced by the inability to disrupt protein synthesis in a cell-free, translation assay under conditions where either natural ricin (positive control) or isolated wt RTA (positive control) did show ribosome inactivation. See Langer, et al. (1996) Anal. Biochem. 243(1):150-153; and Hale (2001) Pharmacol. Toxicol. 88(5):255-260, which are herein incorporated by reference.
• Toxicol. 88(5):255-260 This assay takes advantage of the fact that the N-glycosidase activity of RTA can be monitored with an in vitro translation assay using the rabbit reticulocyte lysate system. See Barbieri et al. (1989) Biochem. J. 257:801-807, which is herein incorporated by reference.
• the assay measures the in vitro translation of the enzyme luciferase, as determined by the fluorescence produced when luciferase reacts with the substrate luciferin.
• a translation lysate was prepared at 4 °C that contained nuclease-treated rabbit reticulocyte lysate, amino acid mixture, Rnasin ribonuclease inhibitor, nuclease-treated deionized water, and luciferase mRNA in a ratio of (v/v): 35:1:1:35:1, respectively (all available from Promega, Madison, Wl).
• mice in each group were injected i.m. with 0.1-ml at the following concentration of test/control articles: a. Group 1: 20 mice injected with 10 ⁇ g of RTA 198 protein. b. Group 2: 10 mice injected with 10 ⁇ g of RTA198 protein + 0.2% alhydrogel. c. Group 3: 20 mice injected with 10 ⁇ g of dgRTA protein. d. Group 4: 20 mice injected with phosphate buffered saline.
• mice in treatment groups 1, 3, & 4 and 10 mice in treatment group 2 were anesthetized and 0.2 to about 0.3 ml of blood was collected by the periorbital sinus method and recorded. The blood was later used to measure the specific antibody concentrations and ricin neutralizing antibody titers.
• mice were weighed and ten mice from treatment groups 1, 3, & 4 were injected intraperitoneally on body weight bases with 0.1 ml of a solution that contained 10 mouse LD 50 of ricin toxin D. The remaining 10 mice from each treatment group were exposed over ten minutes in a dynamic system to a liquid aerosol that supplied 5 to 10 mouse LD 50 of ricin toxin D. After exposure to ricin, daily cage side observations were made for survival rates.
• RTA198 may be used to elicit an immune response in a subject and may be used as a safe and effective ricin vaccine.
• ricin stock solution (5 mg/ml) (Vector Laboratories, h e, Burlington, CA) was diluted 1 : 1000 in ELISA coating buffer and 100 ⁇ l was added to each well of a plate. The plate was stored at about 12 to about 48 hours at about 4 °C. Seven concentrations of mouse anti-ricin serum (Perimmune Inc., Rockville, MD) were prepared and standardized to provide a suitable standard curve for the ELISA. Positive and negative mouse anti- ricin serum controls were prepared and used. Fresh dilutions of unknown sera were prepared by adding 12.5 ⁇ l of serum to 987.5 ⁇ l of MASP buffer.
• Optimal sera dilutions may be determined by conventional methods in the art.
• the plate was washed 3 times with 0.2 ml/well of ELISA wash buffer. To each well, 200 ⁇ l of 5% skim milk buffer was added. The plate was covered and incubated in a moist chamber at about 37 °C for 1 hour. The plate was then washed 3 times with 0.2 ml/well ELISA wash buffer. Then serial dilutions of the unknowns and controls and standards were added to the wells. The plate was incubated at about 37 °C for about 1 hour. The plate was then washed 3 times with 0.2 ml/well ELISA wash buffer.
• DNA Cloning, Sequencing, and Expression of RTA1 -33/44-198 [99] DNA cloning, sequencing, and expression similar to that disclosed in Example 1 were used. Briefly, mutagenesis was done using the QuickChange kit commercially available from Stratagene (La JoUa, CA) based upon the polymerase chain reaction (PCR) method. Starting with the RTA198 construct described in Example 1, a pair of PCR oligonucleotide primers selected to delete 10 amino acids at position 34-43 were used. The sense PCR oligonucleotide used was: 5' CTG TCA GAG GTA GAT TGA CTG TCT TGC CTA ACA GAG TTG G 3' (SEQ LD NO: 14).
• the antisense PCR oligonucleotide used was: 5' CCA ACT CTG TTA GGC AAG ACA GTC AAT CTA CCT CTG ACA G 3' (SEQ ED NO:15). Mutagenesis was conducted under contract by Clinical Research Management (Frederick, MD). [100] After eliminating template DNA, 1 ⁇ l sample of the PCR mixture containing the mutated polynucleotide is used to transform E. coli XLl-Blue competent E. coli cells (Stratagene). The resultant mutated polynucleotide was purified using a commercial Miniprep kit (Qiagen, Valencia, CA) and sequenced using conventional methods to confirm that only the desired changes were made.
• polynucleotide products were purified, ligated to a commercial expression vector based upon the T7 promoter system (Studier, et al. (1990) supra), and then used to transform competent E. coli BL21 DE3 strain (h vitrogen, CA).
• the transformed E. coli BL 21 cells were cultured in TB media comprising 50 ⁇ g/ml of kanamycin until a cell density of 0.4 to 0.6 OD 6 oo was reached.
• Expression of the RTA1 -33/44- 198 polynucleotide was induced using 1 mM EPTG at 25 °C, for about 18 to about 20 hours.
• the cells were centrifuged in a Sorvall GS3 rotor at 6,000 rpm for 15 minutes at 4°C.
• the cell media was decanted and discarded.
• the pellet containing E. coli cells were bathed in TB medium with kanamycin and EPTG to create a cell paste that was frozen at -20°C until further use. Generally, the frozen cell paste was stored for at least about 24 hours prior to protein purification.
• the Mono-Q® 10/10 flow-through was pooled and the buffer was changed by dialysis to 50 mM MES, 2 mM EDTA, pH 6.4.
• the material was syringe filtered (0.2 ⁇ m pore size), and loaded onto an equilibrated Pharmacia Mono-S® 5/5 cation exchange column (50 mM MES, 2 mM EDTA, pH 6.4). Protein was eluted from the column using a NaCl gradient (0-500 mM NaCl in 30 minutes).
• Purified RTAl-33/44-198 began to elute by 20 mM and was completely eluted by about 150 mM NaCl. All fractions showing protein by OD 280 absorbance were analyzed by reducing SDS-PAGE.
• RTAl-33/44-198 as judged by Coomassie blue stained gels, were pooled.
• the buffer was changed to a buffer solution comprising 120 mM NaCl, 2.7 mM KCl, 10 mM NaPO , pH 7.4, by extensive dialysis (2L x 3 changes of greater than about 3 hours at room temperature).
• Purified RTAl-33/44-198 was filtered (0.2 ⁇ m pore size) and stored sterile at 4°C prior to animal studies. Total protein concentrations were determined using the Pierce micro-BCA assay, relative to a bovine serum albumin standard curve. Purity and consistency of lots of RTAl-33/44-198 obtained by this method is demonstrated in Figure 3.
• Figure 3 is an SDS-PAGE result wherein lane 1 shows the molecular weight standards. Lanes 2-5 show about 2 ⁇ g of RTAl-33/44-198 from four different lots that were expressed and purified independent of each other. The gel is 10% (w/v) total acrylamide stained with Coomassie blue. As shown, the each lane produced a clear dark band, which bands were consistently similar to each other. Therefore, the production and purification of the polypeptides of the present invention are reproducible and suitable for vaccine production. [107] To determine whether RTA1 -33/44- 198 exhibited N-glycosidase activity, the assay according to Example ID above was conducted.
• RTAl-33/44-198 When RTAl-33/44-198 was administered, purified RTAl-33/44-198 effectively protected animals from a challenge with about 5 to about 10 LD 5 o of ricin by either intraperitoneal injection, or by whole-body aerosol delivery of ricin toxin.
• mice in each group were injected i.m. with 0.1 -ml at the following concentration of test/control articles: a. Group 1 : 20 mice injected with 10 ⁇ g of RTAl-33/44-198 protein. b. Group 2: 20 mice injected with 10 ⁇ g of RTAl-33/44-198 protein + 0.2% alhydrogel. c. Group 3: 20 mice injected with 10 ⁇ g of dgRTA protein. d. Group 4: 20 mice injected with phosphate buffered saline.
• mice were anesthetized and 0.2 to about 0.3 ml of blood was collected by the periorbital sinus method and recorded. The blood was later used to measure the specific antibody concentrations and ricin neutralizing antibody titers.
• mice One week after blood collection, same mice were weighed and ten mice from each treatment group were injected intraperitoneally on body weight bases with 0.1 ml of a solution that contained 10 mouse LD 5 o of ricin toxin D. The remaining 10 mice from each treatment group were exposed over ten minutes in a dynamic system to a liquid aerosol that supplied 5 to 10 mouse LD 5 o of ricin toxin D. After exposure to ricin, daily cage side observations were made for survival rates.
• RTAl-33/44-198 administered without an adjuvant had no obvious, untoward effects on the mice as noted by survival from three immunizations, weight gain, physical appearance, food consumption, and normal activity in comparison to phosphate buffered saline controls. Vaccinated animals survived without co- administration of an adjuvant (in these experiments, the adjuvant was Alhydrogel® 2% (Al O 2 ) available from Siperfos Biosector). Thus, RTAl-33/44-198 may be used to elicit an immune response in a subject and may be used as a safe and effective ricin vaccine.
• polypeptides of the present invention such as RTA1- 33/44-98 may be used as stable structural scaffolds to present antigenic determinants of ricin for use in immunogenic compositions such as ricin vaccines.
• RTA polypeptides versus dgRTA or recombinant RTA the following experiment may be conducted.
• the amounts and types of aggregates formed by the vaccine candidates may be quantified as a function of protein concentration and time under various conditions of fixed buffer composition, ionic strength, and pH.
• a combination of analytical size- exclusion chromatography (SEC) may be used with on-line multi-angle light scattering (MALS) detection.
• SEC -MALS provides a measure of the molar mass of proteins in solution because the light scattering response is directly proportional to the weight- averaged molar mass (Mw) of the protein sample multiplied by the sample concentration.
• solutions of protein samples at a concentration of about 0.4 to about 1.5 mg/ml in 0.067 M Na/K phosphate, pH 7.5 are separated using standard high performance liquid chromatographic methodology with a flow rate of about 0.5 to about 1.0 ml/min.
• Protein species are detected with an in-line standard UV/VIS HPLC detector, and the relative refractive indices of the sample components are determined from an in-line interferometric refractometer.
• Light scattering data are collected at many wavelengths, averaged, and evaluated using an in-line MALS instrument. Aggregates are quantified by molar mass for each protein sample.
• Example 4 Protein Folding Stability
• Relative protein folding stability is measured indirectly by comparing the amount of energy (proportional to temperature) required to unfold each polypeptide under various conditions of ionic strength or pH.
• the extent of protein unfolding is assessed indirectly by circular dichroism (CD) spectroscopy. Briefly, CD scans of protein samples are performed in a spectropolarimeter, fitted with peltier thermal control unit, using 0.2 mm and 1 cm path length quartz cuvettes, respectively, for near and far UN measurements.
• CD circular dichroism
• Solutions of purified polypeptide are used at a concentration of about 0.5 mg/ml in 0.067 M ⁇ a/K phosphate, pH 7.5.
• the initial scans provide baseline spectra and conoborate that the protein samples are folded.
• protein samples are intentionally and slowly unfolded by increasing the cuvet temperature by means of the thermal control unit.
• Temperature-induced changes in protein secondary structure are assessed indirectly by monitoring the change in mean residue ellipticity at 222 nm. From these data, one may calculate and compare the temperature (Tm) at which 50% of the protein is unfolded. If the novel RTA polypeptides are found to fold and unfold reversibly, then one may also calculate and compare the thermodynamic and enthalpic constants for protein folding.
• Example 5 Hydrophobic Surface Exposure To compare the relative exposure of hydrophobic surfaces among the novel RTA polypeptides of the present invention versus dgRTA, or recombinant RTA the following (indirect) assay may be conducted by measuring the amount of l-anilino-8- naphthalenesulfonic acid (A ⁇ S) bound to each polypeptide under defined conditions of buffer composition, pH, ionic strength and temperature. Increased binding of A ⁇ S, is ⁇ indicative of increased availability of hydrophobic surfaces, and bound dye can be differentiated from unbound dye on the basis of fluorescence.
• a ⁇ S l-anilino-8- naphthalenesulfonic acid
• Example 6 Effects of Storage Time on Biophysical Properties
• the biophysical properties of the novel RTA polypeptides of the present invention may vary as a function of storage time (such as over the course of 24 months) and storage temperature (such as at about 2 to about 8, 25 and 40°C) in different storage formulations the following assay may be conducted.
• the number of possible storage formulations is very large; therefore, only a subset of formulations using a sparse matrix that isolates selected variables are tested. These variables might include the buffer composition, the ionic strength, the pH, or the presence or absence of preferred adjuvants.
• Biophysical properties of the novel RTA polypeptides to be observed under different storage conditions may include, relative molecular mass of polypeptides, apparent isoelectric point of polypeptides (net charge), extent of self-aggregation, protein folding stability, and exposure of hydrophobic surfaces. These can be evaluated as described using SDS-PAGE, native PAGE, isoelectric focusing, light scattering, ANS dye binding, and circular dichroism spectroscopy.
• the structures may be refined by a combination of simulated annealing and molecular dynamics with a maximum likelihood target function, using the CNS program suite. See Adams, P. D., et al. (1997) PNAS USA 94:5018-23; and Brunger, A.T., et al. (1998) Acta Cryst. D54, 905-921.

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