US20180354995A1 - Flagellin-based agents and uses including effective vaccination - Google Patents

Flagellin-based agents and uses including effective vaccination Download PDF

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US20180354995A1
US20180354995A1 US16/017,251 US201816017251A US2018354995A1 US 20180354995 A1 US20180354995 A1 US 20180354995A1 US 201816017251 A US201816017251 A US 201816017251A US 2018354995 A1 US2018354995 A1 US 2018354995A1
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mutant
sequence
flagellin
based agent
vaccine
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Andrei V. Gudkov
Vadim Mett
Vadim Krivokrysenko
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Genome Protection Inc
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    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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Definitions

  • the present invention relates to, inter a/ia, new compositions and methods for vaccination, including adjuvants comprising flagellin-based agents.
  • Vaccines are one of the most effective preventative health tools available against infectious diseases, cancers, and allergies. Vaccination aims to generate a strong immune response to the administrated antigen and provide long-term protection against a disorder. Often, however, an antigen alone is insufficient to stimulate protective immunity.
  • Vaccine adjuvants are compounds that enhance the specific immune responses against antigens in vaccines.
  • Currently, several hundred natural and synthetic compounds are known to have adjuvant activity but only alum salts and AS04 are licensed for use in humans in the United States. This limited list of adjuvants is insufficient for meeting the functional needs of effective vaccination.
  • alum is able to induce a good T H2 response, it has little capacity to stimulate cellular (T H1 ) immune responses which are so important for protection against many pathogens.
  • the present invention provides for improved vaccines and/or adjuvants.
  • the present invention provides a vaccine composition, comprising an adjuvant comprising a flagellin-based agent, such as, for example, CBLB502 (a/k/a entolimod) as well as any of the flagellin-based agents or derivatives described herein (e.g. Table 1), and an aluminum gel or salt and an antigen and, optionally, an additional adjuvant.
  • a flagellin-based agent such as, for example, CBLB502 (a/k/a entolimod) as well as any of the flagellin-based agents or derivatives described herein (e.g. Table 1)
  • an aluminum gel or salt comprising an adjuvant comprising a flagellin-based agent, including CBLB502 as well as any of the flagellin-based agents or derivatives described herein (e.g. Table 1), and an aluminum gel or salt.
  • the vaccine described herein causes an improvement in adjuvant properties relative to a vaccine comprising the antigen and the aluminum gel or salt alone.
  • the vaccine and/or adjuvant described herein causes a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone and/or the adjuvant comprising the aluminum gel or salt alone.
  • the vaccine and/or adjuvant described herein causes immunostimulation of one or more of T H1 and T H2 -mediated immune response (e.g.
  • both of T H1 and T H2 -mediated immune response causes immunostimulation of T H1 -mediated immune response at levels greater than a vaccine comprising the antigen and the aluminum gel or salt alone and/or the adjuvant comprising the aluminum gel or salt alone.
  • the flagellin-based agent may be a flagellin molecule or flagellin-based agent, or variants thereof that has TLR5 agonist activity.
  • the flagellin-based agent may comprise one or more of the sequences of Table 1 (SEQ ID Nos.: 1-252), or variants thereof that have TLR5 agonist activity and can include a variant of Salmonella dublin wild type flagellin (SEQ ID No: 1), CBLB502 (SEQ ID NO: 2) or variants thereof (including closely-related variants such as S33ML (SEQ ID NO: 35), CBLB502-485CT (CBLB533, SEQ ID NO: 71), and CBLB502-S33MX (SEQ ID NO: 150)) and more distantly related variants such as flagellin derivatives from a thermophilic microorganism or flagellin derivatives from a microorganism well-tolerated by human (e.g. SEQ ID NOs: 243-252).
  • the aluminum gel or salt is
  • the present vaccine composition is part of the following vaccines (e.g. the antigens of these vaccines may be used as the antigen of the present vaccines): DTP (diphtheria-tetanus-pertussis vaccine), DTaP (diphtheria-tetanus-acellular pertussis vaccine), Hib (Haemophilus influenzae type b) conjugate vaccines, Pneumococcal conjugate vaccine, Hepatitis A vaccines, Poliomyelitis vaccines, Yellow fever vaccines, Hepatitis B vaccines, combination DTaP, Tdap, Hib, Human Papillomavirus (HPV) vaccine, Anthrax vaccine, and Rabies vaccine.
  • DTP diphtheria-tetanus-pertussis vaccine
  • DTaP diphtheria-tetanus-acellular pertussis vaccine
  • Hib Hemophilus influenzae type b conjugate vaccines
  • Pneumococcal conjugate vaccine He
  • the vaccine described herein is formed, in part, by mixing the flagellin-based agent and aluminum gel or salt to form a stable complex, the ratio (w/w) of flagellin-based agent to aluminum gel or salt being about 1:500 or less (e.g. about 1:500, or about 1:600, or about 1:700, or about 1:800, or about 1:900, or about 1:1000, or about 1:2000, or about 1:5000).
  • the flagellin-based agent and aluminum gel or salt are mixed in a ratio that is substantially below a loading capacity of the aluminum gel or salt.
  • the flagellin-based agent and antigen are adsorbed to the aluminum gel or salt.
  • the present invention provides a method of vaccinating a subject against a disorder, comprising administering an effective amount of a vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with the disorder.
  • the present invention provides a method of immunostimulating a subject in advance of or concurrent with vaccination, comprising administering an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt, wherein both T H1 and T H2 -mediated immune responses are immunostimulated.
  • the disorder is selected from infectious diseases, cancer, allergy, and autoimmune diseases (by way of example, diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, Human Papillomavirus (HPV) infection, various cancers, anthrax, rabies, Japanese Encephalitis, meningitis, measles, mumps, rubella, gastroenteritis, smallpox, typhoid fever, varicella (chickenpox), rotavirus, and shingles).
  • infectious diseases by way of example, diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, Human Papillomavirus (HPV) infection, various cancers, anthrax, rabies, Japanese Encephalitis, meningitis, measles, mumps, rubella, gastroenteritis
  • FIGS. 1A and 1B show the 13 conserved amino acids of flagellin that may be important for TLR5 activity.
  • FIGS. 1A and 1B show a comparison of amino acid sequences of the conserved amino ( FIG. 1A ) and carboxy ( FIG. 1B ) terminus from 21 species of bacteria.
  • the 13 conserved amino acids important for TLR5 activity are indicated by the letter “C” at the bottom of each column.
  • SEQ ID Nos: 253-273 correspond to the sequences listed on the figure from top to bottom, respectively.
  • SEQ ID Nos: 274-294 correspond to the sequences listed on the figure from top to bottom, respectively.
  • FIG. 2 shows ELISA data for the average response of five mouse treatment groups at the time point of 1 week post-boost.
  • Panel A is total IgG
  • Panel B is IgG1
  • Panel C is IgG2a
  • Panel D is IgG2b
  • Panel E is IgG3.
  • Groups 1-5 are defined in TABLE B (Example 2): Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 ⁇ g,” and Group 5 is also called “SA 10 ⁇ g.”
  • FIG. 3 shows ELISA data for the average response of five mouse treatment groups at the time point of 2 weeks post-boost.
  • Panel A is total IgG
  • Panel B is IgG1
  • Panel C is IgG2a
  • Panel D is IgG2b
  • Panel E is IgG3.
  • Groups 1-5 are defined in TABLE B (Example 2): Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 ⁇ g,” and Group 5 is also called “SA 10 ⁇ g.”
  • FIG. 4 shows ELISA data for the average response of five mouse treatment groups at the time point of 4 weeks post-boost.
  • Panel A is total IgG
  • Panel B is IgG1
  • Panel C is IgG2a
  • Panel D is IgG2b
  • Panel E is IgG3.
  • Groups 1-5 are defined in TABLE B (Example 2): Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 ⁇ g,” and Group 5 is also called “SA 10 ⁇ g.”
  • FIG. 5 shows a determination of CBLB502/ALHYDROGEL activity using a cell-based NF-KB activation assay.
  • CBLB502/ALHYDROGEL formulation (5 ⁇ g/100 ⁇ l) and ALHYDROGEL control were serially diluted in assay media 2000- to 13122000-fold (2.6-0.0004 ⁇ g/ml aluminium) and incubated with 293-hTLR5-LacZ cells for 20 hr.
  • Activity of ⁇ -galactosidase was measured after addition of cell lysis buffer and ONPG substrate and expressed as optical density at 414 nm (OD414).
  • FIG. 6 shows NF- ⁇ B-inducing activity of CBLB502/ALHYDROGEL formulated with different CBLB502 doses.
  • 293-hTLR5-LacZ cells were incubated with CBLB502/ALHYDROGEL suspensions prepared at CBLB502 doses ranging from 2.5 to 0.00015 ⁇ g/100 ⁇ l and diluted 200-fold with media.
  • Activity of ⁇ -galactosidase reporter enzyme was measured after 20 hr incubation.
  • FIG. 7 shows dose-response titration curves CBLB502/ALHYDROGEL generated with 293-hTLR5-LacZ reporter cells.
  • CBLB502 was adsorbed to ALHYDROGEL at indicated doses (20 ⁇ g/100 ⁇ l: top panel, 0.31 ⁇ g/100 ⁇ l: middle panel and 0.078 ⁇ g/100 ⁇ l: bottom panel), serially diluted with media and assayed together with soluble CBLB502 standards.
  • FIG. 8 shows the activity of CBLB502/ALHYDROGEL (1 ⁇ g/100 ⁇ l dose) stored at 4° C. for 2 and 6 days. NF- ⁇ B-inducing activity was measured using 293-hTLR5-LacZ reporter cell assay.
  • FIG. 9 shows pharmacokinetics of CBLB502 in mouse serum after subcutaneous injection of a 1 ⁇ g dose of CBLB502 adsorbed to ALHYDROGEL 2%.
  • the graph shows per-group mean serum CBLB502 concentrations and SEM (standard error of the mean) for each time point.
  • FIG. 10 shows induction of KC, G-CSF and IL-6 following s.c. administration of a 1 ⁇ g dose of CBLB502 adsorbed to ALHYDROGEL 2%.
  • Serum concentrations of the cytokines were determined using R&D Systems DuoSet kits DY453 (KC, top panel), DY414 (G-CSF, middle panel) and DY406 (IL-6, bottom panel).
  • the graphs show per-group mean serum cytokine concentrations and SEM (standard error of the mean) for each time point.
  • FIG. 11 shows immunopotentiating effects of a 502 adjuvant.
  • Anti-MA IgG (pg/ml) response panel A
  • anti-502 antibody response panel B
  • alum or entolimod a varying range from 0 to 20 ⁇ g
  • flagellin-based agents including CBLB502 and any of the flagellin-based agents (e.g. agents comprising the sequences of Table 1, including MX-33) described herein, can be mixed with aluminum gels or salts (and, optionally antigens), in ratios well-below the loading/adsorbing capacity of the aluminum gel or salt (e.g. 1:500), and cause a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone and/or the adjuvant comprising the aluminum gel or salt alone.
  • the inventors have also surprisingly discovered that the immunostimulatory effect can influence both the T H1 and T H2 -mediated arms of an immune response.
  • the inventors have also surprisingly discovered that low amounts of flagellin-based agents, including CBLB502 and any of the flagellin-based agents (e.g. agents comprising the sequences of Table 1, including MX-33) relative to antigen are effective for immunostimulation.
  • the invention provides a vaccine composition, comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen and, optionally, an additional adjuvant.
  • the invention provides an adjuvant comprising a flagellin-based agent and an aluminum gel or salt.
  • the described vaccine causes an improvement in adjuvant properties relative to a vaccine comprising the antigen and the aluminum gel or salt alone.
  • the vaccine and/or adjuvant described herein causes a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone (or as compared to the vaccine comprising the antigen and flagellin-based agent alone) and/or the adjuvant comprising the aluminum gel or salt alone (or as compared to the adjuvant comprising the flagellin-based agent alone).
  • the described vaccine and/or described adjuvant causes immunostimulation of one or more of T H1 and T H2 -mediated immune response. In some embodiments, the described vaccine and/or described adjuvant causes immunostimulation of both of T H1 and T H2 -mediated immune response. In some embodiments, the described vaccine and/or described adjuvant causes immunostimulation of T H1 -mediated immune response at levels greater than a vaccine comprising the antigen and the aluminum gel or salt alone or an adjuvant comprising the aluminum gel or salt alone.
  • T H1 -mediated immune response (or “Type 1 response”) largely involves interaction with macrophages and CD8+ T cells and may be linked to interferon- ⁇ , TNF- ⁇ , interleukin-2, and interleukin-10 production.
  • the T H1 -mediated immune response promotes cellular immune system and maximizes the killing efficacy of the macrophages and the proliferation of cytotoxic CD8 + T cells.
  • the T H1 -mediated immune response also promotes the production of opsonizing antibodies (e.g. IgG, IgM and IgA).
  • the Type 1 cytokine IFN- ⁇ increases the production of interleukin-12 by dendritic cells and macrophages, and via positive feedback, IL-12 stimulates the production of IFN- ⁇ in helper T cells, thereby promoting the T H1 profile.
  • Interferon- ⁇ also inhibits the production of cytokines such as interleukin-4, a cytokine associated with the Type 2 response, and thus it also acts to preserve its own response.
  • T H2 -mediated immune response (or “Type 2 response”) largely involves interaction with B-cells, eosinophils, and mast cells and may be linked to interleukin-4, interleukin-5, interleukin-6, interleukin-9, interleukin-10, and interleukin-13.
  • T H2 -mediated immune response promotes humoral immune system and may stimulate B-cells into proliferation, induce B-cell antibody class switching, and increase neutralizing antibody production (e.g. IgG, IgM and IgA as well as IgE antibodies).
  • Other functions of the Type 2 response include promoting its own profile using two different cytokines.
  • Interleukin-4 acts on helper T cells to promote the production of TH2 cytokines (including itself; it is auto-regulatory), while interleukin-10 (IL-10) inhibits a variety of cytokines including interleukin-2 and IFN- ⁇ in helper T cells and IL-12 in dendritic cells and macrophages.
  • IL-10 interleukin-10
  • the combined action of these two cytokines suggests that once the T cell has decided to produce these cytokines, that decision is preserved (and also encourages other T cells to do the same).
  • the stimulation of T H1 and T H2 -mediated immune responses may be measured by assays known in the art, including a number of antibody surrogate assays (e.g. ELISA and the like).
  • assays known in the art including a number of antibody surrogate assays (e.g. ELISA and the like).
  • IgG1 is associated with a T H2 -like response
  • T H1 response is associated with the induction of IgG2a, IgG2b, and IgG3 antibodies.
  • the described vaccine and/or described adjuvant causes an increase in titer of one or more of IgG1, IgG2a, IgG2b, and IgG3 antibodies (e.g. relative to the adjuvant comprising the aluminum gel or salt or flagellin-based agent alone, or relative to the vaccine comprising the antigen and the aluminum gel or salt alone (or flagellin-based agent) alone)).
  • the described vaccine and/or described adjuvant causes a relative increase in the titer of all of IgG1, IgG2a, IgG2b, and IgG3 antibodies.
  • the described vaccine and/or described adjuvant causes a relative increase in the titer of more IgG3 antibodies than the described vaccine and/or described adjuvant in the absence of a flagellin-based agent (or the described vaccine and/or described adjuvant in the absence of an aluminum gel or salt alone).
  • the described vaccine and/or described adjuvant causes a diversified immune response.
  • the total IgG generated by the described vaccines and/or adjuvants is greater than the described vaccines and/or adjuvants without the flagellin-based agent.
  • the flagellin-based agent may be a flagellin molecule or flagellin-related polypeptide, or variants thereof that have TLR5 agonist activity.
  • the flagellin-based agent may be from various sources, including a variety of Gram-positive and Gram-negative bacterial species.
  • the flagellin-based agents may have an amino acid sequence that is derived from any of the flagellins from bacterial species that are depicted in, for example, FIG. 7 of U.S. Patent Publication No. 2003/0044429, the contents of which are incorporated herein by reference in their entirety.
  • the flagellin-based agent may have nucleotide sequences related to those encoding the flagellin polypeptides listed in, for example, FIG. 7 of U.S. 2003/0044429, which are publicly available at sources including the NCBI Genbank database.
  • the flagellin-based agent may be the major component of bacterial flagellum.
  • the flagellin-based agents may be composed of one, or two, or three, or four, or five, or six, or seven domains or fragments thereof (see, e.g., FIG. 10 of U.S. Pat. No. 8,324,163, the contents of which are incorporated herein by reference in their entirety).
  • the domains may be selected from ND0, ND1, ND2, D3, CD2, CD1, and CDO.
  • Domains 0 (D0), 1 (D1), and 2 (D2) may be discontinuous and may be formed when residues in the amino terminus and carboxy terminus are juxtaposed by the formation of a hairpin structure.
  • the amino and carboxy terminus comprising the D1 and D2 domains may be most conserved, whereas the middle hypervariable domain (D3) may be highly variable.
  • the non-conserved D3 domain may be on the surface of the flagellar filament and may contain the major antigenic epitopes.
  • the potent proinflammatory activity of flagellin may reside in the highly conserved N and CD1 and D2 regions.
  • the flagellin-based agents may be from a species of Salmonella, representative examples of which are S. typhimurium and S. dublin (encoded by GenBank Accession Number M84972).
  • the flagellin related-polypeptide may be a fragment, variant, analog, homolog, or derivative of wild type flagellin (SEQ ID NO: 1), or combination thereof.
  • a fragment, variant, analog, homolog, or derivative of flagellin may be obtained by rational-based design based on the domain structure of flagellin and the conserved structure recognized by TLR5.
  • the flagellin-based agent may be related to a flagellin polypeptide from any Gram-positive or Gram-negative bacterial species including, but not limited to, the flagellin polypeptides disclosed in U.S. Pat. Pub. 2003/0044429, the contents of which are incorporated herein, and the flagellin peptides corresponding to the Accession numbers listed in the BLAST results shown in FIG. 7 (panels A-F) of U.S. Patent Pub. 2003/0044429, or variants thereof.
  • the flagellin-based agent comprises or consists of any of the polypeptides or nucleic acids encoding said polypeptides listed in Table 1.
  • the flagellin-based agent is encoded by the nucleotide sequences listed in Table 1.
  • the flagellin-based agent comprises the polypeptides listed in Table 1.
  • the flagellin-based agent comprises one or more of SEQ ID NOs.: 1-252.
  • the flagellin-based agent comprises a flexible linker.
  • the flexible linker comprises SEQ ID NO: 16.
  • the flexible linker comprises SEQ ID NO: 242.
  • the flagellin-based agent is a variant of SEQ ID NO: 1. In variois embodiments, the flagellin-based agent is not SEQ ID NO: 1.
  • the flagellin-based agent comprises or consists of CBLB502 (SEQ ID NO: 2) or is a variant of CBLB502 (SEQ ID NO: 2).
  • CBLB502 provides the advantage of, inter alia, removal of epitopes that generate neutralizing anti-flagellin antibodies and therefore allow for the surprising adjuvant properties seen here in combination with alum.
  • the flagellin-based agent comprises mutations in epitopes recognized by neutralizing anti-CBLB502 antibodies.
  • the flagellin-based agent may comprise one or more mutations in the epitopes recognized by neutralizing anti-CBLB502 antibodies which inhibit or abrogate the ability of the antibodies to neutralize the composition.
  • the flagellin-based agent comprises a truncation and mutations in one or more epitopes.
  • the mutations comprise replacement of the epitope residues with alanine.
  • the mutated epitopes comprise one or more of the following residues: E153, S444, T154, N440, Q142, F131, D443, N68, T447, S110, Q117, R124, D113, E120, N127, and Q128.
  • the flagellin-based agent may comprise insertions, deletions, transposon insertions, and changes to any one of the D0, D1, D2, and the variable D3 domains.
  • the D3 domain may be substituted in part, or in whole, with a hinge or linker polypeptide that allows the D1 and D2 domains to properly fold such that the variant stimulates TLR5 activity.
  • the flagellin-based agent may be a minimal functional core of a flagellin, for example, deleting residues relative to the already shortened CBLB502 molecule.
  • the flagellin-based agent has altered amino acid identity relative to wild type, including deletions, additions and substitutions, that provide for improved activity.
  • the flagellin-based agent is derived from CBLB502 (SEQ ID NO: 2).
  • the flagellin-based agent comprises a truncation in one or more domains.
  • the flagellin-based agent comprises a deletion in an N-terminal domain.
  • the flagellin-based agent comprises a deletion in the NDO domain. In yet a further embodiment, the flagellin-based agent comprises a deletion of the entire NDO domain. In a further embodiment, the flagellin-based agent comprises a deletion in a C-terminal domain. In yet another embodiment, the flagellin-based agent comprises a deletion in the CD0 domain. In yet another embodiment, the flagellin-based agent retains amino acids 470-485 of the CD0 domain. In yet a further embodiment, the flagellin-based agent is CBLB502-S33 (SEQ ID NO: 17).
  • the flagellin-based agent may comprise at least 10, 11, 12, or 13 of the 13 conserved amino acids shown in FIG. 1A and FIG. 1B (positions 89, 90, 91, 95, 98, 101, 115, 422, 423, 426, 431, 436 and 452).
  • the flagellin may be at least 30-99% identical to amino acids 1-174 and 418-505 of SEQ ID NO: 1.
  • the flagellin-based agent comprises a tag.
  • the tag is attached to the N-terminus of the flagellin-based agent.
  • the tag is attached to the C-terminus of the flagellin-based agent.
  • the flagellin-based agent comprises a flexible linker.
  • the flexible linker comprises SEQ ID NO: 16.
  • the flexible linker comprises SEQ ID NO:242.
  • the flagellin-based agent is one or more of the flagellin related composition derived from SEQ ID NO: 2.
  • the flagellin-based agent is one or more of CBLB502-S33ML (SEQ ID NO: 35), CBLB502-485CT (CBLB533, SEQ ID NO: 71), and CBLB502-S33MX (SEQ ID NO: 150).
  • the flagellin-based agent is a flagellin derivative from a thermophilic microorganism. In various embodiments, the flagellin-based agent is a flagellin derivative from a microorganism well-tolerated by human. In some embodiments, the flagellin-based agent is one or more of SEQ ID NOs: 243-252.
  • a variant includes molecules that have TLR5 agonist activity. In some embodiments, a variant includes molecules comprising an amino acid sequence having at least about 70% (e.g.
  • the flagellin-based agent comprises or consists of any of the polypeptides or nucleic acids encoding said polypeptides listed in Table 1.
  • the flagellin-based agent is encoded by the nucleotide sequences listed in Table 1.
  • the flagellin-based agent comprises one or more of the polypeptides listed in Table 1.
  • the flagellin-based agent comprises or consists of polypeptides encoded by either SEQ ID NOs: 69 or 70.
  • the flagellin-based agent comprises or consists of the polypeptides of SEQ ID NO: 71, “CBLB543”.
  • the flagellin-based agent comprises or consists of polypeptides encoded by either SEQ ID NOs: 149 or 151. In some embodiments, the flagellin-based agent comprises or consists of the polypeptides of SEQ ID NO: 150, “CBLB533”.
  • the aluminum gel or salt is selected from aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate, AS04 (which is composed of aluminum salt and MPL), and ALHYDROGEL.
  • the aluminum gel or salt is a formulation or mixture with any of the additional adjuvants described herein.
  • adjuvants in addition to the described flagellin-based agent and an aluminum gel or salt find use in the present invention.
  • the additional adjuvant is selected from, oil-in-water emulsion formulations, saponin adjuvants, ovalbumin, Freunds Adjuvant, cytokines, and chitosans.
  • Illustrative additional adjuvants include, but are not limited to: (1) ovalbumin (e.g. ENDOFIT), which is often used for biochemical studies; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), such as for example (a) MF59 (PCT Publ. No.
  • WO 90/14837 containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as, for example, Model HOy microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, (c) RIBI adjuvant system (RAS), (RIBI IMMUNOCHEM, Hamilton, Mo.) containing 2% Squalene, 0.2% Tween 80, and, optionally, one or more bacterial cell wall components from the group of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), including MPL
  • the additional adjuvant is one or more of a flagellin-based agent (e.g. CBLB502 or any of the agents of Table 1), an aluminium salt or gel, a pattern recognition receptors (PRR) agonist, CpG ODNs and imidazoquinolines.
  • the additional adjuvant is one or more of cyclic [G(3′,5′)pA(3′,5′)p] (e.g. 3′3′-cGAMP VACCIGRADE); cyclic [G(2′,5′)pA(3′,5′)p]2′3′ (e.g.
  • cGAMP VACCIGRADE 2′3′ cGAMP VACCIGRADE
  • cyclic [G(2′,5′)pA(2′,5′)p] e.g. 2′2′-cGAMP VACCIGRADE
  • cyclic diadenylate monophosphate e.g. c-di-AMP VACCIGRADE
  • cyclic diguanylate monophosphate e.g. c-di-GMP VACCIGRADE
  • TLR7 agonist-imidazoquinolines compound e.g. TLR7 agonists, such as, for example, Gardiquimod VACCIGRADE, Imiquimod VACCIGRADE, R848 VACCIGRADE
  • lipopolysaccharides e.g.
  • TLR4 agonists such as that from E. coli 0111:B4 strain (e.g. LPS-EB VACCIGRADE); monophosphoryl lipid A (e.g. MPLA-SM VACCIGRADE and MPLA Synthetic VACCIGRADE); N-glycolylated muramyldipeptide (e.g. N-Glycolyl-MDP VACCIGRADE); CpG ODN, class A and/oror CpG ODN, class B and/or CpG ODN, class C (e.g.
  • the additional adjuvant is a TLR agonist (e.g.
  • NOD nucleotide-binding oligomerization domain
  • STING stimulator of interferon genes
  • the additional adjuvants is one or more of a mineral adjuvant, gel-based adjuvant, tensoactive agent, bacterial product, oil emulsion, particulated adjuvant, fusion protein, and lipopeptide.
  • mineral salt adjuvants besides the aluminum adjuvants described elsewhere, include salts of calcium (e.g. calcium phosphate), iron and zirconium.
  • gel-based adjuvants besides the aluminum gel-based adjuvants described elsewhere, include Acemannan.
  • Tensoactive agents include Quil A, saponin derived from an aqueous extract from the bark of Quillaja saponaria; saponins, tensoactive glycosides containing a hydrophobic nucleus of triterpenoid structure with carbohydrate chains linked to the nucleus, and QS-21.
  • Bacterial products include cell wall peptidoglycan or lipopolysaccharide of Gram-negative bacteria (e.g. from Mycobacterium spp., Corynebacterium parvum, C.
  • MDP N-acetyl muramyl-L-alanyl-D-isoglutamine
  • LPS lipopolysaccharides
  • TDM trehalose dimycolate
  • Oil emulsions include FIA, Montanide, Adjuvant 65, Lipovant, the montanide family of oil-based adjuvants, and various liposomes.
  • poly (DL-lactide-coglycolide) microspheres have been extensively studied and find use herein.
  • cytokines are an adjuvant of the present invention (e.g. IFN- ⁇ and granulocyte-macrophage colony stimulating factor (GM-CSF)).
  • carbohydrate adjuvants e.g. inulin-derived adjuvants, such as, gamma inulin, algammulin (a combination of ⁇ -inulin and aluminum hydroxide), and polysaccharides based on glucose and mannose, such as glucans, dextrans, lentinans, glucomannans and galactomannans
  • inulin-derived adjuvants such as, gamma inulin, algammulin (a combination of ⁇ -inulin and aluminum hydroxide), and polysaccharides based on glucose and mannose, such as glucans, dextrans, lentinans, glucomannans and galactomannans
  • adjuvant formulations are useful in the present invention and include alum salts in combination with other adjuvants such as Lipid A, algammulin, immunostimulatory complexes (ISCOMS), which are virus like particles of 30-40 nm and dodecahedric structure, composed of Quil A, lipids, and cholesterol.
  • adjuvants such as Lipid A, algammulin, immunostimulatory complexes (ISCOMS), which are virus like particles of 30-40 nm and dodecahedric structure, composed of Quil A, lipids, and cholesterol.
  • the additional adjuvants are described in Jennings et al. Adjuvants and Delivery Systems for Viral Vaccines-Mechanisms and Potential. In: Brown F, Haaheim LR, (eds). Modulation of the Immune Response to Vaccine Antigens. Dev. Biol. Stand, Vol. 92. Basel: Karger 1998; 19-28 and/or Sayers et al. J Biomed Biotechnol. 2012; 2012: 831486, and/or Petrovsky and Aguilar, Immunology and Cell Biology (2004) 82, 488-496 the contents of which are hereby incorporated by reference in their entireties.
  • the present adjuvants may be part of live and attenuated, or killed or inactivated, or toxoid, or subunit or conjugate vaccines.
  • the present adjuvants may be part of one or more approved vaccines and/or the antigens of one or more approved vaccines may be the antigens of the present invention.
  • the approved vaccines include: Adenovirus; Anthrax (Biothrax); BCG (Tice); DT (Sanofi); DTaP (Daptacel); DTaP (Infanrix); DTaP-HepB-IPV (Pediarix); DTaP-IPV (Kinrix); DTaP-IPV/Hib (Pentacel); Hib (ActHIB); Hib (Hiberix); Hib (PedvaxHlB); Hib/Hep B (Comvax); Hib/Mening.
  • the present adjuvants may be part of one or more illustrative vaccines and/or the antigens of one or more illustrative vaccines may be the antigens of the present invention.
  • Illustrative vaccines include, by way of example, subunit vaccine and inactivated or “killed” vaccine (e.g.
  • Infanrix-IPV/Hib Bordetella pertussis
  • Infanrix-IPV/Hib Haemophilus influenzae
  • Infanrix-IPV/Hib Polyovirus
  • Infanrix-IPV/Hib Clostridium tetani
  • Infanrix-IPV/Hib Corynebacterium diphtheriae
  • Infanrix-hexa Bordetella pertussis
  • Infanrix-hexa Haemophilus influenzae
  • Infanrix-hexa (Poliovirus), Infanrix-hexa (Hepatitis B virus), Infanrix-hexa ( Clostridium tetani ), Infanrix-hexa ( Corynebacterium diphtheriae ), Infanrix-IPV ( Bordetella pertussis ), Infanrix-IPV ( Clostridium tetani ), Infanrix-hexa ( Coryn
  • 5CVMB Neisseria meningitidis
  • B. pertussis CyaA protein vaccine Bordetella pertussis
  • B. pertussis PTx protein vaccine Bordetella pertussis
  • Cancer VEGFA protein vaccine Cancer
  • E. coli vaccine using intimin polypeptide Escherichia coli
  • Engerix-B Hepatitis B virus
  • pylori VacA protein vaccine Helicobacter pylori
  • HC of type C and D Clostridium botulinum
  • Infanrix/Hib Bordetella pertussis
  • Infanrix/Hib Haemophilus influenzae
  • Infanrix/Hib Clostridium tetani
  • M. gallisepticum TM-1 Protein Subunit Vaccine Mycoplasma gallisepticum
  • MDA-modified human apo B-100 peptide Vaccine Atherosclerosis
  • MSP3-LSP with aluminium hydroxide Plasmodium spp.
  • Mumps HN Protein Subunit Vaccine Mumps virus
  • BoNT/F(Hc) Clostridium botulinum
  • DAPTACEL Corynebacterium diphtheriae
  • Infanrix Bordetella pertussis
  • Infanrix Clostridium tetani
  • KINRIX Clostridium tetani
  • PBT Clostridium botulinum
  • Pediarix Bordetella pertussis
  • inactivated or “killed” vaccines e.g.
  • Avaxim Hepatitis A virus
  • Avaxim-Pediatric Hepatitis A virus
  • FSME-IMMUN Tick-borne Encephalitis Virus (TBEV)
  • Infanrix Corynebacterium diphtheriae
  • Ixiaro Japanese encephalitis virus
  • KINRIX Corynebacterium diphtheriae
  • Pediarix Corynebacterium diphtheriae
  • conjugate vaccines e.g., Arabinomannan-tetanus toxoid conjugate ( Mycobacterium tuberculosis )
  • CCPS-P64kR Neisseria meningitidis
  • COMVAX Haemophilus influenzae
  • Menjugate Neisseria meningitidis
  • Neisvac-C Neisseria meningitidis
  • PedvaxHIB Haemophilus influenzae
  • the present adjuvants are combined in a vaccine targeting a substance abuse.
  • the present adjuvants are used in vaccines against addition to fentanyl, heroin, morphine, opium, oxycodone, hydrocodone, ketamine, PCP, barbiturates, benzodiazepines, flunitrazepam, GHB, methaqualone, hashish, marijuana, LSD, mescaline, psilocybin, amphetamine, cocaine, MDMA, methamphetamine, methylphenidate, and nicotine (e.g.
  • TA-CD those described in US Patent Publication No. 2013/0011432, the contents of which are hereby incorporated by reference (e.g. using 6-(2R,35)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1]octane-2-carbonyloxy-hexanoic acid (GNC) or 6-((2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido)hexanoic acid) (GNE) as the antigen, and TA-NIC (Celtic Pharma)).
  • GNC 6-(2R,35)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1]octane-2-carbonyloxy-hexanoic acid
  • GNE 6-((2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane
  • the present adjuvants e.g. flagellin-based agent and an aluminum gel or salt
  • the present vaccines may comprise any one of the adjuvants or antigens annotated in the VIOLIN or Vaxjo databases (as described in He et al. Nucleic Acids Research. 2014. 42 (D1): D1124-D1132 and Xiang et al. Nucleic Acids Res. 2008 January; 36: D923-8, the contents of which are hereby incorporated by reference in their entirety).
  • the present adjuvants may be part of one or more cancer vaccines and/or the antigens of one or more cancer vaccines may be the antigens of the present invention.
  • Illustrative cancer vaccines include therapeutic and preventative vaccines.
  • cancer vaccines include ONCOPHAGE (ANTIGENICS INC., approved in Russia in 2008 for kidney cancer), APC8015/Sipuleucel-T/PROVENGE (DENDREON, for, e.g.
  • CANCERVAX CANVAXIN
  • GENITOPE CORP MYVAX personalized immunotherapy
  • FAVRILLE INC FAVRILLE INC
  • preventive vaccines which attack the cancer-causing viruses human papillomavirus (e.g. CERVARIX (GSK) and GARDASIL (MERCK)), hepatitis A virus (e.g. CERVARIX (GSK) and GARDASIL (MERCK)), and hepatitis B virus (e.g. RECOMBIVAX HB (MERCK), ENGERIX-B (GSK), ELOVAC B (HUMAN BIOLOGICALS INSTITUTE), GENEVAC B (SERUM INSTITUTE), SHANVAC B, etc.
  • CERVARIX GSK
  • GARDASIL GARDASIL
  • RECOMBIVAX HB MERCK
  • ENGERIX-B ELOVAC B
  • ELOVAC B HUMAN BIOLOGICALS INSTITUTE
  • the present adjuvants may be part of one or more allergen vaccines and/or the antigens of one or more allergen vaccines may be the antigens of the present invention.
  • SCIT subcutaneous immunotherapy
  • allergen compositions and methods are applicable to the present invention (e.g. “allergy shots”).
  • allergy shots vaccinations for allergic rhinitis and conjunctivitis (e.g. pollen (including ragweed), dust mites, animal dander and airborne mold spores); allergic or extrinsic bronchial asthma (e.g.
  • Allergens include pollen (e.g. tree, grass, weed), pet dander (e.g. cat pelt), dust mites, airborne molds, occupational aeroallergens, honey bee venom, yellow jacket venom, hornet venom, wasp venom, and fire ant venom.
  • the antigens of the present vaccines may be the antigens of live and attenuated or killed or inactivated or toxoid or a subunit or conjugate vaccines. In various embodiments, the antigen of the present vaccines is an antigen of any of the vaccines described herein.
  • the present antigen is that of one or more of the following vaccines: DTP (diphtheria-tetanus-pertussis vaccine), DTaP (diphtheria-tetanus-acellular pertussis vaccine), Hib ( Haemophilus influenzae type b) conjugate vaccines, Pneumococcal conjugate vaccine, Hepatitis A vaccines, Poliomyelitis vaccines, Yellow fever vaccines, Hepatitis B vaccines, combination DTaP, Tdap, Hib, Human Papillomavirus (HPV) vaccine, Anthrax vaccine, Bacillus Calmette-Guérin (Tb), and Rabies vaccine.
  • DTP diphtheria-tetanus-pertussis vaccine
  • DTaP diphtheria-tetanus-acellular pertussis vaccine
  • Hib Haemophilus influenzae type b conjugate vaccines
  • Pneumococcal conjugate vaccine Pneumococcal conjugate
  • the flagellin-based agent and antigen are adsorbed to the aluminum gel or salt. In some embodiments, the flagellin-based agent and aluminum gel or salt are mixed to form a stable complex. In some embodiments, the flagellin-based agent and aluminum gel or salt are mixed in a ratio that is substantially below a loading capacity of the aluminum salt. In some embodiments, the flagellin-based agent and aluminum gel or salt are present in a ratio that is substantially below a loading capacity of the aluminum salt.
  • the flagellin-based agent and aluminum gel or salt are mixed or present in a ratio (w/w) of about 1:500, or about 1:600, or about 1:700, or about 1:800, or about 1:900, or about 1:1000, or about 1:2000, or about 1:5000, or about 1:6000, or about 1:7000, or about 1:8000, or about 1:9000, or about 1:10000.
  • the flagellin-based agent and aluminum gel or salt are mixed in a ratio (w/w) of about 1:500 or less.
  • the flagellin-based agent and aluminum gel or salt are present in a ratio that is substantially below a loading capacity of the aluminum salt even in the presence of anitigen.
  • the loading (or adsorption) capacity of the adjuvant can be measured using a variety of analytical methods. In general, it is done by comparing the protein content in the aqueous phase of the agent being loaded or adsorbed (e.g. flagellin-based agent and/or antigen solution) before and after adsorption onto the adjuvant. For instance, the Ramon flocculation test may be used (as is used to determine the adsorption of diphtheria and tetanus toxoid). Further, loading can be measured using immunoprecipitation techniques (e.g. quantitative immunoelectrophoresis or single radial immunodiffusion) or spectrophotometric techniques (e.g.
  • immunoprecipitation techniques e.g. quantitative immunoelectrophoresis or single radial immunodiffusion
  • spectrophotometric techniques e.g.
  • ELISA methods may also be used as can immunoelectrophoresis or HPLC.
  • the aluminum content in the final vaccine can be monitored using a number of known techniques, including spectrometric methods, such as for example atomic adsorption spectrometry.
  • the amount of aluminum gel or salt in the vaccines and/or adjuvants described herein is about 0.05 to about 1.0 mg/dose, or about 0.125 to about 0.625 mg/dose. In some embodiments, the amount of aluminum gel or salt in the vaccines and/or adjuvants described herein is about 0.05, or about 0.10, or about 0.15, or about 0.20, or about 0.25, or about 0.30, or about 0.35, or about 0.40, or about 0.45, or about 0.50, or about 0.55, or about 0.60, or about 0.65, or about 0.70, or about 0.75, or about 0.80, or about 0.85, or about 0.90, or about 0.95, or about 1.0 mg/dose.
  • the amount of flagellin-based agent is about 0.03 to about 5 ⁇ g/dose (e.g. about 0.03 ⁇ g/dose, about 0.1 ⁇ g/dose, about 0.3 ⁇ g/dose, about 0.5 ⁇ g/dose about 1.0 ⁇ g/dose, about 1.5 ⁇ g/dose, about 2.0 ⁇ g/dose, about 2.5 ⁇ g/dose, about 3.0 ⁇ g/dose, about 4.0 ⁇ g/dose, about 4.5 ⁇ g/dose, about 5.0 ⁇ g/dose).
  • 0.03 ⁇ g/dose e.g. about 0.03 ⁇ g/dose, about 0.1 ⁇ g/dose, about 0.3 ⁇ g/dose, about 0.5 ⁇ g/dose about 1.0 ⁇ g/dose, about 1.5 ⁇ g/dose, about 2.0 ⁇ g/dose, about 2.5 ⁇ g/dose, about 3.0 ⁇ g/dose, about 4.0 ⁇ g/dose, about 4.5 ⁇ g/dose, about 5.0 ⁇ g/dose.
  • the present compositions and methods comprise doses of flagellin-based agent that are less than about 5 ⁇ g/dose, or less than 4 ⁇ g/dose, or less than 3 ⁇ g/dose, or less than 2 ⁇ g/dose, or less than 1 ⁇ g/dose, or less than 0.5 ⁇ g/dose. In some embodiments, the present compositions and methods comprise low doses of flagellin-based agent.
  • the present compositions and methods do not involve covalently attaching an antigen to the flagellin-based agent either as a fusion protein or via chemical conjugation.
  • the present compositions do not have either equimolar ratio of antigen to flagellin-based agent (as in a fusion) or several molecules of hapten per one molecule of flagellin-based agent (as in chemical conjugates).
  • the amount of flagellin-based agent in any of the present vaccines is less than the amount of antigen. In various embodiments, the amount of flagellin-based agent in any of the present vaccines is less than the amount of antigen.
  • the amount of flagellin-based agent in any of the present vaccines is substantially less than the amount of antigen. In various embodiments, the amount of flagellin-based agent in any of the present vaccines is about 500-fold, or about 450-fold, or about 400-fold, or about 350-fold, or about 325-fold, or about 300-fold, or about 250-fold, or about 200-fold, or about 150-fold, or about 100-fold, or about 50-fold less than the amount of antigen.
  • the combination of flagellin-based agent and alum do not substatially effect TLR5 interaction by the flagellin-based agent.
  • the present compositions and methods do not induce production of TNF ⁇ .
  • the present combination of flagellin-based agent and alum is subtantially stable at low temperatures for about one week (e.g. at about 4° C. for about 3 days, or about 5 days, or about 6 days, or about 7 days, or about 10 days).
  • the present invention relates to a method of vaccinating a subject against a disorder, comprising administering an effective amount of a vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with the disorder.
  • the invention relates to a use of vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with a disorder for vaccinating a subject against the disorder.
  • the invention relates to a use of an effective amount of vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with a disorder in the manufacture of a medicament for vaccinating a subject against the disorder.
  • the present invention relates to a method of immunostimulating a subject in advance of or concurrent with vaccination, comprising administering an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt, wherein both T H1 and T H2 -mediated immune responses are immunostimulated.
  • the invention relates to a use of an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt for immunostimulating a subject in advance of or concurrent with vaccination.
  • the invention relates to a use of an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt in the manufacture of a medicament for immunostimulating a subject in advance of or concurrent with vaccination.
  • the vaccine described herein causes an improvement in adjuvant properties relative to a vaccine comprising the antigen and the aluminum gel or salt alone (or flagellin-based agent and antigen alone).
  • the vaccine and/or adjuvant described herein causes a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone (or flagellin-based agent and antigen alone) and/or the adjuvant comprising the aluminum gel or salt alone (or the adjuvant comprising the flagellin-based agent alone).
  • the described vaccine and/or described adjuvant causes an increase in titer of 1 or more of, or 2 or more of, or 3 or more of, or all of IgG1, IgG2a, IgG2b, and IgG3 antibodies (e.g. relative to the adjuvant comprising the aluminum gel or salt or flagellin-based agent alone, or relative to the vaccine comprising the antigen and the aluminum gel or salt alone or flagellin-based agent alone)).
  • the described vaccine and/or described adjuvant causes a relative increase in the titer of all of IgG1, IgG2a, IgG2b, and IgG3 antibodies.
  • the described vaccine and/or described adjuvant causes a relative increase in the titer of more IgG3 antibodies than the described vaccine and/or described adjuvant in the absence of a flagellin-based agent (or the described vaccine and/or described adjuvant in the absence of an aluminum gel or salt alone).
  • the antigen is administered simultaneously with or sequentially to the adjuvant.
  • the disorder is selected from infectious diseases, cancer, allergy, and autoimmune diseases.
  • the disorder is selected from diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, Human Papillomavirus (HPV) infection, anthrax, rabies, Japanese Encephalitis, meningitis, measles, mumps, rubella, gastroenteritis, smallpox, typhoid fever, varicella (chickenpox), rotavirus, and shingles.
  • HPV Human Papillomavirus
  • the disorder is a cancer is selected from, but not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer
  • the disorder is an allergy, selected from, by way of non-limiting example, allergic rhinitis and conjunctivitis, allergic or extrinsic bronchial asthma, and insect venom hypersensitivity.
  • the disorder is a substance abuse disorder (e.g. of fentanyl, heroin, morphine, opium, oxycodone, hydrocodone, ketamine, PCP, barbiturates, benzodiazepines, flunitrazepam, GHB, methaqualone, hashish, marijuana, LSD, mescaline, psilocybin, amphetamine, cocaine, MDMA, methamphetamine, methylphenidate, and nicotine).
  • substance abuse disorder e.g. of fentanyl, heroin, morphine, opium, oxycodone, hydrocodone, ketamine, PCP, barbiturates, benzodiazepines, flunitrazepam, GHB, methaqualone, hashish, marijuana, LSD, mescaline, psilocybin, amphetamine, cocaine, MDMA, methamphetamine, methylphenidate, and nicotine).
  • compositions of the present invention can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art.
  • Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.
  • salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzo
  • Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxy
  • compositions of the present invention e.g. the described adjuvants and vaccines
  • compositions of the present invention may comprise a pharmaceutically acceptable carrier or vehicle.
  • Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
  • Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions.
  • suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • Any composition described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the present invention includes the compositions of the present invention (e.g. the described adjuvants and vaccines) in various formulations.
  • Any composition of the present invention can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155).
  • suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
  • compositions of the present invention can also include a solubilizing agent.
  • the agents can be delivered with a suitable vehicle or delivery device as known in the art.
  • Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.
  • Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
  • compositions of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).
  • a carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).
  • any composition of the present invention is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
  • Routes of administration include intramuscular, e.g. by injection or infusion.
  • the described adjuvant of a flagellin-based agent e.g. CBLB502
  • aluminum gel or salt may prevent systemic delivery of the flagellin-based agent and induce localized delivery.
  • routes of administration include nasal, oral, and sublingual delivery.
  • Routes of administration may also be intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, oral, sublingual, intranasal, transdermal, or by inhalation.
  • the administering is effected orally or by parenteral injection.
  • the mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.
  • compositions of the present invention can be administered orally.
  • Such compositions can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local.
  • Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.
  • Dosage forms suitable for parenteral administration include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
  • any composition of the present invention e.g. the described adjuvants and vaccines
  • the dosing schedule can depend on various parameters, including, but not limited to, the disorder being treated, the subject's general health, and the administering physician's discretion.
  • doses may be determined with reference Physicians' Desk Reference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011), the contents of which are incorporated by reference in its entirety.
  • the dosage is normally about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day. Injections may be given up to four times daily.
  • the dosage of any agent described herein is normally about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day. A dosage of up to about 3000 mg per day can be administered.
  • delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
  • a liposome see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
  • composition of the present invention can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543;
  • Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein.
  • the invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.
  • Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).
  • a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may be used.
  • Administration of a composition of the present invention can, independently, be once per patient or may be used in a booster strategy.
  • Administration may be about one to about four times daily or about one to about four times per month or about one to about six times per year or once every two, three, four or five years.
  • Administration can be for the duration of about one day or about one month, about two months, about three months, about six months, about one year, about two years, about three years, and may even be for the life of the subject.
  • the dosage may be administered as a single dose or divided into multiple doses.
  • the dosage regimen utilizing any flagellin related composition (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed.
  • Any flagellin related composition (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily.
  • any flagellin related composition (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen.
  • composition of the present invention may be used in conjunction with one or more additional agents.
  • the invention pertains to co-administration and/or co-formulation. Any of the compositions described herein may be co-formulated and/or co-administered.
  • any composition described herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy.
  • any agent referenced herein may be used in combination with any of the composition described herein.
  • any flagellin-related agent or composition comprising the same may be used with agents that stimulate NOD receptors (e.g. NOD1 and NOD2 agonists, such as peptidoglycan, C12-iE-DAP and L18-MDP) and as described in Infect Immun. October 2013; 81(10): 3855-3864, the contents of which are hereby incorporated by reference in their entirety.
  • NOD1 and NOD2 agonists such as peptidoglycan, C12-iE-DAP and L18-MDP
  • the present invention pertains to chemotherapeutic agents as additional agents.
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin),epirubi
  • irinotecan Camptosar, CPT-11 (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC- ⁇ , Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the methods of treatment can further include the use
  • the flagellin-based agents (and/or additional agents) described herein include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition.
  • derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids.
  • the flagellin-based agents (and/or additional agents) described herein further comprise a cytotoxic agent, comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death.
  • a cytotoxic agent comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death.
  • agents may be conjugated to a composition described herein.
  • flagellin-based agents and/or additional agents described herein may thus be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • Illustrative cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantron
  • cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (0,P′-(DDD)), interferons, and mixtures of these cytotoxic agents.
  • taxol taxol
  • ricin pseudomonas exotoxin
  • gemcitabine cytochalasin B
  • gramicidin D ethidium bromide
  • emetine emetine
  • etoposide tenoposide
  • cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins, taxols, irinotecan, 5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine and vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g.
  • IL-12 or IL-2 IL-12R antagonists
  • Toxin conjugated monoclonal antibodies tumor antigen specific monoclonal antibodies
  • Erbitux Avastin
  • Pertuzumab anti-CD20 antibodies
  • Rituxan ocrelizumab
  • ofatumumab DXL625, HERCEPTIN®, or any combination thereof.
  • Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to the therapeutic agents (e.g. antibodies) to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci.
  • cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104.
  • Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, or binding fragments thereof, with or without the use of a complex-forming agent.
  • radionuclides include beta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67, Yttrium-88, Yttrium-90, Iodine-125, Iodine-131, Samarium-153, Lutetium-177, Rhenium-186 or Rhenium-188, and alpha-emitters such as Astatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.
  • beta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67, Yttrium-88, Yttrium-90, Iodine-125, Iodine-131, Samarium-153, Lutetium-177, Rhenium-186 or Rhenium-188
  • alpha-emitters such as Astatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.
  • Illustrative detectable moieties further include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase and luciferase.
  • Further illustrative fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride.
  • Further illustrative chemiluminescent moieties include, but are not limited to, luminol.
  • Further illustrative bioluminescent materials include, but are not limited to, luciferin and aequorin.
  • Further illustrative radioactive materials include, but are not limited to, Iodine-125, Carbon-14, Sulfur-35, Tritium and Phosphorus-32.
  • the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.
  • the subject and/or animal is a non-mammal, such, for example, a zebrafish.
  • the subject and/or animal may comprise fluorescently-tagged cells (with e.g. GFP).
  • the subject and/or animal is a transgenic animal comprising a fluorescent cell.
  • the subject and/or animal is a human.
  • the human is a pediatric human.
  • the human is an adult human.
  • the human is a geriatric human.
  • the human may be referred to as a patient.
  • the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
  • the subject is a non-human animal, and therefore the invention pertains to veterinary use.
  • the non-human animal is a household pet.
  • the non-human animal is a livestock animal.
  • kits that can simplify the administration of any agent described herein.
  • An illustrative kit of the invention comprises any composition described herein in unit dosage form.
  • the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.
  • the kit can further comprise a label or printed instructions instructing the use of any agent described herein.
  • the kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location.
  • the kit can also further comprise one or more additional agent described herein.
  • the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.
  • the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication.
  • the language “about 50” covers the range of 45 to 55.
  • an “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.
  • something is “decreased” if a read-out of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation.
  • activity is decreased and some downstream read-outs will decrease but others can increase.
  • activity is “increased” if a read-out of activity and/or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.
  • compositional percentages are by weight of the total composition, unless otherwise specified.
  • the word “include,” and its variants is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
  • compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose.
  • therapeutic agents e.g. flagellin-based agents, flagellin-based agents (and/or additional agents) described herein
  • the therapeutic agents are given at a pharmacologically effective dose.
  • a “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease.
  • An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease.
  • administration of therapeutic agents to a patient suffering from cancer provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the disease, e.g., a decrease in tumor burden, a decrease in circulating tumor cells, an increase in progression free survival.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model.
  • Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • a pharmacologically effective amount that will treat cancer will modulate the symptoms typically by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In illustrative embodiments, such modulations will result in, for example, statistically significant and quantifiable changes in the numbers of cancerous cells.
  • Alum adjuvant (IMJECT ALUM, THERMO) was used in combination with CBLB502 to challenge the mice with a model antigen, ovalbumin (OVA).
  • Vaccine was comprised of the following components as shown in TABLE A:
  • Component Amount (per mouse) Ovalbumin 10 mg CBLB502 1 ug/10 ug Alum Adjuvant 500 ug
  • CBLB502 (either 1 ⁇ g or 10 ⁇ g) was mixed with 10 mg of OVA and 500 ⁇ g of Alum adjuvant for 30 minutes at 300 rpm at room temperature.
  • Ovalbumin CBLB502 Alum Adjuvant Group 1 Group 2 10 mg Group 3 10 mg 500 ug Group 4 10 mg 1 ug 500 ug Group 5 10 mg 10 ug 500 ug Two weeks after the initial immunization mice were challenged with an additional booster following the same setup as the original injection. Plasma was collected one, two and four weeks after booster via mandibular vein. Using an ELISA based assay total IgG, IgG1, IgG2a, IgG2b and IgG3 were measured. Results are shown in FIGS. 2-3 . In the figures, Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 ⁇ g,” and Group 5 is also called “SA 10 ⁇ g.”
  • FIG. 2 shows ELISA data for the average response of the five aforementioned mouse treatment groups at the time point of 1 week post-boost.
  • FIG. 3 shows ELISA data for the average response of the five aforementioned mouse treatment groups at the time point of 2 weeks post-boost.
  • FIG. 4 shows ELISA data for the average response of the five aforementioned mouse treatment groups at the time point of 4 weeks post-boost.
  • ALHYDROGEL adjuvant 2% aluminium hydroxide wet gel (colloidal) suspension (INVIVIGEN Catalog #vac-alu-250), was mixed with CBLB502 at different volume ratios, resulting in 10 to 52% ALHYDROGEL suspension in the reaction.
  • CBLB502 concentration was maintained at 10 ⁇ g/ml, corresponding to a 1 ⁇ g/100 ⁇ l inoculation dose (100 ⁇ l inoculation volume is the recommended maximum volume for subcutaneous injection of antigen/adjuvant mixtures per injection site for mice).
  • CBLB502 binding to ALHYDROGEL was also evaluated at different CBLB502 doses.
  • Adsorption of CBLB502 to ALHYDROGEL 52 ⁇ l/100 ⁇ l ALHYDROGEL adjuvant 2%) was determined at CBLB502 doses, ranging from approximately 0.02 to 20 ⁇ g per 100 ⁇ l.
  • the reactions were incubated as described above and unbound CBLB502 was measured by ELISA.
  • the results (TABLE D) demonstrated that almost complete binding of the drug to ALHYDROGEL, 99.82 to 99.99% bound, was accomplished at all tested CBLB502 concentrations.
  • CBLB502 dose Total CBLB502 Unbound Bound Bound ⁇ g/100 ⁇ l ng/ml ng/ml ng/ml (%) 1 20 200000 199.3 199800.7 99.90 2 5 50000 90.6 49909.4 99.82 3 1.25 12500 0.79 12499.2 99.99 4 0.31 3125.0 0.029 3124.97 99.99 5 0.078 781.25 0.023 781.23 99.99 6 0.02 195.31 0.011 195.30 99.99
  • CBLB502 binding to ALHYDROGEL in the presence of TT-SMA vaccine was evaluated. Binding efficiency of CBLB502 to ALHYDROGEL was tested in the presence of a vaccine candidate TT-SMA, succinyl methamphetamine (SMA) hapten conjugated to tetanus toxoid. In a separate study, shown below, this vaccine was administered to mice at a 32 ⁇ g/100 ⁇ l dose to compare efficacy with and without CBLB502.
  • SMA succinyl methamphetamine
  • TT-SMA dose was co-incubated with CBLB502 (0.02 to 20 ⁇ g/100 ⁇ l) and ALHYDROGEL (52 ⁇ l/100 ⁇ l) under standard conditions and unbound CBLB502 was measured by ELISA.
  • the binding efficiency was 99.7 to 99.9% at all tested CBLB502 concentrations, indicating that CBLB502 adsorption to ALHYDROGEL was unaffected with addition of TT-SMA to the reaction mixture
  • CBLB502 dose Total CBLB502 Unbound Bound Bound ⁇ g/100 ⁇ l ng/ml ng/ml ng/ml (%) 1 20 200000 668.88 199331.1 99.67 2 5 50000 50.21 49949.8 99.90 3 1.25 12500 11.44 12488.6 99.91 4 0.31 3125.0 1.65 3123.4 99.95 5 0.078 781.25 0.13 781.1 99.98 6 0.02 195.31 0.06 195.2 99.96
  • CBLB502/ALHYDROGEL using 293-hTLR5-LacZ reporter cells was undertaken.
  • Biological activity of CBLB502 after adsorption to ALHYDROGEL was tested using an in vitro assay which measures activation of a NF- ⁇ B-controlled reporter enzyme, ⁇ -galactosidase, in HEK293-hTLR5::NF- ⁇ N-lacZ (293-hTLR5-LacZ) cells. Since 293-hTLR5-LacZ cells express only one cell membrane-bound toll-like receptor, human TLR5, CBLB502 activity could be determined without interference from other toll-like receptor ligands such as LPS (endotoxin).
  • LPS endotoxin
  • CBLB502/ALHYDROGEL Specific induction of the ⁇ -galactosidase reporter by CBLB502/ALHYDROGEL was demonstrated after incubating the cells with a serially diluted CBLB502/ALHYDROGEL formulation (5 ⁇ g/100 ⁇ l), starting with a 2000-fold dilution in cell growth media and resulting in the concentration range of ALHYDROGEL-adsorbed
  • CBLB502 from 25 to 0.004 ng/ml ( FIG. 5 ).
  • the maximum ⁇ -galactosidase activity was observed at 0.93 ng/ml ALHYDROGEL-adsorbed CBLB502; this value was consistent with typical results of the assay using soluble CBLB502 where the reporter enzyme activity would normally peak around 1 ng/ml.
  • ALHYDROGEL alone tested at same dilutions did not induce the reporter. Therefore, it could be concluded, inter a/ia, that CBLB502 retains its biological activity after adsorption to ALHYDROGEL and becomes readily available for interaction with its target receptor (TLR5) under regular cell culture conditions.
  • TLR5 target receptor
  • CBLB502/ALHYDROGEL suspensions were prepared at CBLB502 inoculation doses ranging from 2.5 to 0.00015 ⁇ g/100 ⁇ l and were added to 293-hTLR5-LacZ cells at 200-fold dilution ( FIG. 6 ).
  • NF- ⁇ B-inducing activity followed a typical dose-response pattern and was measured at sub-nanogram doses of CBLB502/ALHYDROGEL (less than 0.001 ⁇ g/100 ⁇ l), indicating that at such low doses, CBLB502 was not irreversibly bound to the alum adjuvant and remained active.
  • CBLB502/ALHYDROGEL An activity titration of CBLB502/ALHYDROGEL at different CBLB502 doses was undertaken.
  • CBLB502/ALHYDROGEL suspensions were titrated in cell culture media and incubated with 293-hTLR5-LacZ cells.
  • CBLB502 standards were included in the assay to compare the recovered activity.
  • the resulting reporter enzyme activity was plotted against theoretical concentrations of ALHYDROGEL-bound CBLB502, assuming that the protein was completely available ( FIG. 7 ).
  • a formulation of 1 ⁇ g/100 ⁇ l CBLB502 (52 ⁇ l/100 ⁇ l ALHYDROGEL 2%) was prepared and tested for activity after 2 and 6 days storage at 4° C.
  • the results of CBLB502 activity assay using 293-hTLR5-LacZ reporter cells are shown in FIG. 8 .
  • mice were injected subcutaneously with a 1 ⁇ g dose CBLB502 adsorbed to 52 ⁇ l/100 ⁇ l ALHYDROGEL 2% and serum samples were collected between 0.5 and 24 hr post inoculation.
  • Serum concentrations of CBLB502 were determined by ELISA and were found at measurable levels in all groups of animals inoculated with CBLB502/ALHYDROGEL.
  • the pharmacokinetic profile representing per-group mean serum CBLB502 concentrations is shown in FIG. 9 .
  • Half-time elimination of CBLB502 was calculated at 2.4 hr.
  • CBLB502 dissociated rapidly from the complex with ALHYDROGEL after incubation in cell media, containing 10% fetal bovine serum. Therefore, it should be expected to see the protein released from alum into animal bloodstream shortly after inoculation.
  • the serum G-CSF concentration increased about an hour later compared to KC and IL-6 and was maximal between 2 and 4 hr after CBLB502/ALHYDROGEL administration (7141 and 7169 pg/ml per-group mean concentrations at 2 and 4 hr, respectively).
  • CBLB502-induced G-CSF level was decreasing at a slower rate, remaining above baseline at 24 hr time point.
  • the kinetics of cytokine induction after CBLB502/ALHYDROGEL administration was similar to the kinetics observed after injection of the soluble CBLB502 preparations, suggesting consistent effects of this drug formulation.
  • SMA-TT human methamphetamine vaccine
  • Ten groups (n 5) of Balb/c female mice were employed to test 0, 0.03, 0.1, 0.3, 1, 3, 10, 20 ⁇ g of 502 in combination with 32 ⁇ g of TT-SMA and 1.5 mg alum compared to unvaccinated controls and TT-SMA with 502 alone. All vaccinated groups were administered two boosters, at 3 and 6 weeks after the initial vaccination.
  • composition of an adjuvant component for adjuvant-enhanced vaccine is established by testing a range of doses of CBLB502 and CBLB502 optionally combined with an antigen, such as, by way of non-limitation, NOD1 agonist C12-iE-DAP. Additionally, characterization of the effects of these agents on humoral immune response to a model antigen (ovalbumin) is undertaken.
  • an antigen such as, by way of non-limitation, NOD1 agonist C12-iE-DAP.
  • Luciferase detection will be done using a luminescent imager in vivo following luciferin injection at 2, 4, 6 and 18 hours post injection of the adjuvant. For a more accurate and sensitive detection, 3 animals from each group are sacrificed 6 hours post injection of the adjuvant formulation and tissues will be collected from the injection site, lymph nodes nearest to injection site and the liver. The level of luciferase activity indicative of the presence of CBLB 502 is quantitated in these tissue samples following tissue lysis. In addition to functional detection of CBLB 502 based on its expected NF- ⁇ B-inducing activity, an alternative analytical method of an established sensitive ELISA assay for direct detection of CBLB502 in tissue extracts is used.
  • Adjuvant formulations containing the above-described range of doses of CBLB502 are mixed with 10 mg of ovalbumin and vaccination is performed, as described herein, followed by the ELISA determination of titers of different classes of anti-albumin IgG (IgG1, IgG2a, IgG2b and IgG3) antibodies. The results are compared with “Alum only” control. Determination of the effects on different IgG types provides definition of the degrees of engagement of different immunization paths (e.g. T H1 - versus T H2 -mediated routes) by the adjuvant.
  • composition of the adjuvant in terms of anti-ovalbumin antibody inducing efficacy
  • additional enhancement of immunization is tested by adding into the formulation a NOD1 agonist, C12-iE-DAP.
  • mice Female mice are chosen because they have stronger immune responses than male mice and BALB/c mice are a commonly used strain in immunological studies due to their robust immune responses.
  • mice weigh about 25 g and are group-housed (5 per cage) in standard acrylic cages (7′′ ⁇ 11′′ ⁇ 5′′) with corncob bedding polycarbonate and tops. Mice will have ad libitum access to food (Harlan) and water. The vivarium will be maintained at 22 ⁇ 1° C. and on a 12:12 light/dark cycle (lights on at 6 AM). All experimental procedures are approved by the Institutional Animal Care and Use Committee (IACUC) and are within the guidelines delineated in the Guide for the Care and Use of Laboratory Animals.
  • IACUC Institutional Animal Care and Use Committee
  • mice are intramuscularly injected with the vaccine in the gluteal muscle. Each group is optionally immunized with a booster dose with the same vaccine formulation at 3 and 6 weeks post initial immunization. If persistent antibody levels are not maintained for at least 4 weeks, an additional immunization will be given at week 10.
  • ELISA enzyme linked immunosorbent assays
  • ELISA data is analyzed using SigmaPlot (Systat Software Inc.) and background antibody binding to the carrier alone will be subtracted from each sample. Comparisons are made in each plate with a standard curve of purified mouse IgG (Sigma) bound directly in the wells in serial dilution.
  • the CBLB502/alum adjuvant of the above Examples is tested head to head against a flagellin/alum adjuvant using the methods and study design of, for instance, Example 9 (optionally with an antigen). Further, the alum binding and dissociation studies, as well as the mouse evaluation studies, all described in the above Examples, are reapeated with a flagellin/alum adjuvant for comparison to the CBLB502/alum adjuvant. The efficacy of the CBLB502 variant, i.e., CBLB502-S33MX is also tested.
  • TT-SMA methamphetamine vaccine
  • Each immunization dose includes 32 ⁇ g of TT-SMA and adjuvants as follows: Group 1: 0.03 ⁇ g flagellin, Group 2: 0.1 ⁇ g flagellin, Group 3: 0.3 ⁇ g flagellin, Group 4: 1 ⁇ g flagellin, Group 5: 0.03 ⁇ g CBLB502, Group 6: 0.1 ⁇ g CBLB502, Group 7: 0.3 ⁇ g CBLB502, Group 8: 1 pg CBLB502, Group 9: 0.03 g CBLB502-S33MX, Group 10: 0.1 pg CBLB502-S33MX, Group 11: 0.3 ⁇ g CBLB502-S33MX, and Group 12: 1 ⁇ g CBLB502-S33MX.
  • Serum samples are collected and analyzed on days ⁇ 1 (pre-immune serum), 14 (post-prime), 28 (post first boost), and 35 (post second boost). For example, the serum samples are analzyed for the levels of anti-methamphetamine and anti-entolimod antibodies. Analysis indicates that CBLB502/alum and CBLB502-S33MX/alum cause a broader, more diverse, more robust and longer immunostimulatory effect than flagellin/alum. In addition, CBLB502/alum and CBLB502-S33MX/alum activate both T H1 and T H2 -mediated immune response and a greater T H1 -mediated immune response than flagellin/alum.

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Abstract

The present invention relates to, in part, compositions comprising improved flagellin derived constructs and methods of using for vaccination, including adjuvants comprising flagellin-based agents.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. Patent Application No. 15/500,133, filed Jan. 30, 2017, which is a national stage entry of International Patent Application No. PCT/US2015/042887, filed Jul. 30, 2015, which claims the benefit of U.S. Provisional Patent Application Nos. 62/031,116, filed Jul. 30, 2014; 62/110,744, filed Feb. 2, 2015; and 62/117,366, filed Feb. 17, 2015 the entire contents of which are herein incorporated by reference.
    • FIELD OF THE INVENTION
  • The present invention relates to, inter a/ia, new compositions and methods for vaccination, including adjuvants comprising flagellin-based agents.
    • DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
  • The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: CLE019PC SequenceListing.txt; date recorded: Jul. 24, 2015; file size: 261 KB).
    • BACKGROUND
  • Vaccines are one of the most effective preventative health tools available against infectious diseases, cancers, and allergies. Vaccination aims to generate a strong immune response to the administrated antigen and provide long-term protection against a disorder. Often, however, an antigen alone is insufficient to stimulate protective immunity.
  • Vaccine adjuvants are compounds that enhance the specific immune responses against antigens in vaccines. Currently, several hundred natural and synthetic compounds are known to have adjuvant activity but only alum salts and AS04 are licensed for use in humans in the United States. This limited list of adjuvants is insufficient for meeting the functional needs of effective vaccination. For example, although alum is able to induce a good TH2 response, it has little capacity to stimulate cellular (TH1) immune responses which are so important for protection against many pathogens.
  • Accordingly, there remains a need for improved vaccines and/or adjuvants that can effectively stimulate a subject's immune response.
    • SUMMARY OF THE INVENTION
  • Accordingly, in some aspects, the present invention provides for improved vaccines and/or adjuvants.
  • In some aspects, the present invention provides a vaccine composition, comprising an adjuvant comprising a flagellin-based agent, such as, for example, CBLB502 (a/k/a entolimod) as well as any of the flagellin-based agents or derivatives described herein (e.g. Table 1), and an aluminum gel or salt and an antigen and, optionally, an additional adjuvant. In some aspects, the present invention provides an adjuvant composition, comprising an adjuvant comprising a flagellin-based agent, including CBLB502 as well as any of the flagellin-based agents or derivatives described herein (e.g. Table 1), and an aluminum gel or salt. In various embodiments, the vaccine described herein causes an improvement in adjuvant properties relative to a vaccine comprising the antigen and the aluminum gel or salt alone. In various embodiments, the vaccine and/or adjuvant described herein causes a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone and/or the adjuvant comprising the aluminum gel or salt alone. In various embodiments, the vaccine and/or adjuvant described herein causes immunostimulation of one or more of TH1 and TH2-mediated immune response (e.g. both of TH1 and TH2-mediated immune response) and/or the vaccine and/or adjuvant described herein causes immunostimulation of TH1-mediated immune response at levels greater than a vaccine comprising the antigen and the aluminum gel or salt alone and/or the adjuvant comprising the aluminum gel or salt alone.
  • In some embodiments, the flagellin-based agent may be a flagellin molecule or flagellin-based agent, or variants thereof that has TLR5 agonist activity. The flagellin-based agent may comprise one or more of the sequences of Table 1 (SEQ ID Nos.: 1-252), or variants thereof that have TLR5 agonist activity and can include a variant of Salmonella dublin wild type flagellin (SEQ ID No: 1), CBLB502 (SEQ ID NO: 2) or variants thereof (including closely-related variants such as S33ML (SEQ ID NO: 35), CBLB502-485CT (CBLB533, SEQ ID NO: 71), and CBLB502-S33MX (SEQ ID NO: 150)) and more distantly related variants such as flagellin derivatives from a thermophilic microorganism or flagellin derivatives from a microorganism well-tolerated by human (e.g. SEQ ID NOs: 243-252). In some embodiments, the aluminum gel or salt is selected from aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate, and ALHYDROGEL.
  • In various embodiments, the present vaccine composition is part of the following vaccines (e.g. the antigens of these vaccines may be used as the antigen of the present vaccines): DTP (diphtheria-tetanus-pertussis vaccine), DTaP (diphtheria-tetanus-acellular pertussis vaccine), Hib (Haemophilus influenzae type b) conjugate vaccines, Pneumococcal conjugate vaccine, Hepatitis A vaccines, Poliomyelitis vaccines, Yellow fever vaccines, Hepatitis B vaccines, combination DTaP, Tdap, Hib, Human Papillomavirus (HPV) vaccine, Anthrax vaccine, and Rabies vaccine.
  • In various embodiments, the vaccine described herein is formed, in part, by mixing the flagellin-based agent and aluminum gel or salt to form a stable complex, the ratio (w/w) of flagellin-based agent to aluminum gel or salt being about 1:500 or less (e.g. about 1:500, or about 1:600, or about 1:700, or about 1:800, or about 1:900, or about 1:1000, or about 1:2000, or about 1:5000). In some embodiments, the flagellin-based agent and aluminum gel or salt are mixed in a ratio that is substantially below a loading capacity of the aluminum gel or salt. In some embodiments, the flagellin-based agent and antigen are adsorbed to the aluminum gel or salt.
  • In another aspect, the present invention provides a method of vaccinating a subject against a disorder, comprising administering an effective amount of a vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with the disorder. In another aspect, the present invention provides a method of immunostimulating a subject in advance of or concurrent with vaccination, comprising administering an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt, wherein both TH1 and TH2-mediated immune responses are immunostimulated. In various embodiments the disorder is selected from infectious diseases, cancer, allergy, and autoimmune diseases (by way of example, diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, Human Papillomavirus (HPV) infection, various cancers, anthrax, rabies, Japanese Encephalitis, meningitis, measles, mumps, rubella, gastroenteritis, smallpox, typhoid fever, varicella (chickenpox), rotavirus, and shingles).
  • The details of the invention are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIGS. 1A and 1B show the 13 conserved amino acids of flagellin that may be important for TLR5 activity. FIGS. 1A and 1B show a comparison of amino acid sequences of the conserved amino (FIG. 1A) and carboxy (FIG. 1B) terminus from 21 species of bacteria. The 13 conserved amino acids important for TLR5 activity are indicated by the letter “C” at the bottom of each column. The amino add sequences are identified by their accession numbers from TrEMBL (first letter=Q) or Swiss-Prot (first letter=P). With respect to FIG. 1A, SEQ ID Nos: 253-273 correspond to the sequences listed on the figure from top to bottom, respectively. With respect to FIG. 1B, SEQ ID Nos: 274-294 correspond to the sequences listed on the figure from top to bottom, respectively.
  • FIG. 2 shows ELISA data for the average response of five mouse treatment groups at the time point of 1 week post-boost. Panel A is total IgG, Panel B is IgG1, Panel C is IgG2a, Panel D is IgG2b and Panel E is IgG3. Groups 1-5 are defined in TABLE B (Example 2): Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 μg,” and Group 5 is also called “SA 10 μg.”
  • FIG. 3 shows ELISA data for the average response of five mouse treatment groups at the time point of 2 weeks post-boost. Panel A is total IgG, Panel B is IgG1, Panel C is IgG2a, Panel D is IgG2b and Panel E is IgG3. Groups 1-5 are defined in TABLE B (Example 2): Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 μg,” and Group 5 is also called “SA 10 μg.”
  • FIG. 4 shows ELISA data for the average response of five mouse treatment groups at the time point of 4 weeks post-boost. Panel A is total IgG, Panel B is IgG1, Panel C is IgG2a, Panel D is IgG2b and Panel E is IgG3. Groups 1-5 are defined in TABLE B (Example 2): Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 μg,” and Group 5 is also called “SA 10 μg.”
  • FIG. 5 shows a determination of CBLB502/ALHYDROGEL activity using a cell-based NF-KB activation assay. CBLB502/ALHYDROGEL formulation (5 μg/100 μl) and ALHYDROGEL control were serially diluted in assay media 2000- to 13122000-fold (2.6-0.0004 μg/ml aluminium) and incubated with 293-hTLR5-LacZ cells for 20 hr. Activity of β-galactosidase was measured after addition of cell lysis buffer and ONPG substrate and expressed as optical density at 414 nm (OD414).
  • FIG. 6 shows NF-κB-inducing activity of CBLB502/ALHYDROGEL formulated with different CBLB502 doses. 293-hTLR5-LacZ cells were incubated with CBLB502/ALHYDROGEL suspensions prepared at CBLB502 doses ranging from 2.5 to 0.00015 μg/100 μl and diluted 200-fold with media. Activity of β-galactosidase reporter enzyme was measured after 20 hr incubation.
  • FIG. 7 shows dose-response titration curves CBLB502/ALHYDROGEL generated with 293-hTLR5-LacZ reporter cells. CBLB502 was adsorbed to ALHYDROGEL at indicated doses (20 μg/100 μl: top panel, 0.31 μg/100 μl: middle panel and 0.078 μg/100 μl: bottom panel), serially diluted with media and assayed together with soluble CBLB502 standards.
  • FIG. 8 shows the activity of CBLB502/ALHYDROGEL (1 μg/100 μl dose) stored at 4° C. for 2 and 6 days. NF-κB-inducing activity was measured using 293-hTLR5-LacZ reporter cell assay.
  • FIG. 9 shows pharmacokinetics of CBLB502 in mouse serum after subcutaneous injection of a 1 μg dose of CBLB502 adsorbed to ALHYDROGEL 2%. The graph shows per-group mean serum CBLB502 concentrations and SEM (standard error of the mean) for each time point.
  • FIG. 10 shows induction of KC, G-CSF and IL-6 following s.c. administration of a 1 μg dose of CBLB502 adsorbed to ALHYDROGEL 2%. Serum concentrations of the cytokines were determined using R&D Systems DuoSet kits DY453 (KC, top panel), DY414 (G-CSF, middle panel) and DY406 (IL-6, bottom panel). The graphs show per-group mean serum cytokine concentrations and SEM (standard error of the mean) for each time point.
  • FIG. 11 shows immunopotentiating effects of a 502 adjuvant. Anti-MA IgG (pg/ml) response (panel A) and anti-502 antibody response (panel B) by ELISA to TT-SMA vaccine in the presence and absence of alum or entolimod (a varying range from 0 to 20 μg) in pooled sera collected at week 2, 4 and 6 post initial vaccination is shown. Data represent the levels of antibody in the pooled samples from each group (n=5/group).
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The present invention is also based, in part, on the surprising discovery that flagellin-based agents, including CBLB502 and any of the flagellin-based agents (e.g. agents comprising the sequences of Table 1, including MX-33) described herein, can be mixed with aluminum gels or salts (and, optionally antigens), in ratios well-below the loading/adsorbing capacity of the aluminum gel or salt (e.g. 1:500), and cause a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone and/or the adjuvant comprising the aluminum gel or salt alone. The inventors have also surprisingly discovered that the immunostimulatory effect can influence both the TH1 and TH2-mediated arms of an immune response. The inventors have also surprisingly discovered that low amounts of flagellin-based agents, including CBLB502 and any of the flagellin-based agents (e.g. agents comprising the sequences of Table 1, including MX-33) relative to antigen are effective for immunostimulation.
  • In one aspect, the invention provides a vaccine composition, comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen and, optionally, an additional adjuvant. In one aspect, the invention provides an adjuvant comprising a flagellin-based agent and an aluminum gel or salt.
  • In some embodiments, the described vaccine causes an improvement in adjuvant properties relative to a vaccine comprising the antigen and the aluminum gel or salt alone. In various embodiments, the vaccine and/or adjuvant described herein causes a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone (or as compared to the vaccine comprising the antigen and flagellin-based agent alone) and/or the adjuvant comprising the aluminum gel or salt alone (or as compared to the adjuvant comprising the flagellin-based agent alone).
  • In some embodiments, the described vaccine and/or described adjuvant causes immunostimulation of one or more of TH1 and TH2-mediated immune response. In some embodiments, the described vaccine and/or described adjuvant causes immunostimulation of both of TH1 and TH2-mediated immune response. In some embodiments, the described vaccine and/or described adjuvant causes immunostimulation of TH1-mediated immune response at levels greater than a vaccine comprising the antigen and the aluminum gel or salt alone or an adjuvant comprising the aluminum gel or salt alone.
  • TH1-mediated immune response (or “Type 1 response”) largely involves interaction with macrophages and CD8+ T cells and may be linked to interferon-γ, TNF-β, interleukin-2, and interleukin-10 production. The TH1-mediated immune response promotes cellular immune system and maximizes the killing efficacy of the macrophages and the proliferation of cytotoxic CD8+ T cells. The TH1-mediated immune response also promotes the production of opsonizing antibodies (e.g. IgG, IgM and IgA). The Type 1 cytokine IFN-γ increases the production of interleukin-12 by dendritic cells and macrophages, and via positive feedback, IL-12 stimulates the production of IFN-γ in helper T cells, thereby promoting the TH1 profile. Interferon-γ also inhibits the production of cytokines such as interleukin-4, a cytokine associated with the Type 2 response, and thus it also acts to preserve its own response.
  • TH2-mediated immune response (or “Type 2 response”) largely involves interaction with B-cells, eosinophils, and mast cells and may be linked to interleukin-4, interleukin-5, interleukin-6, interleukin-9, interleukin-10, and interleukin-13. TH2-mediated immune response promotes humoral immune system and may stimulate B-cells into proliferation, induce B-cell antibody class switching, and increase neutralizing antibody production (e.g. IgG, IgM and IgA as well as IgE antibodies). Other functions of the Type 2 response include promoting its own profile using two different cytokines. Interleukin-4 acts on helper T cells to promote the production of TH2 cytokines (including itself; it is auto-regulatory), while interleukin-10 (IL-10) inhibits a variety of cytokines including interleukin-2 and IFN-γ in helper T cells and IL-12 in dendritic cells and macrophages. The combined action of these two cytokines suggests that once the T cell has decided to produce these cytokines, that decision is preserved (and also encourages other T cells to do the same).
  • The division is medically relevant to, inter alia and without wishing to be bound by theory, the fact that alum adjuvants are only effective at inducing a TH2-mediated response and not a TH1 mediated response (see, e.g., Smith Korsholm, et al. Immunology. January 2010; 129(1): 75-86, the contents of which are hereby incorporated by reference in their entirety).
  • In various embodiments, the stimulation of TH1 and TH2-mediated immune responses may be measured by assays known in the art, including a number of antibody surrogate assays (e.g. ELISA and the like). For instance, without wishing to be bound by theory, IgG1 is associated with a TH2-like response, while a TH1 response is associated with the induction of IgG2a, IgG2b, and IgG3 antibodies.
  • In some embodiments, the described vaccine and/or described adjuvant causes an increase in titer of one or more of IgG1, IgG2a, IgG2b, and IgG3 antibodies (e.g. relative to the adjuvant comprising the aluminum gel or salt or flagellin-based agent alone, or relative to the vaccine comprising the antigen and the aluminum gel or salt alone (or flagellin-based agent) alone)). In some embodiments, the described vaccine and/or described adjuvant causes a relative increase in the titer of all of IgG1, IgG2a, IgG2b, and IgG3 antibodies. In some embodiments, the described vaccine and/or described adjuvant causes a relative increase in the titer of more IgG3 antibodies than the described vaccine and/or described adjuvant in the absence of a flagellin-based agent (or the described vaccine and/or described adjuvant in the absence of an aluminum gel or salt alone).
  • Accordingly, in some embodiments, the described vaccine and/or described adjuvant causes a diversified immune response. For example, in some embodiments, the total IgG generated by the described vaccines and/or adjuvants is greater than the described vaccines and/or adjuvants without the flagellin-based agent.
  • In some embodiments, the flagellin-based agent may be a flagellin molecule or flagellin-related polypeptide, or variants thereof that have TLR5 agonist activity. The flagellin-based agent may be from various sources, including a variety of Gram-positive and Gram-negative bacterial species. In some embodiments, the flagellin-based agents may have an amino acid sequence that is derived from any of the flagellins from bacterial species that are depicted in, for example, FIG. 7 of U.S. Patent Publication No. 2003/0044429, the contents of which are incorporated herein by reference in their entirety. The flagellin-based agent may have nucleotide sequences related to those encoding the flagellin polypeptides listed in, for example, FIG. 7 of U.S. 2003/0044429, which are publicly available at sources including the NCBI Genbank database.
  • The flagellin-based agent may be the major component of bacterial flagellum. The flagellin-based agents may be composed of one, or two, or three, or four, or five, or six, or seven domains or fragments thereof (see, e.g., FIG. 10 of U.S. Pat. No. 8,324,163, the contents of which are incorporated herein by reference in their entirety). The domains may be selected from ND0, ND1, ND2, D3, CD2, CD1, and CDO. Domains 0 (D0), 1 (D1), and 2 (D2) may be discontinuous and may be formed when residues in the amino terminus and carboxy terminus are juxtaposed by the formation of a hairpin structure. The amino and carboxy terminus comprising the D1 and D2 domains may be most conserved, whereas the middle hypervariable domain (D3) may be highly variable. The non-conserved D3 domain may be on the surface of the flagellar filament and may contain the major antigenic epitopes. The potent proinflammatory activity of flagellin may reside in the highly conserved N and CD1 and D2 regions.
  • The flagellin-based agents may be from a species of Salmonella, representative examples of which are S. typhimurium and S. dublin (encoded by GenBank Accession Number M84972). The flagellin related-polypeptide may be a fragment, variant, analog, homolog, or derivative of wild type flagellin (SEQ ID NO: 1), or combination thereof. A fragment, variant, analog, homolog, or derivative of flagellin may be obtained by rational-based design based on the domain structure of flagellin and the conserved structure recognized by TLR5.
  • The flagellin-based agent may be related to a flagellin polypeptide from any Gram-positive or Gram-negative bacterial species including, but not limited to, the flagellin polypeptides disclosed in U.S. Pat. Pub. 2003/0044429, the contents of which are incorporated herein, and the flagellin peptides corresponding to the Accession numbers listed in the BLAST results shown in FIG. 7 (panels A-F) of U.S. Patent Pub. 2003/0044429, or variants thereof.
  • In some embodiments, the flagellin-based agent comprises or consists of any of the polypeptides or nucleic acids encoding said polypeptides listed in Table 1. In some embodiments, the flagellin-based agent is encoded by the nucleotide sequences listed in Table 1. In a further embodiment, the flagellin-based agent comprises the polypeptides listed in Table 1. In some embodiments, the flagellin-based agent comprises one or more of SEQ ID NOs.: 1-252. In some embodiments, the flagellin-based agent comprises a flexible linker. In a further embodiment, the flexible linker comprises SEQ ID NO: 16. In yet a further embodiment, the flexible linker comprises SEQ ID NO: 242.
  • In some embodiments, the flagellin-based agent is a variant of SEQ ID NO: 1. In variois embodiments, the flagellin-based agent is not SEQ ID NO: 1.
  • In some embodiments, the flagellin-based agent comprises or consists of CBLB502 (SEQ ID NO: 2) or is a variant of CBLB502 (SEQ ID NO: 2). In various embodiments, CBLB502 provides the advantage of, inter alia, removal of epitopes that generate neutralizing anti-flagellin antibodies and therefore allow for the surprising adjuvant properties seen here in combination with alum.
  • In some embodiments, the flagellin-based agent comprises mutations in epitopes recognized by neutralizing anti-CBLB502 antibodies. The flagellin-based agent may comprise one or more mutations in the epitopes recognized by neutralizing anti-CBLB502 antibodies which inhibit or abrogate the ability of the antibodies to neutralize the composition. In yet a further embodiment, the flagellin-based agent comprises a truncation and mutations in one or more epitopes. In a further embodiment, the mutations comprise replacement of the epitope residues with alanine. In a further embodiment, the mutated epitopes comprise one or more of the following residues: E153, S444, T154, N440, Q142, F131, D443, N68, T447, S110, Q117, R124, D113, E120, N127, and Q128.
  • The flagellin-based agent may comprise insertions, deletions, transposon insertions, and changes to any one of the D0, D1, D2, and the variable D3 domains. The D3 domain may be substituted in part, or in whole, with a hinge or linker polypeptide that allows the D1 and D2 domains to properly fold such that the variant stimulates TLR5 activity.
  • In some embodiments, the flagellin-based agent may be a minimal functional core of a flagellin, for example, deleting residues relative to the already shortened CBLB502 molecule. In some embodiments, the flagellin-based agent has altered amino acid identity relative to wild type, including deletions, additions and substitutions, that provide for improved activity. In some embodiments, the flagellin-based agent is derived from CBLB502 (SEQ ID NO: 2). In some embodiments, the flagellin-based agent comprises a truncation in one or more domains. In a further embodiment, the flagellin-based agent comprises a deletion in an N-terminal domain.
  • In yet a further embodiment, the flagellin-based agent comprises a deletion in the NDO domain. In yet a further embodiment, the flagellin-based agent comprises a deletion of the entire NDO domain. In a further embodiment, the flagellin-based agent comprises a deletion in a C-terminal domain. In yet another embodiment, the flagellin-based agent comprises a deletion in the CD0 domain. In yet another embodiment, the flagellin-based agent retains amino acids 470-485 of the CD0 domain. In yet a further embodiment, the flagellin-based agent is CBLB502-S33 (SEQ ID NO: 17).
  • The flagellin-based agent may comprise at least 10, 11, 12, or 13 of the 13 conserved amino acids shown in FIG. 1A and FIG. 1B (positions 89, 90, 91, 95, 98, 101, 115, 422, 423, 426, 431, 436 and 452). The flagellin may be at least 30-99% identical to amino acids 1-174 and 418-505 of SEQ ID NO: 1.
  • In some embodiments, the flagellin-based agent comprises a tag. In yet a further embodiment, the tag is attached to the N-terminus of the flagellin-based agent. In yet another embodiment, the tag is attached to the C-terminus of the flagellin-based agent.
  • In some embodiments, the flagellin-based agent comprises a flexible linker. In a further embodiment, the flexible linker comprises SEQ ID NO: 16. In yet a further embodiment, the flexible linker comprises SEQ ID NO:242.
  • In various embodiments, the flagellin-based agent is one or more of the flagellin related composition derived from SEQ ID NO: 2. In various embodiments, the flagellin-based agent is one or more of CBLB502-S33ML (SEQ ID NO: 35), CBLB502-485CT (CBLB533, SEQ ID NO: 71), and CBLB502-S33MX (SEQ ID NO: 150).
  • In various embodiments, the flagellin-based agent is a flagellin derivative from a thermophilic microorganism. In various embodiments, the flagellin-based agent is a flagellin derivative from a microorganism well-tolerated by human. In some embodiments, the flagellin-based agent is one or more of SEQ ID NOs: 243-252.
  • In some embodiments, a variant includes molecules that have TLR5 agonist activity. In some embodiments, a variant includes molecules comprising an amino acid sequence having at least about 70% (e.g.
  • about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.
  • In some embodiments, the flagellin-based agent comprises or consists of any of the polypeptides or nucleic acids encoding said polypeptides listed in Table 1. In some embodiments, the flagellin-based agent is encoded by the nucleotide sequences listed in Table 1. In a further embodiment, the flagellin-based agent comprises one or more of the polypeptides listed in Table 1. In some embodiments, the flagellin-based agent comprises or consists of polypeptides encoded by either SEQ ID NOs: 69 or 70. In some embodiments, the flagellin-based agent comprises or consists of the polypeptides of SEQ ID NO: 71, “CBLB543”. In some embodiments, the flagellin-based agent comprises or consists of polypeptides encoded by either SEQ ID NOs: 149 or 151. In some embodiments, the flagellin-based agent comprises or consists of the polypeptides of SEQ ID NO: 150, “CBLB533”.
  • TABLE 1
    Illustrative Flagellin-Based Agents
    SEQ DNA/
    ID Construct Name PRT Species Sequence
    0001 Wild type PRT Salmonella MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRN
    dublin ANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFN
    GVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTY
    AAGADKYRVDINSGAVVTDAAAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIA
    GAIKGGKEGDTFDYKGVTFTIDTKTGDDGNGKVSTTINGEKVTLTVADIATGAADVNAATLQSSKN
    VYTSVVNGQFTFDDKTKNESAKLSDLEANNAVKGESKITVNGAEYTANATGDKITLAGKTMFIDKT
    ASGVSTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRI
    EDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR
    0002 CBLB502 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMAQVINTNSLSLLTQNNLNKSQSSLSSAIE
    Sequence RLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSV
    QATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKI
    DVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAI
    QNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLL
    R
    0003 T7 Promoter (forward) DNA Artificial TAATACGACTCACTATAGGGG
    Sequence
    0004 FliC AA74-80 (forward) DNA Artificial ATTGCGCAGACCACTGAAGG
    Sequence
    0005 Thrombin cleavage site PRT Artificial LVPRGS
    Sequence
    0006 Enterokinase cleavage Artificial DDDDK
    site Sequence
    0007 NS (N-terminal spoke PRT Artificial SSGLRINSAKDDA
    region; Ser32-Ala44) Sequence
    0008 CS (C-terminal spoke PRT Artificial EDADYA
    region; Glu464 to Sequence
    Ala469)
    0009 linker PRT Artificial AASAGAGQGGGGSG
    Sequence
    0010 linker PRT Artificial EGKSSGSGSESKST
    Sequence
    0011 linker PRT Artificial GGGRTSSSAASAGAGQGGGGSG
    Sequence
    0012 linker PRT Artificial GPSG
    Sequence
    0013 linker PRT Artificial GSAGSAAGSGEF
    Sequence
    0014 linker PRT Artificial GSPG
    Sequence
    0015 linker PRT Artificial KESGSVSSEQLAQFRSLD
    Sequence
    0016 linker PRT Artificial SPGISGGGGGILDSMG
    Sequence
    0017 Mutant 33-485 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPSGLRINSAKDDAAGQAIANRFTSNIK
    Mutant S33 Sequence GLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRV
    SNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSM
    GTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDA
    DYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR
    0018 Mutant 33-485 DNA Artificial GCAGATTCTGCAGCAGGCTGGTTGATAATCTGGCGCAGGCTAACCAGG
    Forward Primer Sequence
    CBLB485
    0019 Mutant 33-485 DNA Artificial TCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTT
    502 template sequence Sequence
    0020 Mutant 33-485 DNA Artificial CCTGGTTAGCCTGCGCCAGATTATCAACCAGCCTGCTGCAGAATCTGC
    Reverse Primer Sequence
    CBLB485
    0021 Mutant 33-485 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of 485 Sequence AACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    Mutant (T7 Promoter to GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGC
    Stop) GAAGGATCCGTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATT
    GCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGC
    ATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGT
    GCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCA
    GGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGG
    TGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAAC
    CATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAAT
    TCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAA
    GACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTC
    AAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTAC
    CAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACT
    ATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTTGATAA
    0022 Mutant 33-485 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPSGLRINSAKDDAAGQAIANRFTSNIK
    Expressed Mutant 33- Sequence GLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRV
    485 SNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSM
    GTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDA
    DYATEVSNMSKAQILQQAG
    0023 Mutant 450T PRT Artificial MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRN
    Mutant 506T Sequence ANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFN
    GVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDA
    AAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSN
    MSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHGMASMTGGQQMGRDLYDDDDKD
    P
    0024 Mutant 450T DNA Artificial ATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCT
    Mutant 506T Sequence CAGTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAA
    AGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTC
    AGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAAT
    GAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAA
    CTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCG
    CGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATC
    CAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGC
    CTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTA
    GACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCC
    ACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAAT
    TCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGC
    GCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGAT
    TCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCT
    CTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTG
    GACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGTAAGTCGACAAGCTT
    GCG
    0025 Mutant 450T DNA Artificial CGAAAGACCATATGGCAGGCCAGGCGATTGC
    Forward F45CT Sequence
    0026 Mutant 450T DNA Artificial CGCAAGCTTGTCGACTTACGGATCCTTATCGTC
    Reverse R45CT Sequence
    0027 Mutant 450T DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT
    Sequence of 450T Sequence TAACTTTAAGAAGGAGATATACATATGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAAT
    construct ATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACT
    GAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGC
    CACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCT
    GGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGA
    CAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAA
    AATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGG
    TGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGA
    AAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTT
    CTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAA
    CCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATA
    TGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTT
    CCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTATG
    GCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCC
    GTAAGTCGAC
    0028 Mutant 450T PRT Artificial MAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLK
    Expressed Mutant 450T Sequence SIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNS
    PGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNT
    VTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHGM
    ASMTGGQQMGRDLYDDDDKDP
    0029 Mutant 33GP5 DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Expressed Mutant 33ML Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCGGACCATCA
    GGTCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTT
    AACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGG
    TGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAA
    TGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGT
    GGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCT
    TGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAA
    CGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCG
    CAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCAT
    CATCATCATGGTTAA
    0030 Mutant 33GPS PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Expressed Mutant 33ML Sequence ATNGTNSDSDLKSIGPSGQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDL
    QKIDVKSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSAR
    SRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0031 Mutant 33GP5 DNA Artificial GATATACATATGAGCGGGTTACGGATCAACAG
    Forward primer FSY3CT Sequence
    0032 Mutant 33GP5 DNA Artificial AGATCTCCCGGGGAATTAACATTGAACCC
    Reverse primer RMIMxN Sequence
    0033 Mutant 33GP5 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    mutant 33GP5 GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTGGACCATCAGGTGAAATTCAGC
    AACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGT
    CTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATC
    TGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAAGTA
    CCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTC
    TGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATC
    TGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTA
    AAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAA
    AACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0034 Mutant 33GP5 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Expressed Mutant Sequence ATGPSGEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFN
    33GP5 VNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSN
    MSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0035 Mutant 33ML PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant 330T (Fixed A) Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQ
    NRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVP
    RGSHHHHHHG
    0036 Mutant 33ML DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Mutant 330T (Fixed A) Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACG
    CTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAG
    TGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACC
    TTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCA
    ACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCG
    CAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCAT
    CATCATCATGGTTAA
    0037 Mutant 33ML DNA Artificial TCTAGACCCGGGAAGTACCGCTAACCCACTGGCTTCAATTG
    Forward primer F502ML Sequence
    0038 Mutant 33ML DNA Artificial CCAGTCATGTCGACTTAACCATGATGATGATGATGATGAG
    Reverse primer R33CT Sequence
    0039 Mutant 33ML DNA Artificial CTCATCATCATCATCATCATGGTTAAGTCGACAAGCTTGCGGCCGCAGAGCTCGC
    502 template sequence Sequence
    0040 Mutant 33ML DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    33ML construct Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0041 Mutant 33ML DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Expressed Mutant 33ML Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTA
    ATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAG
    GTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGT
    TAA
    0042 Mutant 33ML PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant 33ML Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0043 Mutant 370T DNA Artificial ATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCT
    delta NDO mutant based Sequence CAGTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACGGCGCGAA
    on CBLB506T AGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTC
    AGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAAT
    GAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAA
    CTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCG
    CGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATC
    CAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGC
    CTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTA
    GACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCC
    ACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAAT
    TCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGC
    GCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGAT
    TCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCT
    CTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTG
    GACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGTAAGTCGACAAGCTT
    GCG
    0044 Mutant 370T PRT Artificial MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINGAKDDAAGQAIANRFTSNIKGLTQASRN
    delta NDO mutant based Sequence ANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFN
    on CBLB506T GVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDA
    AAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSN
    MSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHGMASMTGGQQMGRDLYDDDDKD
    P
    0045 Mutant 370T DNA Artificial CTCTGGTCATATGATCAACAGCGCGAAAGACGATGC
    Forward F37CT Sequence
    0046 Mutant 370T DNA Artificial TCTAGAGTCGACTATTAAGCCATACCATGATGATGATGATGATGAG
    Reverse R37CT Sequence
    0047 Mutant 370T DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT
    370T construct Sequence TAACTTTAAGAAGGAGATATACATATGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGC
    GATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGA
    CGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGC
    GTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTA
    TCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTA
    ACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGT
    GAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAAT
    GTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATC
    AATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGC
    ATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGC
    CATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATG
    CTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTT
    CCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGT
    TCTCATCATCATCATCATCATGGTATGGCTTAATAGTCGAC
    0048 Mutant 370T PRT Artificial MINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNG
    Mutant 370T Sequence TNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLG
    LDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFD
    SAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSH
    HHHHHGMA
    0049 Mutant 445 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPMAQVINTNSLSLLTQNNLNKSQSSLS
    502-SY1 Sequence SAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVR
    ELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITID
    LQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSS
    LGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVL
    SLLR
    0050 Mutant 445 DNA Artificial ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATG
    502-SY1 Sequence GGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGCGAAGGATCCGATGGCACAAGTCA
    TTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCTCAGTCCTCACTGA
    GTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCA
    GGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAAC
    GCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAA
    CCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATC
    TGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGA
    CTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTA
    ACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATG
    GGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGT
    ACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATT
    GATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGTTTT
    GATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATC
    GAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCT
    GGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTAA
    0051 Mutant 445 DNA Artificial GGCAATTCAAAACCGTTTTGATTAAGCCATTACCAACCTTGG
    Forward Primer Sequence
    CBLB445
    0052 Mutant 445 DNA Artificial CCAAGGTTGGTAATGGCTTAATCAAAACGGTTTTGAATTGCC
    everse Primer Sequence
    CBLB445
    0053 Mutant 445 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    mutant 445 Sequence AACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGC
    GAAGGATCCGATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCT
    GAACAAATCTCAGTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAA
    CAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAG
    GTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGT
    GCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAA
    CGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGA
    AATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCA
    GATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGA
    TGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTG
    GTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTA
    CCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTC
    TGGGGGCAATTCAAAACCGTTTTGATTAA
    0054 Mutant 445 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPMAQVINTNSLSLLTQNNLNKSQSSLS
    mutant 445 Sequence SAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVR
    ELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITID
    LQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSS
    LGAIQNRFD
    0055 Mutant 461 DNA Artificial CAATCTGAACTCCGCGCGTTGACGTATCTAAGATGCTGACTATGC
    Forward Primer Sequence
    CBLB461
    0056 Mutant 461 DNA Artificial GCATAGTCAGCATCTTAGATACGTCAACGCGCGGAGTTCAGATTG
    Reverse Primer Sequence
    CBLB461
    0057 Mutant 461 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Mutant 461 Sequence AACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGC
    GAAGGATCCGATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCT
    GAACAAATCTCAGTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAA
    CAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAG
    GTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGT
    GCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAA
    CGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGA
    AATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCA
    GATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGA
    TGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTG
    GTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTA
    CCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTC
    TGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATC
    TGAACTCCGCGCGTTGACGTATCTAA
    0058 Mutant 461 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPMAQVINTNSLSLLTQNNLNKSQSSLS
    Mutant 461 Sequence SAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVR
    ELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITID
    LQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSS
    LGAIQNRFDSAITNLGNTVTNLNSAR
    0059 Mutant 467 DNA Artificial CGTAGCCGTATCGAAGATGCTTAATAGGCAACGGAAGTTTCTAATATG
    Forward Primer Sequence
    CBLB467
    0060 Mutant 467 DNA Artificial CATATTAGAAACTTCCGTTGCCTATTAAGCATCTTCGATACGGCTACG
    Reverse Primer Sequence
    CBLB467
    0061 Mutant 467 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Mutant 467 Sequence AACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGC
    GAAGGATCCGATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCT
    GAACAAATCTCAGTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAA
    CAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAG
    GTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGT
    GCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAA
    CGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGA
    AATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCA
    GATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGA
    TGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTG
    GTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTA
    CCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTC
    TGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATC
    TGAACTCCGCGCGTAGCCGTATCGAAGATGCTTAATAG
    0062 Mutant 467 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPMAQVINTNSLSLLTQNNLNKSQSSLS
    Mutant 467 Sequence SAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVR
    ELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITID
    LQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSS
    LGAIQNRFDSAITNLGNTVTNLNSARSRIEDA
    0063 Mutant 4700T DNA Artificial ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATG
    CBLB502 Sequence GGTCGGGATCTGTACGACGATGACGATAAGGATCCGATGGCACAAGTCATTAATACAAACAG
    CCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCTCAGTCCTCACTGAGTTCCGCTATTGA
    GCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATT
    GCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGC
    ATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGT
    GCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCA
    GGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGG
    TGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAAC
    CATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAAT
    TCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAA
    GACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTC
    AAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTAC
    CAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACT
    ATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTC
    TGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTAA
    0064 Mutant 4700T DNA Artificial CGATAAGGATCATATGGCACAAGTCATTAATAC
    Forward Primer F4700T Sequence
    0065 Mutant 4700T DNA Artificial AGATCTGTCGACTTAACCATGATGATGATGATGATGAGAACCCCGCGGAACCAGTGCATAGT
    Reverse Primer R4700T Sequence CAGCATCTTCGATACG
    0066 Mutant 4700T DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Mutant 4700T Sequence AACTTTAAGAAGGAGATATACATATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGA
    CCCAGAATAACCTGAACAAATCTCAGTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTG
    GTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCAC
    TTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCA
    GACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTG
    TTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGC
    AACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGT
    CTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATC
    TGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTT
    CCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCA
    GCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCA
    GTTCGTTCTTCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAAT
    ACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTCC
    GCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0067 Mutant 4700T PRT Artificial MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRN
    Mutant 4700T Sequence ANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFN
    GVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDA
    AAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYALVPRG
    SHHHHHHG
    0068 Mutant 4850T DNA Artificial AGATCTCCGCGGAACCAGACCAGCCTGCTGCAGAATCTGC
    Reverse primer R485M0 Sequence
    0069 Mutant 4850T DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    4850T GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTCTGGTT
    CCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0070 Mutant 4850T DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Mutant 4850T Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTA
    ATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTCTGGTTCCGCGGGGTTCTCATCATCAT
    CATCATCATGGTTAA
    0071 Mutant 4850T PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant 4850T Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGLVPRGSHHHHHHG
    0072 Mutant 485D DNA Artificial AACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGG
    DNA template for Sequence GCAATTCAAAACCGTTTTGATTCAGCCATTACCGCCCTTGGCAATACGGTAACCAAT
    deletion mutations from
    Mutant 4850T variant
    0073 Mutant 485D PRT Artificial NPLASIDSALSKVDAVRSSLGAIQNRFDSAITALGNTVTN
    PRT sequence for Sequence
    deletion mutations from
    Mutant 4850T variant
    0074 Mutant 485D DNA Artificial GTTCGTTCTTCTCTGGGGGCAATTGATTCAGCCATTACCGCCCTTG
    Forward Primer F485D Sequence
    0075 Mutant 485D DNA Artificial CAAGGGCGGTAATGGCTGAATCAATTGCCCCCAGAGAAGAACGAAC
    Reverse Primer R485D Sequence
    0076 Mutant 485D DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    4850T_Delta construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTGATTCAGCCATTACCGCCCTTG
    GCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACG
    GAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTCTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0077 Mutant 485D PRT Artificial MSGLRINSAKDDAAGQIAINRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant 485D (CT_Delta Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    439-442) KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIDSAITALGNTVTNLNSARSRIEDADYA
    TEVSNMSKAQILQQAGLVPRGSHHHHHHG
    0078 Mutant CGD1 DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Mutant SY3CT Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTCCGCGG
    GGTTCTCATCATCATCATCATCATGGTTAA
    0079 Mutant CGD1 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant SY3CT Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYALVPRGSHHHHHHG
    0080 Mutant CGD1 DNA Artificial ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGAT
    GFPuv4 Sequence GTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTT
    ACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTC
    TGACGTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAA
    GAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACT
    ACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAG
    GTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACA
    CAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCA
    CAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGA
    TGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTTGAAAGATCC
    CAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGG
    CATGGATGAACTATACAAA
    0081 Mutant CGD1 PRT Artificial MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
    GFPuv4 Sequence GVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK
    EDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPD
    NHYLSTQSALLKDPNEKRDHMVLLEFVTAAGITHGMDELYK
    0082 Mutant CGD1 DNA Artificial ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGAT
    GFPuv4 mutation of wt Sequence GTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTT
    Ndel site ACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTC
    TGACGTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAA
    GAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACT
    ACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAG
    GTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACA
    CAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCA
    CAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGA
    TGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTTGAAAGATCC
    CAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGG
    CATGGATGAACTATACAAATAA
    0083 Mutant CGD1 DNA Artificial TCTAGACGGCCGATCTCAGGTAAGAATGGAATCAAAGCTAACTTCAAAATTCGC
    Forward primer FCGFP Sequence
    0084 Mutant CGD1 PRT Artificial NVYIPISGKNGIKANFKIRH
    PRT altered GFPuv4 Sequence
    sequence
    0085 Mutant CGD1 DNA Artificial AGATCTCCGCGGTTTGTATAGTTCATCCATGCCATGTGTAATCCC
    Reverse RCGFP Sequence
    0086 Mutant CGD1 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of CGD1 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    onstruct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCACTGGTTCCGCCGATCTCAGGTAAGAATGGAATCAAAGCTAACTTCAAAATTCGC
    CACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGC
    GATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTTGAAAGAT
    CCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACAT
    GGCATGGATGAACTATACAAACCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0087 Mutant CGD1 DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Expressed Mutant CGD1 Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTCCGCCG
    ATCTCAGGTAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCC
    GTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCA
    GACAACCATTACCTGTCGACACAATCTGCCCTTTTGAAAGATCCCAACGAAAAGCGTGACCAC
    ATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA
    CCGCGGGGTTCTCATCATCATCATCATCATGGTTAA
    0088 Mutant CGD1 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Expressed Mutant CGD1 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYALVPPISGKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALLKDPNE
    KRDHMVLLEFVTAAGITHGMDELYKPRGSHHHHHHG
    0089 Mutant CPM194 DNA Artificial ATGAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGT
    Mutant CPM194 Sequence TCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTA
    ACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCATCCCCGGGAAGCGG
    GTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTT
    CTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGA
    CCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTT
    CAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAA
    CGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCT
    CAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTCTGGTTCCGCGGGGTTCTCA
    TCATCATCATCATCATGGTTAA
    0090 Mutant CPM194 PRT Artificial MSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYASPGSGLRIN
    Mutant CPM194 Sequence SAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNS
    DSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGLVPRGSHHHHHHG
    0091 Mutant CPM194 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Mutant CPM194 Sequence AACTTTAAGAAGGAGATATACATATGAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATT
    GTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCAT
    TACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTG
    ACTATGCATCCCCGGGAAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCA
    GGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAA
    CGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGC
    AGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAAT
    CTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAAT
    TTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATG
    GTCTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0092 Mutant CPM194 DNA Artificial TCTAGACATATGAGTACCGCTAACCCACTGGCTTCAATTG
    Forward primer FCD1 Sequence
    0093 Mutant CPM194 DNA Artificial GCTTCCCGGGGATGCATAGTCAGCATCTTCGATACGGC
    Reverse primer RCD1J Sequence
    0094 Mutant CPM194 DNA Artificial GCATCCCCGGGAAGCGGGTTACGGATCAACAGCG
    Forward primer FND1J Sequence
    0095 Mutant CPM194 DNA Artificial AGATCTCCGCGGAACCAGACCATCGTTAGCACCAACCTGGATTTTCATCT
    Reverse primer RND1 Sequence
    0096 Mutant CPM217 DNA Artificial ATGAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGT
    Mutant CPM217 Sequence TCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTA
    ACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCATCCCCGGGAAGCGG
    GTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTT
    CTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGA
    CCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTT
    CAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAA
    CGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCT
    CAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCT
    GCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATCTGGTTCCGCGGGG
    TTCTCATCATCATCATCATCATGGTTAA
    0097 Mutant CPM217 PRT Artificial MSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYASPGSGLRIN
    Mutant CPM217 Sequence SAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNS
    DSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDG
    FNVNLVPRGSHHHHHHG
    0098 Mutant CPM217 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Mutant CPM217 Sequence AACTTTAAGAAGGAGATATACATATGAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATT
    GTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCAT
    TACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTG
    ACTATGCATCCCCGGGAAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCA
    GGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAA
    CGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGC
    AGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAAT
    CTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAAT
    TTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATG
    GTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCA
    ATGTTAATCTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0099 Mutant CPM217 DNA Artificial AGATCTCCGCGGAACCAGATTAACATTGAACCCATCAAGGCCAAG
    Reverse primer Sequence
    RCPM217
    0100 Mutant GD1G DNA Artificial CCCGTTATCCGGATCACATGAAACGGCATGACTTTTTC
    Forward Primer FGFP77 Sequence
    0101 Mutant GD1G DNA Artificial GAAAAAGTCATGCCGTTTCATGTGATCCGGATAACGGG
    Reverse Primer RGFP77 Sequence
    0102 Mutant GD1G DNA Artificial CTGTTCCATGGCCAACACTTG
    FGFP54 Sequence
    0103 Mutant GD1G DNA Artificial TCTAGACATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCC
    Forward primer FNGFP Sequence
    0104 Mutant GD1G DNA Artificial GGCCTATGCGGCCGCAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAA
    altered GFP DNA Sequence
    sequence
    0105 Mutant GD1G DNA Artificial AGATCTATTAATGCGGCCTGATAGGCCTTGTTTGTCTGCCGTGATGTATACATTGTG
    Reverse RNGFP Sequence
    0106 Mutant GD1G PRT Artificial SHNVYITADKQGLSGRNM
    altered GFP PRT Sequence
    sequence
    0107 Mutant GD1G DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of GD1G Sequence AACTTTAAGAAGGAGATATACATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAA
    construct TTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAG
    GTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCC
    ATGGCCAACACTTGTCACTACTCTGACGTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCA
    CATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTAT
    ATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCT
    TGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAA
    CTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAGGCCTATCAGGC
    CGCATTATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTG
    CTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCA
    TTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTG
    CGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAG
    GATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGT
    GTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAAC
    CATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAAT
    TCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGC
    AGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAA
    TACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTC
    CGCCGATCTCAGGTAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATG
    GATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTT
    TACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTTGAAAGATCCCAACGAAAAGCGTG
    ACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAACTAT
    ACAAACCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0108 Mutant GD1G DNA Artificial ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGAT
    Expressed Mutant Sequence GTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTT
    D1G ACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTC
    TGACGTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAA
    GAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACT
    ACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAG
    GTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACA
    CAATGTATACATCACGGCAGACAAACAAGGCCTATCAGGCCGCATTATGAGCGGGTTACGGA
    TCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATC
    AAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGA
    AGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCA
    CTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGG
    AAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACA
    ACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAA
    TTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACC
    CACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAA
    TTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCG
    CGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTCCGCCGATCTCAGGTAAGAATGGA
    ATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCAT
    TATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCG
    ACACAATCTGCCCTTTTGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTT
    GTAACTGCTGCTGGGATTACACATGGCATGGATGAACTATACAAACCGCGGGGTTCTCATCAT
    CATCATCATCATGGTTAA
    0109 Mutant GD1G PRT Artificial MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
    Expressed Mutant Sequence GVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK
    GD1G EDGNILGHKLEYNYNSHNVYITADKQGLSGRIMSGLRINSAKDDAAGQAIANRFTSNIKGLTQASR
    NANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQF
    NGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGSTANPLASIDSALSKVDAV
    RSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYALVPPISGKNGIKANFKIRHNIEDGSVQLAD
    HYQQNTPIGDGPVLLPDNHYLSTQSALLKDPNEKRDHMVLLEFVTAAGITHGMDELYKPRGSHHH
    HHHG
    0110 Mutant MF227C DNA Artificial CTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTACA
    mutant 4700T template Sequence TTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTG
    0111 Mutant MF2270 PRT Artificial LGLDGFNVNSPGISGGGGGITLINEDAAAAKKSTANPLASI
    mutant 4700T template Sequence
    0112 Mutant MF227C DNA Artificial AGATCTCCGCGGAACCAGTAAAGAGAGGACGTTTTGCGGAACCTGGTTTGCATAGTCAGCAT
    Reverse Primer R2YY Sequence CTTCGATACG
    0113 Mutant MF227C DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    Mutant MF227C GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCAT
    CATCATCATCATGGTTAAGTCGAC
    0114 Mutant MF227C DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Mutant MF227C Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAAACCAGGTTCCG
    CAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTTAA
    0115 Mutant MF2270 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant MF2270 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYANQVPQNVLSLLVPRGSHHHHHHG
    0116 Mutant MF227N DNA Artificial AGATCTCCCGGGGAACCATCGTTAGCACCAACCTGGATTTTC
    Reverse Primer Sequence
    RMF227N
    0117 Mutant MF227N DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    mutant MF227N GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAA
    AAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCA
    ACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTAT
    GCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTG
    GCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCA
    TCATCATCATCATGGTTAAGTCGAC
    0118 Mutant MF227N DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    mutant MF227N Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTTCCCCGGGAAG
    TACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTC
    TCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAA
    TCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTC
    TAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGC
    AAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTTAA
    0119 Mutant MF227N PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    mutant MF227N Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGSPGSTANPLA
    SIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGT
    SVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0120 Mutant MF233 DNA Artificial AGATCTCCGCGGAACCAGCAGGTTATTCTGGGTCAACAGCGACAGGCTGTTTGTATTAATGA
    Reverse primer RMF233 Sequence CTTGTGCATAGTCAGCATCTTCGATACG
    0121 Mutant MF233 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct MF233 GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGCTGGTT
    CCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0122 Mutant MF233 DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    MF233 Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCACAAGTCATTAATA
    CAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGCTGGTTCCGCGGGGTTCTCATCATCAT
    CATCATCATGGTTAA
    0123 Mutant MF233 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    MF233 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYAQVINTNSLSLLTQNNLLVPRGSHHHHHHG
    0124 Mutant MF471 DNA Artificial GCTGACTATGCAACGGCAGTTTCTGCTATGTCTGCAGCGCAGATTCTGC
    Forward primer F471-77 Sequence
    0125 Mutant MF471 DNA Artificial GCAGAATCTGCGCTGCAGACATAGCAGAAACTGCCGTTGCATAGTCAGC
    Reverse Primer R471-77 Sequence
    0126 Mutant MF471 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct MF471 GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGCAGTTTCTGCTATGTCTGCAGCGCAGATTCTGCAGCAGGCTGGTCTGGT
    TCCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0127 Mutant MF471 DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    MF471 Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGCAGTTTCT
    GCTATGTCTGCAGCGCAGATTCTGCAGCAGGCTGGTCTGGTTCCGCGGGGTTCTCATCATCA
    TCATCATCATGGTTAA
    0128 Mutant MF471 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    MF471 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATAVSAMSAAQILQQAGLVPRGSHHHHHHG
    0129 Mutant MF479 DNA Artificial GTTTCTAATATGTCTAAAGCGGCGATTCTGGGAGCGGCTGGTCTGGTTCCGCGG
    Forward primer F479-83 Sequence
    0130 Mutant MF479 DNA Artificial CCGCGGAACCAGACCAGCCGCTCCCAGAATCGCCGCTTTAGACATATTAGAAAC
    Reverse Primer R479-83 Sequence
    0131 Mutant MF479 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct MF479 GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGGCGATTCTGGGAGCGGCTGGTCTGGT
    TCCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0132 Mutant MF479 DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Mutant MF479 Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTA
    ATATGTCTAAAGCGGCGATTCTGGGAGCGGCTGGTCTGGTTCCGCGGGGTTCTCATCATCAT
    CATCATCATGGTTAA
    0133 Mutant MF479 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant MF479 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAAILGAAGLVPRGSHHHHHHG
    0134 Mutant N45 DNA Artificial TCTAGAGGATCCGGCAGGCCAGGCG
    Forward primer N45_F Sequence
    0135 Mutant N45 DNA Artificial CGCAAGCTTGTCGACTTAACGC
    Reverse R502D0 Sequence
    0136 Mutant N45 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of Sequence AACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    Mutant N45 GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGC
    GAAGGATCCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTC
    AGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAAT
    GAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAA
    CTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCG
    CGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATC
    CAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGC
    CTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTA
    GACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCC
    ACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAAT
    TCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGC
    GCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGAT
    TCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCT
    CTTTACTGCGTTAAGTCGACAAGCTTGCGG
    0137 Mutant N45 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPAGQAIANRFTSNIKGLTQASRNAND
    Mutant N45 Sequence GISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKV
    LSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAK
    KSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKA
    QILQQAGTSVLAQANQVPQNVLSLLR
    0138 Mutant NGD1 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of NGD1 Sequence AACTTTAAGAAGGAGATATACATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAA
    construct TTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAG
    GTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCC
    ATGGCCAACACTTGTCACTACTCTGACGTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCA
    CATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTAT
    ATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCT
    TGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAA
    CTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAGGCCTATCAGGC
    CGCATTATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTG
    CTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCA
    TTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTG
    CGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAG
    GATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGT
    GTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAAC
    CATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAAT
    TCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGC
    AGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAA
    TACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTC
    CGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0139 Mutant NGD1 DNA Artificial ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGAT
    Expressed Mutant 5Y3- Sequence GTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTT
    GFP ACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTC
    TGACGTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAA
    GAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACT
    ACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAG
    GTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACA
    CAATGTATACATCACGGCAGACAAACAAGGCCTATCAGGCCGCATTATGAGCGGGTTACGGA
    TCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATC
    AAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGA
    AGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCA
    CTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGG
    AAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACA
    ACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAA
    TTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACC
    CACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAA
    TTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCG
    CGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTCCGCGGGGTTCTCATCATCATCAT
    CATCATGGTTAA
    0140 Mutant NGD1 PRT Artificial MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
    Expressed Mutant SY3- Sequence GVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK
    GFP/Mutant NGD1 EDGNILGHKLEYNYNSHNVYITADKQGLSGRIMSGLRINSAKDDAAGQAIANRFTSNIKGLTQASR
    NANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQF
    NGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGSTANPLASIDSALSKVDAV
    RSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYALVPRGSHHHHHHG
    0141 Mutant S33 DNA Artificial TCTAGAGGATCCGTCTGGTCTGCGTATCAACAGCGC
    Forward F502_533 Sequence
    0142 Mutant S33 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of Sequence AACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    Mutant S33 GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGC
    GAAGGATCCGTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATT
    GCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGC
    ATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGT
    GCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCA
    GGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGG
    TGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAAC
    CATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAAT
    TCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAA
    GACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTC
    AAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTAC
    CAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACT
    ATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTC
    TGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTAAGTCGAC
    0143 Mutant S33 DNA Artificial ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATG
    Mutant S33 Sequence GGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGCGAAGGATCCGTCTGGTCTGCGTA
    TCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATC
    AAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGA
    AGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCA
    CTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGG
    AAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACA
    ACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAA
    TTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTG
    GTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAA
    AGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCT
    TCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACC
    AATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATG
    TCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCC
    GCAAAACGTCCTCTCTTTACTGCGTTAA
    0144 Mutant S33 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPSGLRINSAKDDAAGQAIANRFTSNIK
    Mutant S33 Sequence GLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRV
    SNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSM
    GTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDA
    DYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR
    0145 Mutant SY3CT DNA Artificial AGATCTCCGCGGAACCAGTGCATAGTCAGCATCTTCGATACGGC
    Reverse primer RSY3CT Sequence
    0146 Mutant SY3CT DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    SY3CT construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0147 Mutant SY3CT DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Expressed Mutant Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    SY3CT TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCACTGGTTCCGCGG
    GGTTCTCATCATCATCATCATCATGGTTAA
    0148 Mutant SY3CT PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Expressed Mutant Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    SY3CT KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYALVPRGSHHHHHHG
    0149 Mutant 33MX DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Mutant 33MX Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTGCAGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTGCCGGGGCTAACGCTGATGCCGCTCTGAAAGCTATCCAGGCTGA
    AATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTCAGCAGACTCAAGCTGCCGCTGTTAA
    AGTCCTGTCTCAGGACAACGCAATGGCAATCCAGGTTGGTGCTAACGATGGTGCCGCTATTA
    CCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCC
    CGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTC
    AAATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAG
    GTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGT
    TAA
    0150 Mutant 33MX PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNAADGISIAQTTEGALNEINNNLQRVRELSVQA
    Mutant 33MX Sequence TAGANADAALKAIQAEIQQRLEEIDRVSQQTQAAAVKVLSQDNAMAIQVGANDGAAITIDLQKIDVK
    SLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDA
    DYATEVSQMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0151 Mutant 33MX DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of 33MX Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTGCAGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACA
    ACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTGCCGGGGCTAACGCTGATGC
    CGCTCTGAAAGCTATCCAGGCTGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTCA
    GCAGACTCAAGCTGCCGCTGTTAAAGTCCTGTCTCAGGACAACGCAATGGCAATCCAGGTTG
    GTGCTAACGATGGTGCCGCTATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCC
    TTGATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTG
    CATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAG
    CCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGAT
    GCTGACTATGCAACGGAAGTTTCTCAAATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACT
    TCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGG
    TTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0152 Mutant 485MX DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA Sequence of Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    85MX construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTGCAGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACA
    ACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTGCCGGGGCTAACGCTGATGC
    CGCTCTGAAAGCTATCCAGGCTGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTCA
    GCAGACTCAAGCTGCCGCTGTTAAAGTCCTGTCTCAGGACAACGCAATGGCAATCCAGGTTG
    GTGCTAACGATGGTGCCGCTATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCC
    TTGATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTG
    CATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAG
    CCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGAT
    GCTGACTATGCAACGGAAGTTTCTCAAATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTCT
    GGTTCCGCGGGGTTCTCATCATCATCATCATCATGGTTAAGTCGAC
    0153 Mutant 485MX DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    485MX construct Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTGCAGACGGCATTTCTA
    TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTGCCGGGGCTAACGCTGATGCCGCTCTGAAAGCTATCCAGGCTGA
    AATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTCAGCAGACTCAAGCTGCCGCTGTTAA
    AGTCCTGTCTCAGGACAACGCAATGGCAATCCAGGTTGGTGCTAACGATGGTGCCGCTATTA
    CCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCC
    CGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTC
    AAATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTCTGGTTCCGCGGGGTTCTCATCATCAT
    CATCATCATGGTTAA
    0154 Mutant 485MX PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNAADGISIAQTTEGALNEINNNLQRVRELSVQA
    485MX construct Sequence TAGANADAALKAIQAEIQQRLEEIDRVSQQTQAAAVKVLSQDNAMAIQVGANDGAAITIDLQKIDVK
    SLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDA
    DYATEVSQMSKAQILQQAGLVPRGSHHHHHHG
    0155 Mutant MIM4 DNA Artificial ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATG
    CBLB502 variant Sequence GGTCGGGATCTGTACGACGATGACGATAAGGATCCGATGGCACAAGTCATTAATACAAACAG
    CCTGTCGCTGTTGACCCAGAATAACCTGCAGAAATCTCAGTCCTCACTGAGTTCCGCTATTGA
    GCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATT
    GCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGC
    ATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGT
    GCGTGAGTTGTCTGTTCAGGCCACTCAAGGGACTAACTCTGATTCCGATCTGAAATCTATCCA
    GGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTCAGCAGACTCAATTTAACGG
    TGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAAC
    CATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAAT
    TCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAA
    GACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTC
    AAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGTTTTGATTCAGCCATTAC
    CAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACT
    ATGCAACGGAAGTTTCTCAAATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTC
    TGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTAA
    0156 Mutant MIM4 DNA Artificial AACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGG
    Primers design (for Sequence GCAATTCAAAACCGTTTTGATTCAGCCATTACCGCCCTTGGCGCTACGGTAACCGCTCTGGC
    deletion aa Gln439; CTCCGCGCGTAGCGCTATCGAAGATGCTGACTATGCAACGGAAGTTTCTCAAATG
    Asn440; Arg441;
    Phe442):
    0157 Mutant MIM4 PRT Artificial NPLASIDSALSKVDAVRSSLGAIQNRFDSAITALGATVTALASARSAIEDADYATEVSNM
    Primers design (for Sequence
    deletion aa Gln439;
    Asn440; Arg441;
    Phe442):
    0158 Mutant MIM4 DNA Artificial GCAGTTCGTTCTTCTCTGGGGGCAATTGATTCAGCCATTACCGCCCTTGG
    Forward Primer MIM4 Sequence
    0159 Mutant MIM4 DNA Artificial CCAAGGGCGGTAATGGCTGAATCAATTGCCCCCAGAGAAGAACGAACTGC
    Reverse Primer MIM4 Sequence
    0160 Mutant MIM4 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT
    MIM4 Sequence TAACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGAT
    GGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCTCA
    GTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAG
    ACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAG
    GCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGA
    AATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACT
    CTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCG
    TTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCA
    GGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCT
    TGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAG
    ACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCA
    CTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATT
    GATTCAGCCATTACCGCCCTTGGCGCTACGGTAACCGCTCTGGCCTCCGCGGCTAGCCGTAT
    CGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGC
    TGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTA
    A
    0161 Mutant MIM4 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMAQVINTNSLSLLTQNNLNKSQSSLSSAIE
    MIM4 Sequence RLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSV
    QATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKI
    DVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAI
    DSAITALGATVTALASAASRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR
    0162 Mutant MIM5 DNA Artificial AACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGG
    Primers design Sequence GCAATTGCAAAGGCTTTTGATTCAGCCATTACCGCCCTTGGCGCTACGGTAACCGCTCTGGC
    (mutations Gln439Ala; CTCCGCGCGTAGCGCTATCGAAGATGCTGACTATGCAACGGAAGTTTCTCAAATG
    Asn440Lys; Arg441Ala):
    0163 Mutant MIM5 PRT Artificial NPLASIDSALSKVDAVRSSLGAIAKAFDSAITALGATVTALASARSAIEDADYATEVSNM
    Primers design Sequence
    (mutationsGln439Ala;
    Asn440Lys;Arg441Ala):
    0164 Mutant MIM5 DNA Artificial CGTTCTTCTCTGGGGGCAATTGCAAAGGCTTTTGATTCAGCCATTACCGC
    Forward Primer MIM5 Sequence
    0165 Mutant MIM5 DNA Artificial GCGGTAATGGCTGAATCAAAAGCCTTTGCAATTGCCCCCAGAGAAGAACG
    Reverse Primer MIM5 Sequence
    0166 Mutant MIM5 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT
    MIM5 Sequence TAACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGAT
    GGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCTCA
    GTCCTCACTGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAG
    ACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAG
    GCTTCCCGTAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGA
    AATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACT
    CTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCG
    TTTCTAATCAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCA
    GGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCT
    TGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAG
    ACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCA
    CTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATT
    GCAAAGGCTTTTGATTCAGCCATTACCGCCCTTGGCGCTACGGTAACCGCTCTGGCCTCCGC
    GGCTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGAT
    TCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCT
    CTTTACTGCGTTAA
    0167 Mutant MIM5 PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMAQVINTNSLSLLTQNNLNKSQSSLSSAIE
    MIM5 Sequence RLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSV
    QATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKI
    DVKSLGLDGFNVNSPGISGGGGGILDSMGTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAI
    AKAFDSAITALGATVTALASAASRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR
    0168 Mutant MIMX DNA Artificial ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATG
    Mutant 33MIMX Sequence GGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGCGAAGGATCCGTCTGGTCTGCGTA
    TCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATC
    AAAGGTCTGACTCAGGCTTCCCGTAACGCTGCAGACGGCATTTCTATTGCGCAGACCACTGA
    AGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCA
    CTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGG
    AAGAAATCGATCGCGTTTCTAATCAGACTCAAGCTAACGGTGTTAAAGTCCTGTCTCAGGACA
    ACGCAATGAAAATCCAGGTTGGTGCTAACGATGGTGCCGCTATTACCATCGATCTGCAAAAAA
    TTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCCGGGAATTTCCGGTGGTG
    GTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGAAGACGCTGCCGCAGCCAAGAAA
    AGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCT
    TCTCTGGGGGCAATTCAAGCTCGTTTTGCCGCGGCCATTGCTAACCTTGGCAATACGGTAAC
    CAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATAT
    GTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTC
    CGCAAAACGTCCTCTCTTTACTGCGTTAA
    0169 Mutant MIMX PRT Artificial MRGSHHHHHHGMASMTGGQQMGRDLYDLVPRGSAKDPSGLRINSAKDDAAGQAIANRFTSNIK
    MIMX Sequence GLTQASRNAADGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRV
    SNQTQANGVKVLSQDNAMKIQVGANDGAAITIDLQKIDVKSLGLDGFNVNSPGISGGGGGILDSM
    GTLINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQARFAAAIANLGNTVTNLNSARSRIEDA
    DYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR
    0170 Mutant MIMX DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of Sequence AACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTA
    33MIMx GCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACCTGGTTCCGCGCGGTAGCGC
    GAAGGATCCGTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATT
    GCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTGCAGACGG
    CATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGT
    GCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCA
    GGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAAGCTAACGG
    TGTTAAAGTCCTGTCTCAGGACAACGCAATGAAAATCCAGGTTGGTGCTAACGATGGTGCCG
    CTATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAA
    TTCCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTACATTAATCAATGA
    AGACGCTGCCGCAGCCAAGAAAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGT
    CAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAGCTCGTTTTGCCGCGGCCATT
    GCTAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGA
    CTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGT
    TCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTAAGTCGAC
    0171 Mutant MIXC DNA Artificial AGATCTGTCGACTTAACCATGATGATGATGATGATGAGAACCCCGCGGAACCAGTAAAGAGA
    Reverse primer RMIXC Sequence GGACGTTTTGCGGAACC
    0172 Mutant MIXC DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of MIXC Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAGCTCGTTTTGCCGCGGCC
    ATTGCTAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0173 Mutant MIXC PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    MIXC Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQARFAAAIANLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0174 Mutant MIXN DNA Artificial AGATCTCATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGC
    Forward primer FMIMxN Sequence
    0175 Mutant MIXN DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    DNA sequence of MIX.N Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTGCAGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACA
    ACAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCC
    GATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAAT
    CAGACTCAAGCTAACGGTGTTAAAGTCCTGTCTCAGGACAACGCAATGAAAATCCAGGTTGGT
    GCTAACGATGGTGCCGCTATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTT
    GATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCA
    TTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCC
    ATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0176 Mutant MIXN PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNAADGISIAQTTEGALNEINNNLQRVRELSVQA
    Expressed Mutant MIX.N Sequence TNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQANGVKVLSQDNAMKIQVGANDGAAITIDLQKIDVK
    SLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDA
    DYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0177 Mutants MIM1; MIM2 DNA Artificial AACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGG
    and MIM3 Sequence GCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACT
    502 Mutants MIM1; CCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTCAAATGTCTAAAGCG
    MIM2 and MIM3- CAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGT
    terminal part of CCTCTCTTTACTGCGTTAA
    CBLB502
    0178 Mutants MIM1; MIM2 DNA Artificial ATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGAT
    and MIM3 Sequence
    Primers design (mutant
    MIM1)
    0179 Mutants MIM1; MIM2 PRT Artificial ITNLGNTVTNLNSARSRIED
    and MIM3 Sequence
    Primers design (mutant
    MIM1)
    0180 Mutants MIM1; MIM2 DNA Artificial CCTTGGCAATACGGTAACCGCTCTGGCCTCCGCGCGTAGCCGTATC
    and MIM3 Sequence
    Forward Primer 455-57
    0181 Mutants MIM1; MIM2 DNA Artificial GATACGGCTACGCGCGGAGGCCAGAGCGGTTACCGTATTGCCAAGG
    and MIM3 Sequence
    Reverse Primer 455-57
    0182 Mutants MIM1; MIM2 DNA Artificial ACGGTAACCGCTCTGGCCTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAA
    and MIM3 Sequence
    Primers design (mutant
    MIM2_MIM1 plus
    R460A)
    0183 Mutants MIM1; MIM2 PRT Artificial TVTALASARSRIEDADYATE
    and MIM3 Sequence
    Primers design (mutant
    MIM2_MIM1 plus
    R460A)
    0184 Mutants MIM1; MIM2 DNA Artificial GCTCTGGCCTCCGCGGCTAGCCGTATCGAAGATG
    and MIM3 Sequence
    Forward Primer 460
    0185 Mutants MIM1; MIM2 DNA Artificial CATCTTCGATACGGCTAGCCGCGGAGGCCAGAGC
    and MIM3 Sequence
    Reverse Primer 460
    0186 Mutants MIM1; MIM2 DNA Artificial CAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCGCTCTGGCCTCC
    and MIM3 Sequence
    Primers design (mutant
    MIM3_MIM2 plus
    N448A; N451A)
    0187 Mutants MIM1; MIM2 PRT Artificial QNRFDSAITNLGNTVTALAS
    and MIM3 Sequence
    Primers design (mutant
    MIM3_MIM2 plus
    N448A; N451A)
    0188 Mutants MIM1; MIM2 DNA Artificial GTTTTGATTCAGCCATTACCGCCCTTGGCGCTACGGTAACCGCTCTGG
    and MIM3 Sequence
    Forward Primer 448-51
    0189 Mutants MIM1; MIM2 DNA Artificial CCAGAGCGGTTACCGTAGCGCCAAGGGCGGTAATGGCTGAATCAAAAC
    and MIM3 Sequence
    Reverse Primer 448-51
    0190 Mutant ME42 DNA Artificial CAACAGCGCGAAAGCCGATGCGGGAGGCCAGGCGATTGC
    Forward Primer ME42 Sequence
    0191 Mutant ME42 DNA Artificial GCAATCGCCTGGCCTCCCGCATCGGCTTTCGCGCTGTTG
    Reverse Primer ME42 Sequence
    0192 Mutant ME42 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME42 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGCCGATGCG
    construct GGAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0193 Mutant ME42 PRT Artificial MSGLRINSAKADAGGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME42 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0194 Mutant ME110 DNA Artificial GTCTGTTCAGGCCACTGCCGGGGCTAACTCTGATTCCGATCTG
    Forward Primer ME100 Sequence
    0195 Mutant ME110 DNA Artificial CAGATCGGAATCAGAGTTAGCCCCGGCAGTGGCCTGAACAGAC
    Reverse Primer ME100 Sequence
    0196 Mutant ME110 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME100 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTGCCGGGGCTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0197 Mutant ME110 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME110 Sequence ATAGANSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0198 Mutant ME100/110 DNA Artificial CTGATTCCGATCTGAAAGCTATCCAGGCTGAAATTCAGCAACGTC
    Forward Primer ME110 Sequence
    0199 Mutant ME100/110 DNA Artificial GACGTTGCTGAATTTCAGCCTGGATAGCTTTCAGATCGGAATCAG
    Reverse Primer ME110 Sequence
    0200 Mutant ME100/110 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME100/110 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTGCCGGGGCTAACTCTGATTCCG
    ATCTGAAAGCTATCCAGGCTGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0201 Mutant ME104N DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Intermediate Mutant Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    ME100/110 TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTGCCGGGGCTAACTCTGATTCCGATCTGAAAGCTATCCAGGCTGA
    AATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTA
    ATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAG
    GTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGT
    TAA
    0202 Mutant ME100/110 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME110/110 Sequence ATAGANSDSDLKAIQAEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0203 Mutant ME104 DNA Artificial GCCACTAACGGGACTAACGCTGATGCCGCTCTGAAATCTATCCAG
    Forward Primer ME104 Sequence
    0204 Mutant ME104 DNA Artificial CTGGATAGATTTCAGAGCGGCATCAGCGTTAGTCCCGTTAGTGGC
    Reverse Primer ME104 Sequence
    0205 Mutant ME104 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME104 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACGCTGATGCCG
    CTCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0206 Mutant ME104 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME104 Sequence ATNGTNADAALKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0207 Mutant ME104N DNA Artificial GCCACTGCCGGGGCTAACGCTGATGCCGCTCTGAAAGCTATCCAG
    Primer FME104New Sequence
    0208 Mutant ME104N DNA Artificial CTGGATAGCTTTCAGAGCGGCATCAGCGTTAGCCCCGGCAGTGGC
    Primer RME104New Sequence
    0209 Mutant ME104N DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of construct Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    ME104New GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTGCCGGGGCTAACGCTGATGCC
    GCTCTGAAAGCTATCCAGGCTGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAAT
    CAGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGT
    GCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTT
    GATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCA
    TTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCC
    ATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0210 Mutant ME104N PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME104N Sequence ATAGANADAALKAIQAEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0211 Mutant ME110 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME110 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAAGCTATCCAGGCTGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0212 Mutant ME110 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME110 Sequence ATNGTNSDSDLKAIQAEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0213 Mutant ME117 DNA Artificial CTATCCAGGATGAAATTCAGGCACGTCTGGCAGAAATCGATCGCG
    Forward Primer ME117 Sequence
    0214 Mutant ME117 DNA Artificial CGCGATCGATTTCTGCCAGACGTGCCTGAATTTCATCCTGGATAG
    Reverse Primer ME117 Sequence
    0215 Mutant ME117 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of 33ML Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    (should this say TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    ME117?) CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGGCACGTCTGGCAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0216 Mutant ME117 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME117 Sequence ATNGTNSDSDLKSIQDEIQARLAEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0217 Mutant ME124 DNA Artificial GGAAGAAATCGATGCCGTTTCTGCTGCGACTCAATTTAACGGTGTTAAAGTCCTGTCTC
    Forward Primer ME104 Sequence
    0218 Mutant ME124 DNA Artificial GAGACAGGACTTTAACACCGTTAAATTGAGTCGCAGCAGAAACGGCATCGATTTCTTCC
    Reverse Primer ME104 Sequence
    0219 Mutant ME124 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME124 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATGCCGTTTCTGCTG
    CGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0220 Mutant ME124 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME124 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDAVSAATQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0221 Mutant ME124P DNA Artificial CAGCAACGTCTGGAAGAAATCGATGCCGTTTCTAATCAGACTCAATTTAACGG
    Forward Primer ME124P Sequence
    0222 Mutant ME124P DNA Artificial CCGTTAAATTGAGTCTGATTAGAAACGGCATCGATTTCTTCCAGACGTTGCTG
    Reverse Primer ME124P Sequence
    0223 Mutant ME124 DNA Artificial ATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCGGCAGGCCAGGCGATTGCTAACC
    Expressed Mutant Sequence GCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGCATTTCTA
    ME124P TTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAACAACCTGCAGCGTGTGCGTGAG
    TTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAA
    ATTCAGCAACGTCTGGAAGAAATCGATGCCGTTTCTAATCAGACTCAATTTAACGGTGTTAAA
    GTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTAC
    CATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTGATGGGTTCAATGTTAATTCCCC
    GGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTC
    GTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCATTACCAACCTTGGCAATACGG
    TAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTA
    ATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAG
    GTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTCTCATCATCATCATCATCATGGT
    TAA
    0224 Mutant ME124P DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    ME124P Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATGCCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0225 Mutant ME124P PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    ME124P Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDAVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0226 Mutant ME132 DNA Artificial CGTTTCTAATCAGACTCAATTTGCCGCTGTTAAAGTCCTGTCTCAGGACAACC
    Forward Primer ME132 Sequence
    0227 Mutant ME132 DNA Artificial GGTTGTCCTGAGACAGGACTTTAACAGCGGCAAATTGAGTCTGATTAGAAACG
    Reverse Primer ME132 Sequence
    0228 Mutant ME132 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME132 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTGCCGCTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0229 Mutant ME132 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME117 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFAAVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    (ME132?) KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0230 Mutant ME142 DNA Artificial GTTAAAGTCCTGTCTCAGGACAACGCGATGGCAATCCAGGTTGGTGCTAACG
    Forward Primer ME142 Sequence
    0231 Mutant ME142 DNA Artificial CGTTAGCACCAACCTGGATTGCCATCGCGTTGTCCTGAGACAGGACTTTAAC
    Reverse Primer ME142 Sequence
    0232 Mutant ME142 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME142 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACGCGATGGCAATCCAGGTTGGT
    GCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTT
    GATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCA
    TTGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCC
    ATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0233 Mutant ME142 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME142 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNAMAIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0234 Mutant ME150 DNA Artificial GATGAAAATCCAGGTTGGTGCTAGCGCTGCTGAAACCATTACCATCGATCTGC
    Forward Primer ME150 Sequence
    0235 Mutant ME150 DNA Artificial GCAGATCGATGGTAATGGTTTCAGCAGCGCTAGCACCAACCTGGATTTTCATC
    Reverse Primer ME150 Sequence
    0236 Mutant ME150 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME150 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAGCGCTGCTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTCC
    GTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTTC
    TCATCATCATCATCATCATGGTTAAGTCGAC
    0237 Mutant ME150 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME150 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGASAAETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0238 Mutant ME468 DNA Artificial GCCGTATCGAAGATGCTGACGCTGGAGCGGAAGTTGCTAATATGTCTAAAGCGCAG
    Forward Primer ME468 Sequence
    0239 Mutant ME468 DNA Artificial CTGCGCTTTAGACATATTAGCAACTTCCGCTCCAGCGTCAGCATCTTCGATACGGC
    everse Primer ME468 Sequence
    0240 Mutant ME468 DNA Artificial TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAATAATTTTGTTT
    Sequence of ME468 Sequence AACTTTAAGAAGGAGATATACATATGAGCGGGTTACGGATCAACAGCGCGAAAGACGATGCG
    construct GCAGGCCAGGCGATTGCTAACCGCTTCACTTCTAATATCAAAGGTCTGACTCAGGCTTCCCG
    TAACGCTAACGACGGCATTTCTATTGCGCAGACCACTGAAGGTGCGCTGAATGAAATCAACAA
    CAACCTGCAGCGTGTGCGTGAGTTGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCG
    ATCTGAAATCTATCCAGGATGAAATTCAGCAACGTCTGGAAGAAATCGATCGCGTTTCTAATC
    AGACTCAATTTAACGGTGTTAAAGTCCTGTCTCAGGACAACCAGATGAAAATCCAGGTTGGTG
    CTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATGTGAAAAGCCTTGGCCTTG
    ATGGGTTCAATGTTAATTCCCCGGGAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCAT
    TGTCAAAAGTGGACGCAGTTCGTTCTTCTCTGGGGGCAATTCAAAACCGCTTTGATTCAGCCA
    TTACCAACCTTGGCAATACGGTAACCAATCTGAACTCCGCGCGTAGCCGTATCGAAGATGCT
    GACGCTGGAGCGGAAGTTGCTAATATGTCTAAAGCGCAGATTCTGCAGCAGGCTGGTACTTC
    CGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGGTTCCGCGGGGTT
    CTCATCATCATCATCATCATGGTTAAGTCGAC
    0241 Mutant ME468 PRT Artificial MSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQ
    Mutant ME468 Sequence ATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQDNQMKIQVGANDGETITIDLQKIDV
    KSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIED
    ADAGAEVANMSKAQILQQAGTSVLAQANQVPQNVLSLLVPRGSHHHHHHG
    0242 Linker PRT Artificial SPG
    Sequence
    0243 Lru283 PRT Artificial mghhhhhhsgMEEFNMRINTNVAAMNTYSRLTAANTAKSNSLAKLSSGLRINKAGDDAAGLAISEKM
    Sequence KSQIGGLTQAKRNAQDGISLVQTAEGALNETHSILERMRDLAVQGSNGTLTSSDRGSINKELKALH
    QELTRISNTTEFNTQKLFSQTKQKSVTFTFQIGANAGQTLSVAITAMSGEALLVSTDAKFSLNAAGT
    NAGAMIKSIDAAIAKVSDQRADLGAVQNRLEHTINNLTATNENLSDANSRIRDVDMAEEMMTFTKS
    NILSQAATSMLAQANAMPNSVLNLLQG
    0244 Tpe270 PRT Artificial mghhhhhhsgMRINHNISALNAWRNIDQTQYSMSKTLERLSSGLRINRAGDDAAGLAISEKMRGQIK
    Sequence GLNMAIKNAQDAISLIQTAEGALTEVHSILQRMRELAVQAASDTNTNVDREQIQKEIDQLREEIDRIA
    RTTEFNTKKLLDGKLEGFRSQVDAKVVTGGNINVQLGTVSSKAVEGTYVIEVGaAERAIMVVDAAI
    HRVSTARAALGAIQNRLEHTISNLGVAAENLTAAESRIRDADMAKEMMEFTKQQILLQSSMAMLAQ
    SNTLPQNVLQLMR
    0245 Tpe159w PRT Artificial mghhhhhhsGLNMAIKNAQDAISLIQTAEGALTEVHSILQRMRELAVQAASDTNTNVDREQIQKEIDQ
    Sequence LREEIDRIARTTEFNTKKLLDGKLEGFRSQVDAKVVTGGNINVQLGTVSSKAVEGTYVIEVGaAERA
    IMVVDAAIHRVSTARAALGAIQNRLEHTISNLG
    0246 Chy275 PRT Artificial mghhhhhhsgMSLRINNNIEALNAWRALNSTSNALQKSMEKLSSGLRINRAGDDAAGLAISEKLRAQI
    Sequence RGLNQAIRNAQDGISLIQTAEGGLSEIQNILQRMRELGVQAANGTLNNQDISAITTELNQLFNEIDRI
    AGATEFNTKNLLAVSTGLVVTLQVGANAGQVIAFTIDNAGTASLGLSSADLAINDNASASAFISKVD
    SALQKVSTYRANLGSIQNRLEHTIANLGIASENLSASESRIRDVDMAAEMMNFTKNQILQQAGVAIL
    AQANQAPQAVLQLLR
    0247 Chy162w PRT Artificial mghhhhhhsGLNQAIRNAQDGISLIQTAEGGLSEIQNILQRMRELGVQAANGTLNNQDISAITTELNQ
    Sequence LFNEIDRIAGATEFNTKNLLAVSTGLVVTLQVGANAGQVIAFTIDNAGTASLGLSSADLAINDNASAS
    AFISKVDSALQKVSTYRANLGSIQNRLEHTIANLG
    0248 ChyU137 PRT Artificial mghhhhhhsGLNQAIRNAQDGISLIQTAEGGLSEIQNILQRMRELGVQAANGTLNNQDISAITTELNQ
    Sequence LFNEIDRIAGATEFNTKNLLAAGTASLGLSSADLAINDNASASAFISKVDSALQKVSTYRANLGSIQN
    RLEHTIANLG
    0249 ChyN108 PRT Artificial mghhhhhhSASAFISKVDSALQKVSTYRANLGSIQNRLEHTIANLGpdGLNQAIRNAQDGISLIQTAEG
    Sequence GLSEIQNILQRMRELGVQAANGTLNNQDISAITTELNQLFNEIDRIA
    0250 ChyZ94 PRT Artificial mghhhhhhsNNQDISAITTELNQLFNEIDRIAGATgsGGLSEIQNILQRMRELGVQAANGTLNggSASA
    Sequence FISKVDSALQKVSTYRANLGSIQNRLEHTIANLG
    0251 Fir161B PRT Artificial mghhhhhhsGLAQASRNAQDAISIAQTAEGALDETQSILQRVRELGVQGANGTLTADDINALQAEVD
    Sequence QLIAEIDRIAGATEFNTQNLLDGSFTTKAFQVGANSGQNMTLTIGKMDTTTLGLSSADLAINDNAFA
    NGAISTVDSALQKVSAERAKLGAIQNRLEHTIANLG
    0252 Fir161MNB PRT Artificial mghhhhhhsGLAQASRQAQDAISIAQTAEGALDETQSILQRVRELGVQGADGTLTADDIDALQAEVD
    Sequence QLIAEIDRIAGATEFATQKLLDGSFTTKAFQVGAASGQDVTLTIGKVDTTTLGLSSADLAIDSAAFAD
    GAISTVDSALQKVSAERAKLGAIQNRLEHTIAQLG
  • In some embodiments, the aluminum gel or salt is selected from aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate, AS04 (which is composed of aluminum salt and MPL), and ALHYDROGEL. In some embodiments, the aluminum gel or salt is a formulation or mixture with any of the additional adjuvants described herein.
  • In some embodiments, adjuvants in addition to the described flagellin-based agent and an aluminum gel or salt find use in the present invention. In some embodiments, the additional adjuvant is selected from, oil-in-water emulsion formulations, saponin adjuvants, ovalbumin, Freunds Adjuvant, cytokines, and chitosans. Illustrative additional adjuvants include, but are not limited to: (1) ovalbumin (e.g. ENDOFIT), which is often used for biochemical studies; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), such as for example (a) MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as, for example, Model HOy microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, (c) RIBI adjuvant system (RAS), (RIBI IMMUNOCHEM, Hamilton, Mo.) containing 2% Squalene, 0.2% Tween 80, and, optionally, one or more bacterial cell wall components from the group of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), including MPL+CWS (DETOX™); and (d) ADDAVAX (Invitrogen); (3) saponin adjuvants, such as STIMULON (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (by way of non-limiting example, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; (6) chitosans and other derivatives of chitin or poly-N-acetyl-D-glucosamine in which the greater proportion of the N-acetyl groups have been removed through hydrolysis (see, e.g., European Patent Application 460 020, which is hereby incorporated by reference in its entirety, disclosing pharmaceutical formulations including chitosans as mucosal absorption enhancers; and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition, e.g., monophosphoryl lipid A. In other embodiments, the additional adjuvant is one or more of a flagellin-based agent (e.g. CBLB502 or any of the agents of Table 1), an aluminium salt or gel, a pattern recognition receptors (PRR) agonist, CpG ODNs and imidazoquinolines. In some embodiments, the additional adjuvant is one or more of cyclic [G(3′,5′)pA(3′,5′)p] (e.g. 3′3′-cGAMP VACCIGRADE); cyclic [G(2′,5′)pA(3′,5′)p]2′3′ (e.g. 2′3′ cGAMP VACCIGRADE); cyclic [G(2′,5′)pA(2′,5′)p] (e.g. 2′2′-cGAMP VACCIGRADE), cyclic diadenylate monophosphate (e.g. c-di-AMP VACCIGRADE); cyclic diguanylate monophosphate (e.g. c-di-GMP VACCIGRADE); TLR7 agonist-imidazoquinolines compound (e.g. TLR7 agonists, such as, for example, Gardiquimod VACCIGRADE, Imiquimod VACCIGRADE, R848 VACCIGRADE); lipopolysaccharides (e.g. TLR4 agonists), such as that from E. coli 0111:B4 strain (e.g. LPS-EB VACCIGRADE); monophosphoryl lipid A (e.g. MPLA-SM VACCIGRADE and MPLA Synthetic VACCIGRADE); N-glycolylated muramyldipeptide (e.g. N-Glycolyl-MDP VACCIGRADE); CpG ODN, class A and/oror CpG ODN, class B and/or CpG ODN, class C (e.g. ODN 1585 VACCIGRADE, ODN 1826 VACCIGRADE, ODN 2006 VACCIGRADE, ODN 2395 VACCIGRADE), a triacylated lipoprotein (e.g. Pam3CSK4 VACCIGRADE); Polyinosine-polycytidylic acid (e.g. Poly(I:C) (HMW) VACCIGRADE); and cord factor (i.e. mycobacterial cell wall component trehalose 6,6′ dimycolate (TDM,)) or an analog thereof (e.g. TDB VACCIGRADE, TDB-HS15 VACCIGRADE). In some emobodiments, the additional adjuvant is a TLR agonist (e.g. TLR1, and/or TLR2, and/or TLR3, and/or TLR4, and/or TLR5, and/or TLR6, and/or TLR7, and/or TLR8, and/or TLR9, and/or TLR10, and/or TLR11, and/or TLR12, and/or TLR13), a nucleotide-binding oligomerization domain (NOD) agonist, a stimulator of interferon genes (STING) ligand, or related agent.
  • In some embodiments, the additional adjuvants is one or more of a mineral adjuvant, gel-based adjuvant, tensoactive agent, bacterial product, oil emulsion, particulated adjuvant, fusion protein, and lipopeptide. Other mineral salt adjuvants, besides the aluminum adjuvants described elsewhere, include salts of calcium (e.g. calcium phosphate), iron and zirconium. Other gel-based adjuvants, besides the aluminum gel-based adjuvants described elsewhere, include Acemannan. Tensoactive agents include Quil A, saponin derived from an aqueous extract from the bark of Quillaja saponaria; saponins, tensoactive glycosides containing a hydrophobic nucleus of triterpenoid structure with carbohydrate chains linked to the nucleus, and QS-21. Bacterial products include cell wall peptidoglycan or lipopolysaccharide of Gram-negative bacteria (e.g. from Mycobacterium spp., Corynebacterium parvum, C. granulosum, Bordetella pertussis and Neisseria meningitidis), N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), different compounds derived from MDP (e.g. threonyl-MDP), lipopolysaccharides (LPS) (e.g. from the cell wall of Gram-negative bacteria), trehalose dimycolate (TDM), and DNA containing CpG motifs. Oil emulsions include FIA, Montanide, Adjuvant 65, Lipovant, the montanide family of oil-based adjuvants, and various liposomes. Among particulated and polymeric systems, poly (DL-lactide-coglycolide) microspheres have been extensively studied and find use herein.
  • Further, in some embodiments, cytokines are an adjuvant of the present invention (e.g. IFN-γ and granulocyte-macrophage colony stimulating factor (GM-CSF)). Also carbohydrate adjuvants (e.g. inulin-derived adjuvants, such as, gamma inulin, algammulin (a combination of γ-inulin and aluminum hydroxide), and polysaccharides based on glucose and mannose, such as glucans, dextrans, lentinans, glucomannans and galactomannans) find use in the present invention. In some embodiments, adjuvant formulations are useful in the present invention and include alum salts in combination with other adjuvants such as Lipid A, algammulin, immunostimulatory complexes (ISCOMS), which are virus like particles of 30-40 nm and dodecahedric structure, composed of Quil A, lipids, and cholesterol.
  • In some embodiments, the additional adjuvants are described in Jennings et al. Adjuvants and Delivery Systems for Viral Vaccines-Mechanisms and Potential. In: Brown F, Haaheim LR, (eds). Modulation of the Immune Response to Vaccine Antigens. Dev. Biol. Stand, Vol. 92. Basel: Karger 1998; 19-28 and/or Sayers et al. J Biomed Biotechnol. 2012; 2012: 831486, and/or Petrovsky and Aguilar, Immunology and Cell Biology (2004) 82, 488-496 the contents of which are hereby incorporated by reference in their entireties.
  • In various embodiments, the present adjuvants (e.g. flagellin-based agent and an aluminum gel or salt) may be part of live and attenuated, or killed or inactivated, or toxoid, or subunit or conjugate vaccines.
  • In various embodiments, the present adjuvants (e.g. flagellin-based agent and an aluminum gel or salt) may be part of one or more approved vaccines and/or the antigens of one or more approved vaccines may be the antigens of the present invention. In some embodiments, the approved vaccines include: Adenovirus; Anthrax (Biothrax); BCG (Tice); DT (Sanofi); DTaP (Daptacel); DTaP (Infanrix); DTaP-HepB-IPV (Pediarix); DTaP-IPV (Kinrix); DTaP-IPV/Hib (Pentacel); Hib (ActHIB); Hib (Hiberix); Hib (PedvaxHlB); Hib/Hep B (Comvax); Hib/Mening. CY (MenHibrix); Hep A (Havrix); Hep A (Havrix); Hep B (Engerix-B); Hep B (Recombivax); Hep A/Hep B (Twinrix); Human Papillomavirus (HPV) (Cerverix); Human Papillomavirus (HPV) (Gardasil); Influenza (Afluria); Influenza (Agriflu); Influenza (Fluarix); Influenza (Flublok); Influenza (Flucelvax); Influenza (Fluvirin); Influenza (Flulevel); Influenza (Fluzone: Standard, High-Dose, & Intradermal); Influenza (FluMist); Japanese Encephalitis (Ixiaro); Meningococcal (MCV4-Menactra); Meningococcal (MCV4-Menveo); Meningococcal (MPSV4-Menomune); MMR (MMR-II); MMRV (ProQuad); Pneumococcal (PCV13—Prevnar 13); Pneumococcal (PPSV-23—Pneumovax); Polio (IPV—Ipol); Rabies (Imovax); Rabies (RabAvert); Rotavirus (RotaTeq); Rotavirus (Rotarix); Smallpox (Vaccinia—ACAM2000); Td (Decavac); Td (Tenivac); Td (Mass Biologics); Tdap (Adacel); Tdap (Boostrix); Typhoid (inactivated—Typhim Vi); Typhoid (oral—Ty21a); Varicella (Varivax); Yellow Fever (YF-Vax); and Zoster (Shingles—Zostavax).
  • In various embodiments, the present adjuvants (e.g. flagellin-based agent and an aluminum gel or salt) may be part of one or more illustrative vaccines and/or the antigens of one or more illustrative vaccines may be the antigens of the present invention. Illustrative vaccines include, by way of example, subunit vaccine and inactivated or “killed” vaccine (e.g. Infanrix-IPV/Hib (Bordetella pertussis), Infanrix-IPV/Hib (Haemophilus influenzae), Infanrix-IPV/Hib (Poliovirus), Infanrix-IPV/Hib (Clostridium tetani), Infanrix-IPV/Hib (Corynebacterium diphtheriae), Infanrix-hexa (Bordetella pertussis), Infanrix-hexa (Haemophilus influenzae), Infanrix-hexa (Poliovirus), Infanrix-hexa (Hepatitis B virus), Infanrix-hexa (Clostridium tetani), Infanrix-hexa (Corynebacterium diphtheriae), Infanrix-IPV (Bordetella pertussis), Infanrix-IPV (Poliovirus), Infanrix-IPV (Clostridium tetani), Infanrix-IPV (Corynebacterium diphtheriae), Infanrix/Hib (Corynebacterium diphtheriae), Pediarix (Clostridium tetani), Pediarix (Poliovirus), Pediarix (Hepatitis B virus), ViVaxim (Salmonella spp.), ViVaxim (Hepatitis A virus); subunit vaccines (e.g. 5CVMB (Neisseria meningitidis), B. pertussis CyaA protein vaccine (Bordetella pertussis), B. pertussis PTx protein vaccine (Bordetella pertussis), Cancer VEGFA protein vaccine (Cancer), E. coli vaccine using intimin polypeptide (Escherichia coli), Engerix-B (Hepatitis B virus), H. pylori VacA protein vaccine (Helicobacter pylori), HC of type C and D (Clostridium botulinum), Infanrix/Hib (Bordetella pertussis), Infanrix/Hib (Haemophilus influenzae), Infanrix/Hib (Clostridium tetani), M. gallisepticum TM-1 Protein Subunit Vaccine (Mycoplasma gallisepticum), MDA-modified human apo B-100 peptide Vaccine (Atherosclerosis), MSP3-LSP with aluminium hydroxide (Plasmodium spp.), Mumps HN Protein Subunit Vaccine (Mumps virus), N. miningitidis TBP2 Protein Vaccine (Neisseria meningitidis), P. aeruginosa Oprl Protein Vaccine (Pseudomonas aeruginosa), P. falciparum Subunit SE36 Protein Vaccine (Plasmodium spp.), Phleum pratense Allergy Phl p 12 Subunit Vaccine (Allergy), Recombivax HB (Hepatitis B virus), S. pneumoniae ClpP protein Vaccine (Streptococcus pneumoniae); toxoid vaccine (e.g. BoNT/F(Hc) (Clostridium botulinum), DAPTACEL (Corynebacterium diphtheriae), Infanrix (Bordetella pertussis), Infanrix (Clostridium tetani), KINRIX (Clostridium tetani), PBT (Clostridium botulinum), Pediarix (Bordetella pertussis), inactivated or “killed” vaccines (e.g. Avaxim (Hepatitis A virus), Avaxim-Pediatric (Hepatitis A virus), FSME-IMMUN (Tick-borne Encephalitis Virus (TBEV)), Infanrix (Corynebacterium diphtheriae), Ixiaro (Japanese encephalitis virus), KINRIX (Corynebacterium diphtheriae), and Pediarix (Corynebacterium diphtheriae)); and conjugate vaccines (e.g., Arabinomannan-tetanus toxoid conjugate (Mycobacterium tuberculosis)), CCPS-P64kR (Neisseria meningitidis), COMVAX (Haemophilus influenzae), Menjugate (Neisseria meningitidis), Neisvac-C (Neisseria meningitidis), and PedvaxHIB (Haemophilus influenzae)).
  • In some embodiments, the present adjuvants (e.g. flagellin-based agent and an aluminum gel or salt) are combined in a vaccine targeting a substance abuse. For example, in one embodiment, the present adjuvants are used in vaccines against addition to fentanyl, heroin, morphine, opium, oxycodone, hydrocodone, ketamine, PCP, barbiturates, benzodiazepines, flunitrazepam, GHB, methaqualone, hashish, marijuana, LSD, mescaline, psilocybin, amphetamine, cocaine, MDMA, methamphetamine, methylphenidate, and nicotine (e.g. TA-CD (Celtic Pharma), those described in US Patent Publication No. 2013/0011432, the contents of which are hereby incorporated by reference (e.g. using 6-(2R,35)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1]octane-2-carbonyloxy-hexanoic acid (GNC) or 6-((2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido)hexanoic acid) (GNE) as the antigen, and TA-NIC (Celtic Pharma)).
  • In some embodiments, the present adjuvants (e.g. flagellin-based agent and an aluminum gel or salt) and/or the present vaccines may comprise any one of the adjuvants or antigens annotated in the VIOLIN or Vaxjo databases (as described in He et al. Nucleic Acids Research. 2014. 42 (D1): D1124-D1132 and Xiang et al. Nucleic Acids Res. 2008 January; 36: D923-8, the contents of which are hereby incorporated by reference in their entirety).
  • In various embodiments, the present adjuvants (e.g. flagellin-based agent and an aluminum gel or salt) may be part of one or more cancer vaccines and/or the antigens of one or more cancer vaccines may be the antigens of the present invention. Illustrative cancer vaccines include therapeutic and preventative vaccines. For instance, cancer vaccines include ONCOPHAGE (ANTIGENICS INC., approved in Russia in 2008 for kidney cancer), APC8015/Sipuleucel-T/PROVENGE (DENDREON, for, e.g. metastatic hormone-refractory prostate cancer), CANCERVAX (CANVAXIN), GENITOPE CORP (MYVAX personalized immunotherapy), and FAVRILLE INC (FAVID), preventive vaccines which attack the cancer-causing viruses human papillomavirus (e.g. CERVARIX (GSK) and GARDASIL (MERCK)), hepatitis A virus (e.g. CERVARIX (GSK) and GARDASIL (MERCK)), and hepatitis B virus (e.g. RECOMBIVAX HB (MERCK), ENGERIX-B (GSK), ELOVAC B (HUMAN BIOLOGICALS INSTITUTE), GENEVAC B (SERUM INSTITUTE), SHANVAC B, etc.
  • In various embodiments, the present adjuvants (e.g. flagellin-based agent and an aluminum gel or salt) may be part of one or more allergen vaccines and/or the antigens of one or more allergen vaccines may be the antigens of the present invention. For instance, subcutaneous immunotherapy (SCIT) allergen compositions and methods are applicable to the present invention (e.g. “allergy shots”). For example, vaccinations for allergic rhinitis and conjunctivitis (e.g. pollen (including ragweed), dust mites, animal dander and airborne mold spores); allergic or extrinsic bronchial asthma (e.g. house dust mites, pollen, animal dander, mold (Cladosporium), latex); and insect venom hypersensitivity. Allergens include pollen (e.g. tree, grass, weed), pet dander (e.g. cat pelt), dust mites, airborne molds, occupational aeroallergens, honey bee venom, yellow jacket venom, hornet venom, wasp venom, and fire ant venom.
  • In various embodiments, the antigens of the present vaccines may be the antigens of live and attenuated or killed or inactivated or toxoid or a subunit or conjugate vaccines. In various embodiments, the antigen of the present vaccines is an antigen of any of the vaccines described herein. For example, in some embodiments, the present antigen is that of one or more of the following vaccines: DTP (diphtheria-tetanus-pertussis vaccine), DTaP (diphtheria-tetanus-acellular pertussis vaccine), Hib (Haemophilus influenzae type b) conjugate vaccines, Pneumococcal conjugate vaccine, Hepatitis A vaccines, Poliomyelitis vaccines, Yellow fever vaccines, Hepatitis B vaccines, combination DTaP, Tdap, Hib, Human Papillomavirus (HPV) vaccine, Anthrax vaccine, Bacillus Calmette-Guérin (Tb), and Rabies vaccine.
  • In various embodiments, the flagellin-based agent and antigen are adsorbed to the aluminum gel or salt. In some embodiments, the flagellin-based agent and aluminum gel or salt are mixed to form a stable complex. In some embodiments, the flagellin-based agent and aluminum gel or salt are mixed in a ratio that is substantially below a loading capacity of the aluminum salt. In some embodiments, the flagellin-based agent and aluminum gel or salt are present in a ratio that is substantially below a loading capacity of the aluminum salt. In various embodiments, the flagellin-based agent and aluminum gel or salt are mixed or present in a ratio (w/w) of about 1:500, or about 1:600, or about 1:700, or about 1:800, or about 1:900, or about 1:1000, or about 1:2000, or about 1:5000, or about 1:6000, or about 1:7000, or about 1:8000, or about 1:9000, or about 1:10000. In some embodiments, the flagellin-based agent and aluminum gel or salt are mixed in a ratio (w/w) of about 1:500 or less. In some embodiments, the flagellin-based agent and aluminum gel or salt are present in a ratio that is substantially below a loading capacity of the aluminum salt even in the presence of anitigen.
  • The loading (or adsorption) capacity of the adjuvant (e.g. the aluminum gel or salt) can be measured using a variety of analytical methods. In general, it is done by comparing the protein content in the aqueous phase of the agent being loaded or adsorbed (e.g. flagellin-based agent and/or antigen solution) before and after adsorption onto the adjuvant. For instance, the Ramon flocculation test may be used (as is used to determine the adsorption of diphtheria and tetanus toxoid). Further, loading can be measured using immunoprecipitation techniques (e.g. quantitative immunoelectrophoresis or single radial immunodiffusion) or spectrophotometric techniques (e.g. the BCA method) ELISA methods may also be used as can immunoelectrophoresis or HPLC. The aluminum content in the final vaccine can be monitored using a number of known techniques, including spectrometric methods, such as for example atomic adsorption spectrometry.
  • In some embodiments, the amount of aluminum gel or salt in the vaccines and/or adjuvants described herein is about 0.05 to about 1.0 mg/dose, or about 0.125 to about 0.625 mg/dose. In some embodiments, the amount of aluminum gel or salt in the vaccines and/or adjuvants described herein is about 0.05, or about 0.10, or about 0.15, or about 0.20, or about 0.25, or about 0.30, or about 0.35, or about 0.40, or about 0.45, or about 0.50, or about 0.55, or about 0.60, or about 0.65, or about 0.70, or about 0.75, or about 0.80, or about 0.85, or about 0.90, or about 0.95, or about 1.0 mg/dose.
  • In some embodiments, the amount of flagellin-based agent is about 0.03 to about 5 μg/dose (e.g. about 0.03 μg/dose, about 0.1 μg/dose, about 0.3 μg/dose, about 0.5 μg/dose about 1.0 μg/dose, about 1.5 μg/dose, about 2.0 μg/dose, about 2.5 μg/dose, about 3.0 μg/dose, about 4.0 μg/dose, about 4.5 μg/dose, about 5.0 μg/dose). In various embodiments, the present compositions and methods comprise doses of flagellin-based agent that are less than about 5 μg/dose, or less than 4 μg/dose, or less than 3 μg/dose, or less than 2 μg/dose, or less than 1 μg/dose, or less than 0.5 μg/dose. In some embodiments, the present compositions and methods comprise low doses of flagellin-based agent.
  • In various embodiments, the present compositions and methods do not involve covalently attaching an antigen to the flagellin-based agent either as a fusion protein or via chemical conjugation. In various embodiments, the present compositions do not have either equimolar ratio of antigen to flagellin-based agent (as in a fusion) or several molecules of hapten per one molecule of flagellin-based agent (as in chemical conjugates). In various embodiments, the amount of flagellin-based agent in any of the present vaccines is less than the amount of antigen. In various embodiments, the amount of flagellin-based agent in any of the present vaccines is less than the amount of antigen. In various embodiments, the amount of flagellin-based agent in any of the present vaccines is substantially less than the amount of antigen. In various embodiments, the amount of flagellin-based agent in any of the present vaccines is about 500-fold, or about 450-fold, or about 400-fold, or about 350-fold, or about 325-fold, or about 300-fold, or about 250-fold, or about 200-fold, or about 150-fold, or about 100-fold, or about 50-fold less than the amount of antigen.
  • In various embodiments, the combination of flagellin-based agent and alum do not substatially effect TLR5 interaction by the flagellin-based agent.
  • In various embodiments, the present compositions and methods do not induce production of TNFα.
  • In various embodiments the present combination of flagellin-based agent and alum is subtantially stable at low temperatures for about one week (e.g. at about 4° C. for about 3 days, or about 5 days, or about 6 days, or about 7 days, or about 10 days).
  • In another aspect, the present invention relates to a method of vaccinating a subject against a disorder, comprising administering an effective amount of a vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with the disorder. In another aspect, the invention relates to a use of vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with a disorder for vaccinating a subject against the disorder. In another aspect, the invention relates to a use of an effective amount of vaccine comprising an adjuvant comprising a flagellin-based agent and an aluminum gel or salt and an antigen associated with a disorder in the manufacture of a medicament for vaccinating a subject against the disorder.
  • In another aspect, the present invention relates to a method of immunostimulating a subject in advance of or concurrent with vaccination, comprising administering an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt, wherein both TH1 and TH2-mediated immune responses are immunostimulated. In another aspect, the invention relates to a use of an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt for immunostimulating a subject in advance of or concurrent with vaccination. In another aspect, the invention relates to a use of an effective amount of an adjuvant comprising a flagellin-based agent and an aluminum gel or salt in the manufacture of a medicament for immunostimulating a subject in advance of or concurrent with vaccination.
  • In various embodiments, the vaccine described herein causes an improvement in adjuvant properties relative to a vaccine comprising the antigen and the aluminum gel or salt alone (or flagellin-based agent and antigen alone). In various embodiments, the vaccine and/or adjuvant described herein causes a broader, more diverse, more robust and longer lasting immunostimulatory effect than the vaccine comprising the antigen and the aluminum gel or salt alone (or flagellin-based agent and antigen alone) and/or the adjuvant comprising the aluminum gel or salt alone (or the adjuvant comprising the flagellin-based agent alone).
  • In some embodiments, the described vaccine and/or described adjuvant causes an increase in titer of 1 or more of, or 2 or more of, or 3 or more of, or all of IgG1, IgG2a, IgG2b, and IgG3 antibodies (e.g. relative to the adjuvant comprising the aluminum gel or salt or flagellin-based agent alone, or relative to the vaccine comprising the antigen and the aluminum gel or salt alone or flagellin-based agent alone)). In some embodiments, the described vaccine and/or described adjuvant causes a relative increase in the titer of all of IgG1, IgG2a, IgG2b, and IgG3 antibodies. In some embodiments, the described vaccine and/or described adjuvant causes a relative increase in the titer of more IgG3 antibodies than the described vaccine and/or described adjuvant in the absence of a flagellin-based agent (or the described vaccine and/or described adjuvant in the absence of an aluminum gel or salt alone).
  • In some embodiments, the antigen is administered simultaneously with or sequentially to the adjuvant.
  • In some embodiments, the disorder is selected from infectious diseases, cancer, allergy, and autoimmune diseases.
  • In some embodiments, the disorder is selected from diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, Human Papillomavirus (HPV) infection, anthrax, rabies, Japanese Encephalitis, meningitis, measles, mumps, rubella, gastroenteritis, smallpox, typhoid fever, varicella (chickenpox), rotavirus, and shingles.
  • In some embodiments, the disorder is a cancer is selected from, but not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
  • In some embodiments, the disorder is an allergy, selected from, by way of non-limiting example, allergic rhinitis and conjunctivitis, allergic or extrinsic bronchial asthma, and insect venom hypersensitivity.
  • In some embodiments, the disorder is a substance abuse disorder (e.g. of fentanyl, heroin, morphine, opium, oxycodone, hydrocodone, ketamine, PCP, barbiturates, benzodiazepines, flunitrazepam, GHB, methaqualone, hashish, marijuana, LSD, mescaline, psilocybin, amphetamine, cocaine, MDMA, methamphetamine, methylphenidate, and nicotine).
  • In some embodiments, the compositions of the present invention (e.g. the described adjuvants and vaccines) can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.
  • Pharmaceutically acceptable salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, a-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.
  • The term “pharmaceutically acceptable salt” also refers to a salt of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.
  • In some embodiments, the compositions of the present invention (e.g. the described adjuvants and vaccines) described herein are in the form of a pharmaceutically acceptable salt.
  • In some embodiments, the compositions of the present invention (e.g. the described adjuvants and vaccines) may comprise a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
  • Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any composition described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • The present invention includes the compositions of the present invention (e.g. the described adjuvants and vaccines) in various formulations. Any composition of the present invention can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
  • Where necessary, the compositions of the present invention can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
  • The formulations comprising the compositions of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).
  • In one embodiment, any composition of the present invention is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
  • Routes of administration include intramuscular, e.g. by injection or infusion. In some embodiments, the described adjuvant of a flagellin-based agent (e.g. CBLB502) and aluminum gel or salt may prevent systemic delivery of the flagellin-based agent and induce localized delivery. In other embodiments, routes of administration include nasal, oral, and sublingual delivery.
  • Routes of administration may also be intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, oral, sublingual, intranasal, transdermal, or by inhalation. In some embodiments, the administering is effected orally or by parenteral injection. The mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.
  • Any composition of the present invention (e.g. the described adjuvants and vaccines) can be administered orally. Such compositions can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.
  • Dosage forms suitable for parenteral administration (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
  • The dosage of any composition of the present invention (e.g. the described adjuvants and vaccines) as well as the dosing schedule can depend on various parameters, including, but not limited to, the disorder being treated, the subject's general health, and the administering physician's discretion.
  • In vitro or in vivo assays can be employed to help identify optimal dosage ranges. For example, doses may be determined with reference Physicians' Desk Reference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011), the contents of which are incorporated by reference in its entirety.
  • For administration of a composition of the present invention (e.g. the described adjuvants and vaccines) by parenteral injection, the dosage is normally about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day. Injections may be given up to four times daily. Generally, when orally or parenterally administered, the dosage of any agent described herein is normally about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day. A dosage of up to about 3000 mg per day can be administered.
  • In another embodiment, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
  • Any composition of the present invention (e.g. the described adjuvants and vaccines) can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543;
  • 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein. The invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.
  • Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
  • In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).
  • In another embodiment, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533) may be used.
  • Administration of a composition of the present invention (e.g. the described adjuvants and vaccines) can, independently, be once per patient or may be used in a booster strategy. Administration may be about one to about four times daily or about one to about four times per month or about one to about six times per year or once every two, three, four or five years. Administration can be for the duration of about one day or about one month, about two months, about three months, about six months, about one year, about two years, about three years, and may even be for the life of the subject. The dosage may be administered as a single dose or divided into multiple doses.
  • The dosage regimen utilizing any flagellin related composition (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed. Any flagellin related composition (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, any flagellin related composition (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen.
  • In some embodiments, the composition of the present invention (e.g. the described adjuvants and vaccines) may be used in conjunction with one or more additional agents. In some embodiments, the invention pertains to co-administration and/or co-formulation. Any of the compositions described herein may be co-formulated and/or co-administered.
  • In some embodiments, any composition described herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy. In various embodiments, any agent referenced herein may be used in combination with any of the composition described herein.
  • In some embodiments, the present invention pertains to additional agents described elsewhere herein. In one embodiment, any flagellin-related agent or composition comprising the same may be used with agents that stimulate NOD receptors (e.g. NOD1 and NOD2 agonists, such as peptidoglycan, C12-iE-DAP and L18-MDP) and as described in Infect Immun. October 2013; 81(10): 3855-3864, the contents of which are hereby incorporated by reference in their entirety.
  • In some embodiments, the present invention pertains to chemotherapeutic agents as additional agents.
  • Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; def of amine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation. In addition, the methods of treatment can further include the use of photodynamic therapy.
  • In some embodiments, the flagellin-based agents (and/or additional agents) described herein, include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids.
  • In still other embodiments, the flagellin-based agents (and/or additional agents) described herein further comprise a cytotoxic agent, comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents may be conjugated to a composition described herein.
  • The flagellin-based agents (and/or additional agents) described herein may thus be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • Illustrative cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (0,P′-(DDD)), interferons, and mixtures of these cytotoxic agents.
  • Further cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins, taxols, irinotecan, 5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine and vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, Toxin conjugated monoclonal antibodies, tumor antigen specific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies, Rituxan, ocrelizumab, ofatumumab, DXL625, HERCEPTIN®, or any combination thereof. Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to the therapeutic agents (e.g. antibodies) to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).
  • Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, or binding fragments thereof, with or without the use of a complex-forming agent. Such radionuclides include beta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67, Yttrium-88, Yttrium-90, Iodine-125, Iodine-131, Samarium-153, Lutetium-177, Rhenium-186 or Rhenium-188, and alpha-emitters such as Astatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.
  • Illustrative detectable moieties further include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase and luciferase. Further illustrative fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride. Further illustrative chemiluminescent moieties include, but are not limited to, luminol. Further illustrative bioluminescent materials include, but are not limited to, luciferin and aequorin. Further illustrative radioactive materials include, but are not limited to, Iodine-125, Carbon-14, Sulfur-35, Tritium and Phosphorus-32.
  • In some embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In some embodiments, the subject and/or animal may comprise fluorescently-tagged cells (with e.g. GFP). In some embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell.
  • In some embodiments, the subject and/or animal is a human. In some embodiments, the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient.
  • In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
  • In other embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.
  • The invention provides kits that can simplify the administration of any agent described herein. An illustrative kit of the invention comprises any composition described herein in unit dosage form. In one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.
  • The following definitions are used in connection with the invention disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.
  • As used herein, “a,” “an,” or “the” can mean one or more than one.
  • Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.
  • An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.
  • As used herein, something is “decreased” if a read-out of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in some embodiments, activity is decreased and some downstream read-outs will decrease but others can increase.
  • Conversely, activity is “increased” if a read-out of activity and/or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.
  • As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”
  • As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
  • The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents (e.g. flagellin-based agents, flagellin-based agents (and/or additional agents) described herein) for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. For example, administration of therapeutic agents to a patient suffering from cancer provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the disease, e.g., a decrease in tumor burden, a decrease in circulating tumor cells, an increase in progression free survival. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • In certain embodiments, a pharmacologically effective amount that will treat cancer will modulate the symptoms typically by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In illustrative embodiments, such modulations will result in, for example, statistically significant and quantifiable changes in the numbers of cancerous cells.
  • This invention is further illustrated by the following non-limiting examples.
    • EXAMPLES Example 1 Preparation of the Model Vaccine
  • Alum adjuvant (IMJECT ALUM, THERMO) was used in combination with CBLB502 to challenge the mice with a model antigen, ovalbumin (OVA). Vaccine was comprised of the following components as shown in TABLE A:
  • Component Amount (per mouse)
    Ovalbumin  10 mg
    CBLB502 1 ug/10 ug
    Alum Adjuvant
    500 ug
  • To achieve excellent absorption CBLB502 (either 1 μg or 10 μg) was mixed with 10 mg of OVA and 500 μg of Alum adjuvant for 30 minutes at 300 rpm at room temperature.
    • Example 2 Mouse Immunization Study
  • C57BI/6 male mice (10 weeks of age, n=6 for each experiment) were used for injection. Mice were divided into five groups with six animals in each group (n=6). Mice were injected with 100 μl of above described vaccine in each of the hind legs (200 μl total per mouse) as shown in TABLE B.
  • Ovalbumin CBLB502 Alum Adjuvant
    Group
    1
    Group 2 10 mg
    Group
    3 10 mg 500 ug
    Group
    4 10 mg  1 ug 500 ug
    Group
    5 10 mg 10 ug 500 ug

    Two weeks after the initial immunization mice were challenged with an additional booster following the same setup as the original injection. Plasma was collected one, two and four weeks after booster via mandibular vein. Using an ELISA based assay total IgG, IgG1, IgG2a, IgG2b and IgG3 were measured. Results are shown in FIGS. 2-3. In the figures, Group 1 is also called “Untreated;” Group 2 is also called “OVA,” Groups 3 is also called “OVA+Alum,” Group 4 is also called “SA 1 μg,” and Group 5 is also called “SA 10 μg.”
  • FIG. 2 shows ELISA data for the average response of the five aforementioned mouse treatment groups at the time point of 1 week post-boost. FIG. 3 shows ELISA data for the average response of the five aforementioned mouse treatment groups at the time point of 2 weeks post-boost. FIG. 4 shows ELISA data for the average response of the five aforementioned mouse treatment groups at the time point of 4 weeks post-boost.
    • Example 3 Evaluation of CBLB502 Binding to ALHYDROGEL
  • The binding capacity of ALHYDROGEL for CBLB502 was evaluated. ALHYDROGEL adjuvant 2%, aluminium hydroxide wet gel (colloidal) suspension (INVIVIGEN Catalog #vac-alu-250), was mixed with CBLB502 at different volume ratios, resulting in 10 to 52% ALHYDROGEL suspension in the reaction. CBLB502 concentration was maintained at 10 μg/ml, corresponding to a 1 μg/100 μl inoculation dose (100 μl inoculation volume is the recommended maximum volume for subcutaneous injection of antigen/adjuvant mixtures per injection site for mice). After overnight incubation at 4° C., the tubes were centrifuged to sediment ALHYDROGEL and unbound CBLB502, remaining in solution was measured by ELISA. The amounts of adsorbed CBLB502 were calculated as difference between total protein added to the mixture and unbound CBLB502 (TABLE C). The maximum adsorption of CBLB502, more than 99.9% bound, was observed when the reactions were formulated with 40 to 52 μl ALHYDROGEI/100 μl; at lower ratios, the binding efficiency decreased gradually to about 83% at 10 μml/100 μl ALHYDROGEL suspension.
  • TABLE C
    CBLB502 adsorption at different ALHYDROGEL 2% volume ratios. The binding reactions were
    composed of CBLB502, PBS and the indicated volumes of ALHYDROGEl adjuvant 2%.
    ALHYDROGEL 2% CBLB502 dose Total CBLB502 Unbound Bound Bound
    μl/100 μl μg/100 μl ng/ml ng/ml ng/ml (%)
    1 52 1 10000 2.1 9997.9 99.98
    2 50 1 10000 1.6 9998.4 99.98
    3 40 1 10000 2.9 9997.1 99.97
    4 30 1 10000 22.7 9977.3 99.77
    5 20 1 10000 173.4 9826.6 98.27
    6 10 1 10000 1692.5 8307.5 83.07
  • CBLB502 binding to ALHYDROGEL was also evaluated at different CBLB502 doses. Adsorption of CBLB502 to ALHYDROGEL (52 μl/100 μl ALHYDROGEL adjuvant 2%) was determined at CBLB502 doses, ranging from approximately 0.02 to 20 μg per 100 μl. The reactions were incubated as described above and unbound CBLB502 was measured by ELISA. The results (TABLE D) demonstrated that almost complete binding of the drug to ALHYDROGEL, 99.82 to 99.99% bound, was accomplished at all tested CBLB502 concentrations.
  • TABLE D
    Efficiency of CBLB502 binding to ALHYDROGEl in PBS.
    The incubations were performed with CBLB502 at the indicated
    doses in PBS and 52 μl/100 μl ALHYDROGEL adjuvant 2%.
    CBLB502 dose Total CBLB502 Unbound Bound Bound
    μg/100 μl ng/ml ng/ml ng/ml (%)
    1 20 200000 199.3 199800.7 99.90
    2 5 50000 90.6 49909.4 99.82
    3 1.25 12500 0.79 12499.2 99.99
    4 0.31 3125.0 0.029 3124.97 99.99
    5 0.078 781.25 0.023 781.23 99.99
    6 0.02 195.31 0.011 195.30 99.99
  • CBLB502 binding to ALHYDROGEL in the presence of TT-SMA vaccine was evaluated. Binding efficiency of CBLB502 to ALHYDROGEL was tested in the presence of a vaccine candidate TT-SMA, succinyl methamphetamine (SMA) hapten conjugated to tetanus toxoid. In a separate study, shown below, this vaccine was administered to mice at a 32 μg/100 μl dose to compare efficacy with and without CBLB502. This TT-SMA dose was co-incubated with CBLB502 (0.02 to 20 μg/100 μl) and ALHYDROGEL (52 μl/100 μl) under standard conditions and unbound CBLB502 was measured by ELISA. The binding efficiency was 99.7 to 99.9% at all tested CBLB502 concentrations, indicating that CBLB502 adsorption to ALHYDROGEL was unaffected with addition of TT-SMA to the reaction mixture
  • TABLE E
    CBLB502 binding to ALHYDROGEL in the presence of TT-SMA.
    The incubations were performed with CBLB502 at the indicated
    doses in PBS, 52 μl/100 μl ALHYDROGEL adjuvant 2% and
    32 μg/100 μl TT-SMA conjugate.
    CBLB502 dose Total CBLB502 Unbound Bound Bound
    μg/100 μl ng/ml ng/ml ng/ml (%)
    1 20 200000 668.88 199331.1 99.67
    2 5 50000 50.21 49949.8 99.90
    3 1.25 12500 11.44 12488.6 99.91
    4 0.31 3125.0 1.65 3123.4 99.95
    5 0.078 781.25 0.13 781.1 99.98
    6 0.02 195.31 0.06 195.2 99.96
    • Example 4 Analysis of In Vitro CBLB502 Activity After Adsorption to ALHYDROGEL
  • An activity assay of CBLB502/ALHYDROGEL using 293-hTLR5-LacZ reporter cells was undertaken. Biological activity of CBLB502 after adsorption to ALHYDROGEL was tested using an in vitro assay which measures activation of a NF-κB-controlled reporter enzyme, β-galactosidase, in HEK293-hTLR5::NF-κN-lacZ (293-hTLR5-LacZ) cells. Since 293-hTLR5-LacZ cells express only one cell membrane-bound toll-like receptor, human TLR5, CBLB502 activity could be determined without interference from other toll-like receptor ligands such as LPS (endotoxin).
  • Specific induction of the β-galactosidase reporter by CBLB502/ALHYDROGEL was demonstrated after incubating the cells with a serially diluted CBLB502/ALHYDROGEL formulation (5 μg/100 μl), starting with a 2000-fold dilution in cell growth media and resulting in the concentration range of ALHYDROGEL-adsorbed
  • CBLB502 from 25 to 0.004 ng/ml (FIG. 5). The maximum β-galactosidase activity was observed at 0.93 ng/ml ALHYDROGEL-adsorbed CBLB502; this value was consistent with typical results of the assay using soluble CBLB502 where the reporter enzyme activity would normally peak around 1 ng/ml. ALHYDROGEL alone tested at same dilutions did not induce the reporter. Therefore, it could be concluded, inter a/ia, that CBLB502 retains its biological activity after adsorption to ALHYDROGEL and becomes readily available for interaction with its target receptor (TLR5) under regular cell culture conditions.
  • Next, CBLB502/ALHYDROGEL suspensions were prepared at CBLB502 inoculation doses ranging from 2.5 to 0.00015 μg/100 μl and were added to 293-hTLR5-LacZ cells at 200-fold dilution (FIG. 6). NF-κB-inducing activity followed a typical dose-response pattern and was measured at sub-nanogram doses of CBLB502/ALHYDROGEL (less than 0.001 μg/100 μl), indicating that at such low doses, CBLB502 was not irreversibly bound to the alum adjuvant and remained active.
  • An activity titration of CBLB502/ALHYDROGEL at different CBLB502 doses was undertaken. CBLB502/ALHYDROGEL suspensions were titrated in cell culture media and incubated with 293-hTLR5-LacZ cells. CBLB502 standards were included in the assay to compare the recovered activity. The resulting reporter enzyme activity was plotted against theoretical concentrations of ALHYDROGEL-bound CBLB502, assuming that the protein was completely available (FIG. 7).
  • The results of titration assay demonstrated that at a formulation dose of 20 μg/100 μl, the activity of CBLB502 followed the expected concentrations, essentially matching the activity of soluble standards. However, reduction of the dose 64- and 256-fold to respectively, about 0.31 and 0.078 μg/100 μl, resulted in lower relative recovery of NF-κB-inducing activity to approximately 15-30%. This apparent loss of CBLB502 activity could be due to stronger binding of the particular amounts of the protein relative to the amounts of alum in the formulation. These fixed amounts could represent a small fraction of total CBLB502 at the higher doses such as 20 μg/100 μl and would not affect significantly the measured activity. However, at lower doses, this tightly adsorbed fraction may become substantial compared to the total protein, leading to a lower measurable activity of CBLB502
    • Example 5 Assessment of CBLB502/ALHYDROGEL Short-Term Storage Stability at 4° C.
  • A formulation of 1 μg/100 μl CBLB502 (52 μl/100 μl ALHYDROGEL 2%) was prepared and tested for activity after 2 and 6 days storage at 4° C. The results of CBLB502 activity assay using 293-hTLR5-LacZ reporter cells are shown in FIG. 8. Based on comparison with the soluble CBLB502 standard, in can be concluded that NF-κB-inducing activity of ALHYDROGEL-adsorbed CBLB502 remained unchanged after 6 days under the storage conditions.
    • Example 6 Evaluation of CBLB502 Dissociation from ALHYDROGEL in PBS and Cell Media
  • Aliquots of CBLB502/ALHYDROGEL (1 μg/100 μl dose; 52 μl/100 μl ALHYDROGEL 2%) were re-suspended in PBS or cell media (DMEM supplemented with 10% FBS) and incubated for 3 hr at 22° C. and 37° C. After centrifugation to remove ALHYDROGEL, dissociated CBLB502 was measured in supernatants by ELISA. The calculation of CBLB502 amounts that were recovered during incubation demonstrated that the protein remained stably associated with ALHYDROGEL in PBS; however, in cell media, 28 and 53% CBLB502 was released from the complex at 22° C. and 37° C., respectively (TABLE F). Such rapid dissociation from ALHYDROGEL in media may be facilitated by serum proteins which could displace CBLB502 from the complex with alum, explaining how the protein becomes readily available in the cell-based activity assay.
  • TABLE F
    CBLB502 recovery from ALHYDROGEL after incubation in PBS and cell media
    22° C. 37° C.
    Incubation CBLB502 dose Total CBLB502 Recovered Desorption Recovered Desorption
    conditions μg/100 μl ng/ml ng/ml (%) ng/ml (%)
    PBS 1 10000 2.3 0.02 33.2 0.33
    Media 1 10000 2807.6 28.1 5300.9 53.0
    • Example 7 Quantitation of CBLB502 and Cytokine Levels in Mouse Serum After Subcutaneous Administration of CBLB502/ALHYDROGEL
  • An animal study was conducted to evaluate feasibility of methods to determine the levels of CBLB502 and cytokines in serum after administration of a CBLB502/ALHYDROGEL formulation. The study design is presented in TABLE G. In this study, mice were injected subcutaneously with a 1 μg dose CBLB502 adsorbed to 52 μl/100 μl ALHYDROGEL 2% and serum samples were collected between 0.5 and 24 hr post inoculation.
  • TABLE G
    Study design.
    Time
    Age Injection Point,
    Group Group Strain volume, blood
    No. Size Sex Treatment μl collection Evaluation
    1 3 8 wk old ALHYDROGEL/PBS, sc 100  1 hr Serum prepared from
    2 3 C57BL/6 ALHYDROGEL/PBS/CBLB502, 100 30 min collected blood and
    3 3 Female (1 μg/100 μl CBLB502; 52 μl/ 100  1 h analyzed for
    4 3 100 μl ALHYDROGEL 2%) 100  2 h entolimod
    5 3 sc 100  4 h concentration and
    6 3 100  8 h cytokine levels by
    7 3 100 24 h ELISA
  • Serum concentrations of CBLB502 were determined by ELISA and were found at measurable levels in all groups of animals inoculated with CBLB502/ALHYDROGEL. The pharmacokinetic profile representing per-group mean serum CBLB502 concentrations is shown in FIG. 9. The maximum mean CBLB502 concentration, 0.516 ng/ml, was observed at the first blood collection time point, 0.5 hr post administration; the minimum concentration was 0.012 ng/ml at 24 hr. Half-time elimination of CBLB502 was calculated at 2.4 hr. As discussed above, CBLB502 dissociated rapidly from the complex with ALHYDROGEL after incubation in cell media, containing 10% fetal bovine serum. Therefore, it should be expected to see the protein released from alum into animal bloodstream shortly after inoculation.
  • The concentrations of three cytokines, KC, G-CSF and IL-6, were measured in the collected mouse serum samples by ELISA (FIG. 10). Rapid induction of KC and IL-6 was detected 1 hr after CBLB502/ALHYDROGEL administration; the maximum per-group mean concentrations, 29624 and 479 pg/ml, respectively, were determined at 2 hr time point. After reaching maximum, the blood levels of these cytokines decreased to baseline at 4 to 8 hr post injection. The serum G-CSF concentration increased about an hour later compared to KC and IL-6 and was maximal between 2 and 4 hr after CBLB502/ALHYDROGEL administration (7141 and 7169 pg/ml per-group mean concentrations at 2 and 4 hr, respectively). However, CBLB502-induced G-CSF level was decreasing at a slower rate, remaining above baseline at 24 hr time point. In general, the kinetics of cytokine induction after CBLB502/ALHYDROGEL administration was similar to the kinetics observed after injection of the soluble CBLB502 preparations, suggesting consistent effects of this drug formulation.
    • Example 8 CBLB502 Adjuvant With Methamphetamine Vaccine
  • This study evaluated CBLB502 adjuvant at a 32 μg dose of human methamphetamine vaccine (SMA-TT) to achieve high and long lasting levels of anti-MA antibody in mice. A low to high dose of CBLB502 ranging from 0.03 to 20 μg with TT-SMA and alum was studied. Ten groups (n=5) of Balb/c female mice were employed to test 0, 0.03, 0.1, 0.3, 1, 3, 10, 20 μg of 502 in combination with 32 μg of TT-SMA and 1.5 mg alum compared to unvaccinated controls and TT-SMA with 502 alone. All vaccinated groups were administered two boosters, at 3 and 6 weeks after the initial vaccination. Levels of anti-MA IgG were assessed by ELISA in pooled sera samples collected at 2, 4, and 6 weeks after initial vaccination. The antibody levels were higher at 0.03 to 0.3 μg 502 in comparison to the higher doses (1 to 20 μg) of 502. In this experiment, antibody levels were 2 fold higher in the groups with 0.1 μg of 502 in combination with TT-SMA and alum (group 4) than in the comparison group with TT-SMA and alum alone (group 1) and approx. 4 fold higher when compared to a group with TT-SMA and 502 alone (group 2), as demonstrated in FIG. 11 (panel A). Anti-502 antibodies in the same sera sample were also evaluated. As shown in FIG. 11 (panel B), the level of anti-502 antibodies was minimal at the low dose range of 502 and increases at higher doses of 502.
    • Example 9 Generation of Vaccines With Various Antigens
  • In this Example, the composition of an adjuvant component for adjuvant-enhanced vaccine is established by testing a range of doses of CBLB502 and CBLB502 optionally combined with an antigen, such as, by way of non-limitation, NOD1 agonist C12-iE-DAP. Additionally, characterization of the effects of these agents on humoral immune response to a model antigen (ovalbumin) is undertaken.
  • As shown above, aluminum hydroxide and CBLB502 form a complex which, without wishing to be bound by theory, is stabilized by electrostatic forces as substances with opposite charges in aqueous medium. To test relative efficacy of different compositions of the adjuvant component of the vaccine, a standard dose of aluminum is used (1.5 μg per injection) loaded with a range of doses of CBLB502 (up to 30 μg and including 10 μg, 3 μg, 1 μg, 0.3 μg and 0.1 μg). CBLB502 in aqueous solution is added to the alum suspension in PBS and stirred for 10 min at room temperature. Aluminum hydroxide is then spun down by centrifugation and traces of CBLB502 in supernatants are determined using two quantitative analytical assays: (i) ELISA-based and (ii) reporter cell based (TLR5-positive 293 cells carrying NF-κB responsive reporter and calibrated for detection of CBLB 502 in the medium). In light of, inter alia, the above Examples, it is expected that a substantial fraction of CBLB 502 will be stably bound to alum under these conditions and within the chosen range of doses of TLR5 agonists.
  • To determine whether this adjuvant remains localized at the vaccine injection site, adult BALB/c female mice (n=10) carrying in their germ line firefly luciferase cDNA under the control of NF-κB-responsive promoter will be used. These “NF-κB reporter mice” are a sensitive tool for the detection of release of CBLB502 from the injection site by monitoring the luciferase activity in TLR5-positive tissues such as liver or small intestine. Only one dose of CBLB502-containing adjuvant formulation will be used that corresponds to the highest dose that demonstrated stability as a composition with Alum in vitro (presumably 30 μg). Two doses of free CBLB502 (1 and 3 μg in aqueous solution, s.c. injection) will be used for comparison as positive controls of systemically distributed TLR5 agonist. Luciferase detection will be done using a luminescent imager in vivo following luciferin injection at 2, 4, 6 and 18 hours post injection of the adjuvant. For a more accurate and sensitive detection, 3 animals from each group are sacrificed 6 hours post injection of the adjuvant formulation and tissues will be collected from the injection site, lymph nodes nearest to injection site and the liver. The level of luciferase activity indicative of the presence of CBLB 502 is quantitated in these tissue samples following tissue lysis. In addition to functional detection of CBLB 502 based on its expected NF-κB-inducing activity, an alternative analytical method of an established sensitive ELISA assay for direct detection of CBLB502 in tissue extracts is used.
  • Adjuvant formulations containing the above-described range of doses of CBLB502 are mixed with 10 mg of ovalbumin and vaccination is performed, as described herein, followed by the ELISA determination of titers of different classes of anti-albumin IgG (IgG1, IgG2a, IgG2b and IgG3) antibodies. The results are compared with “Alum only” control. Determination of the effects on different IgG types provides definition of the degrees of engagement of different immunization paths (e.g. TH1- versus TH2-mediated routes) by the adjuvant.
  • After determination of the composition of the adjuvant (in terms of anti-ovalbumin antibody inducing efficacy), additional enhancement of immunization is tested by adding into the formulation a NOD1 agonist, C12-iE-DAP.
  • Also, determination of vaccine efficacy at different CBLB 502 doses to generate antibodies to antigen is undertaken in mice. Female mice are chosen because they have stronger immune responses than male mice and BALB/c mice are a commonly used strain in immunological studies due to their robust immune responses. At the beginning of the study, mice weigh about 25 g and are group-housed (5 per cage) in standard acrylic cages (7″×11″×5″) with corncob bedding polycarbonate and tops. Mice will have ad libitum access to food (Harlan) and water. The vivarium will be maintained at 22±1° C. and on a 12:12 light/dark cycle (lights on at 6 AM). All experimental procedures are approved by the Institutional Animal Care and Use Committee (IACUC) and are within the guidelines delineated in the Guide for the Care and Use of Laboratory Animals.
  • Mice are intramuscularly injected with the vaccine in the gluteal muscle. Each group is optionally immunized with a booster dose with the same vaccine formulation at 3 and 6 weeks post initial immunization. If persistent antibody levels are not maintained for at least 4 weeks, an additional immunization will be given at week 10.
  • Blood is drawn at 14, 28, 42, 56 and 84 days post initial immunization and allowed to clot at room temperature for 2 hours. Serum is collected after centrifugation at 4000 rpm for 15 min. Samples are stored at −80 degrees until ready to run the enzyme linked immunosorbent assays (ELISA). In the context of a vaccine with antigen X, anti-antigen X specific antibodies are assessed by ELISAs. To measure the antibodies, ELISA plates (Immulon 2HB, Daigger, Vernon Hills, Ill.) are coated overnight in carbonate buffer (0.05 M; pH 9.6) using fish gelatin, which is a heterologous carrier protein, as the conjugate partner for SMA. Pooled or individual serum samples are added to plates in 2 fold serial dilutions starting at 1K in phosphate buffered saline (PBS)-Tween (0.1%) and incubated for 2 hours. Plates are washed with PBS-Tween prior to adding goat anti-mouse IgG conjugated to horse-radish peroxidase (HRP) (Southern Biotech, Birmingham, Ala.). Plates are incubated for another 30 min and washed before adding substrate (Tetramethylbenzidine, Sigma, St. Louis, Mo.). Plates are incubated for 45 min in the dark prior to stopping the reaction with 1M HCl. The optical density (OD) of the resulting dark spots on the plates from the antibody with antigen X linking are used to measure titer levels on a microplate reader (iMark Microplate Absorbance Reader) using Microplate Manager v 6.1 software.
  • ELISA data is analyzed using SigmaPlot (Systat Software Inc.) and background antibody binding to the carrier alone will be subtracted from each sample. Comparisons are made in each plate with a standard curve of purified mouse IgG (Sigma) bound directly in the wells in serial dilution.
    • Example 10 Comparison of CBLB502-Based Adjuvants/Vaccines With Flagellin-Based Adjuvants/Vaccines
  • The CBLB502/alum adjuvant of the above Examples is tested head to head against a flagellin/alum adjuvant using the methods and study design of, for instance, Example 9 (optionally with an antigen). Further, the alum binding and dissociation studies, as well as the mouse evaluation studies, all described in the above Examples, are reapeated with a flagellin/alum adjuvant for comparison to the CBLB502/alum adjuvant. The efficacy of the CBLB502 variant, i.e., CBLB502-S33MX is also tested.
  • In one comparison experiment, a group of mice (n=6) is immunized with TT-SMA, which is a methamphetamine vaccine and is used illustratively, co-adsorbed onto Alhydrogel with either flagellin, CBLB502, or CBLB502-S33MX. Immunication is carried out on days 0, 14, and 28 of the study. Each immunization dose includes 32 μg of TT-SMA and adjuvants as follows: Group 1: 0.03 μg flagellin, Group 2: 0.1 μg flagellin, Group 3: 0.3 μg flagellin, Group 4: 1 μg flagellin, Group 5: 0.03 μg CBLB502, Group 6: 0.1 μg CBLB502, Group 7: 0.3 μg CBLB502, Group 8: 1 pg CBLB502, Group 9: 0.03 g CBLB502-S33MX, Group 10: 0.1 pg CBLB502-S33MX, Group 11: 0.3 μg CBLB502-S33MX, and Group 12: 1 μg CBLB502-S33MX.
  • Serum samples are collected and analyzed on days −1 (pre-immune serum), 14 (post-prime), 28 (post first boost), and 35 (post second boost). For example, the serum samples are analzyed for the levels of anti-methamphetamine and anti-entolimod antibodies. Analysis indicates that CBLB502/alum and CBLB502-S33MX/alum cause a broader, more diverse, more robust and longer immunostimulatory effect than flagellin/alum. In addition, CBLB502/alum and CBLB502-S33MX/alum activate both TH1 and TH2-mediated immune response and a greater TH1-mediated immune response than flagellin/alum.
    • Equivalents
  • While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.
  • Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
    • Incorporation by Reference
  • All patents and publications referenced herein are hereby incorporated by reference in their entireties.
  • The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
  • As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.
    • REFERENCES
    • 1. Yoon S I, Kurnasov O, Natarajan V, Hong M, Gudkov A V, Osterman A L, Wilson I A., 2012. Structural Basis of TLR5-Flagellin Recognition and Signaling. Science 335:859-864 (PMID: 22344444)
    • 2. Smith K D, Andersen-Nissen E, Hayashi F, Strobe K, Bergman M A, Barrett S L, Cookson B T, Aderem A. 2003. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol. 4:1247-53 (PMID: 14625549)
    • 3. Mizel, S. B., A. P. West, R. R. Hantgan. 2003. Identification of a sequence in human Toll-like receptor 5 required for the binding of Gram-negative flagellin. J. Biol. Chem. 278:23624-23629 (PMID: 12711596)
    • 4. Murthy, K. G., Deb, A., Goonesekera, S., Szabo, C. & Salzman, A. L. (2004) J. Biol. Chem. 279:5667-5675 (PMID: 14634022)
    • 5. Andersen-Nissen E., Smith K. D., Strobe K. L., Barrett S. L., Cookson B. T., Logan S. M., Aderem A.(2005) Evasion of Toll-like receptor 5 by flagellated bacteria. Proc. Natl. Acad. Sci. U.S.A. 102: 9247-9252 (PMID: 15956202)
    • 6. Andersen-Nissen E, Smith K D, Bonneau R, Strong R K, Aderem A. 2007. A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin. J Exp Med. 204:393-403 (PMID: 17283206)
    • 7. Burdelya L G, Krivokrysenko V I, Tallant T C, Strom E, Gleiberman A S, Gupta D, Kurnasov O V, Fort F L, Osterman A L, Didonato J A, Feinstein E, Gudkov A V., 2008. An agonist of Toll-like receptor 5 has radioprotective activity in mouse and primate models. Science 320:226-230 (PMID: 18403709).
    • 8. Huleatt J W, Nakaar V, Desai P, Huang Y, Hewitt D, Jacobs A, Tang J, McDonald W, Song L, Evans R K et al. 2008. Potent immunogenicity and eff0icacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TRL5 ligand flagellin. Vaccine. 26:201-214.

Claims (22)

1.-24. (canceled)
25. A method of vaccinating a subject against a substance abuse disorder, comprising administering an effective amount of a vaccine comprising:
(a) an adjuvant comprising:
a flagellin-based agent comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 2, and an aluminum gel or salt,
wherein the ratio (w/w) of flagellin-based agent to aluminum gel or salt is about 1:500 or less and
(b) an antigen, the antigen being a constituent of an infectious agent selected from a live and attenuated, killed, inactivated or toxoid, infectious agent.
26. The method of claim 25, wherein the substance abuse disorder is addiction to one or more of fentanyl, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), heroin, morphine, opium, oxycodone, hydrocodone, ketamine, phencyclidine (PCP), barbiturates, benzodiazepines, flunitrazepam, γ-hydroxybutyric acid (GHB), methaqualone, hashish, marijuana, lysergic acid diethylamide (LSD), mescaline, psilocybin, amphetamine, cocaine, methylphenidate, and nicotine.
27. The method of claim 26, wherein the substance abuse disorder is addiction to methamphetamine.
28. The method of claim 26, wherein the substance abuse disorder is addiction to fentanyl.
29. The method of claim 25, wherein the antigen is the antigen of any one of the following vaccines: DTP (diphtheria-tetanus-pertussis vaccine), DTaP (diphtheria-tetanus-acellular pertussis vaccine), Hib (Haemophilus influenzae type b) conjugate vaccines, Pneumococcal conjugate vaccine, Hepatitis A vaccines, Poliomyelitis vaccines, Yellow fever vaccines, Hepatitis B vaccines, combination DTaP, Tdap, Hib, Human Papillomavirus (HPV) vaccine, Anthrax vaccine, and Rabies vaccine.
30. The method of claim 25, wherein the antigen further comprises of any one of fentanyl, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), heroin, morphine, opium, oxycodone, hydrocodone, ketamine, phencyclidine (PCP), barbiturates, benzodiazepines, flunitrazepam, γ-hydroxybutyric acid (GHB), methaqualone, hashish, marijuana, lysergic acid diethylamide (LSD), mescaline, psilocybin, amphetamine, cocaine, methylphenidate, and nicotine.
31. The method of claim 25, wherein the antigen is diphtheria antigen.
32. The method of claim 25, wherein the antigen is tetanus antigen.
33. The method of claim 25, wherein the flagellin-based agent comprises the amino acid sequence of SEQ ID NO: 2.
34. The method of claim 25, wherein the flagellin-based agent comprises the amino acid sequence of SEQ ID NO: 2 and the antigen comprises succinyl methamphetamine (SMA) hapten conjugated to tetanus toxoid.
35. The method of claim 25, wherein the flagellin-based agent inhibits and abrogates the ability of neutralizing anti-flagellin antibodies to neutralize the adjuvant.
36. The method of claim 25, wherein the aluminum gel or salt is selected from aluminum hydroxide, aluminum phosphate, and aluminum sulfate.
37. The method of claim 25, wherein the flagellin-based agent and/or antigen is adsorbed to the aluminum gel or salt.
38. The method of claim 25, wherein one or more of the flagellin-based agent and aluminum gel or salt are mixed to form a stable complex.
39. The method of claim 25, wherein the flagellin-based agent and aluminum gel or salt are mixed in a ratio that is substantially below a loading capacity of the aluminum salt.
40. The method of claim 38, wherein the flagellin-based agent and aluminum gel or salt are mixed in a ratio (w/w) of about 1:500, or about 1:600, or about 1:700, or about 1:800, or about 1:900, or about 1:1000, or about 1:2000, or about 1:5000, or about 1:10000.
41. The method of claim 25, wherein the vaccine further comprises an additional adjuvant selected from oil-in-water emulsion formulations, saponin adjuvants, ovalbumin, Freund's Adjuvant, cytokines, and chitosans.
42. The method of claim 25, wherein the vaccine and/or adjuvant causes immunostimulation of one or more of a TH1 and TH2-mediated immune response.
43. The method of claim 25, wherein the administering is orally or by parenteral injection.
44. The method of claim 25, wherein the administering takes place by controlled-release or sustained-release.
45. The method of claim 25, wherein the vaccine is administered once per subject or is used in a booster strategy.
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