WO1997012977A1 - Novel g-csf receptor agonists - Google Patents

Novel g-csf receptor agonists Download PDF

Info

Publication number
WO1997012977A1
WO1997012977A1 PCT/US1996/015935 US9615935W WO9712977A1 WO 1997012977 A1 WO1997012977 A1 WO 1997012977A1 US 9615935 W US9615935 W US 9615935W WO 9712977 A1 WO9712977 A1 WO 9712977A1
Authority
WO
WIPO (PCT)
Prior art keywords
leu
ala
gin
ser
gly
Prior art date
Application number
PCT/US1996/015935
Other languages
French (fr)
Inventor
Linda L. Zurfluh
Barbara K. Klein
Charles A. Mcwherter
Yiqing Feng
John P. Mckearn
Sarah Ruth Braford-Goldberg
Original Assignee
G.D. Searle & Co.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by G.D. Searle & Co. filed Critical G.D. Searle & Co.
Priority to AU73900/96A priority Critical patent/AU717733B2/en
Priority to JP51445997A priority patent/JP2002515729A/en
Priority to EP96936191A priority patent/EP0859843A1/en
Priority to US08/833,167 priority patent/US6100070A/en
Publication of WO1997012977A1 publication Critical patent/WO1997012977A1/en
Priority to US09/344,837 priority patent/US6358505B1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to human G-CSF receptor agonists with activity on hematopoietic cell differentiation and expansion.
  • the human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets) .
  • white blood cells including neutrophils, macrophages, and basophils/mast cells
  • red blood cells erythrocytes
  • clot-forming cells megakaryocytes/platelets
  • U.S. Patent 4,810,643 relates to DNA and methods of making G-CSF and Cys to Ser substitution variants of G-CSF.
  • Kuga et al . ( Bi ochem. + Biophys . Res . Comm . 159:103- 111, 1988) made a series of G-CSF variants to partially define the structure-function relationship.
  • Kuga et al . found that internal and C- erminal deletions abolished activity, while N-terminal deletions of up to 11 ammo acids and amino acid substitutions at positions 1, 2 and 3 were active.
  • Watanabe et al . ( Anal . Biochem . 195:38-44, 1991) made a variant to study G-CSF receptor binding in which amino acids 1 and 3 were changed to Tyr for radioiodination of the protein. Watanabe et al . found this T r 1 , Tyr ⁇ G-CSF variant to be active.
  • WO 95/27732 describes, but does not show that the molecule has biological activity, a circularly permuted G- CSF ligand with a breakpoint at positions 68/69 creating a circularly permuted G-CSF ligand with a new N-termmus at the original position 69 of G-CSF and a new C-termmus at the original position 68 of G-CSF.
  • WO 95/27732 also discloses circularly permuted GM-C ⁇ F, IL-2 and IL-4.
  • the new sequence is joined, either directly or through an additional portion of sequence (linker) , to an ammo acid that is at or near the original ⁇ -termmus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the ammo acid that was ⁇ -termmal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
  • This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg __ Creighton, J. Mol . Biol . 165:407-413, 1983; Li & Coffino, Mol . Cell . Biol . 13:2377-2383, 1993) .
  • the proteins examined have represented a broad range of structural classes, including proteins that contain predominantly ⁇ -helix ( ⁇ nterleukm-4; Kreitman et al . , Cytokine 7 :311-318, 1995), ⁇ -sheet (interleukin-1 ; Horlick et al . , Protein Eng. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate lsomerase; Luger et al . , Sci ence 243:206-210, 1989) .
  • Broad categories of protein function are represented in these sequence reorganization studies :
  • sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus ⁇ -glucanase, mterleuk -l ⁇ , ⁇ spectrm SH3 domain, peps ogen, ⁇ nterleukm-4) .
  • the positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N- terminus to the breakpoint is ca. 10 to 80% of the total sequence length) .
  • the linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang & Schachman, Proc. Natl . Acad. Sci . U. S. A . 90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original -terminus.
  • modified human G-CSF receptor agonists of the present invention can be represented by the Formula:
  • L is a linker
  • the constituent amino acids residues of human G-CSF are numbered sequentially 1 through J from the amino to the carboxyl terminus.
  • a pair of adjacent amino acids within this protein may be numbered n and n+1 respectively where n is an integer ranging from 1 to J-l.
  • the r.sidue n+1 becomes the new N-terminus of the new G-CSF receptor agonist and the residue n becomes the new C- terminus of the new G-CSF receptor agonist.
  • the present invention relates to novel G-CSF receptor agonists of the following formula:
  • Xaa at position 1 is Thr, Ser, Arg, Tyr or Gly;
  • Xaa at position 2 is Pro or Leu
  • Xaa at position 3 is Leu, Arg, Tyr or Ser; Xaa at position 13 is Phe, Ser, His, Thr or Pro;
  • Xaa at position 16 is Lys, Pro, Ser, Thr or His;
  • Xaa at position 17 is Cys, Ser, Gly, Ala, lie, Tyr or Arg;
  • Xaa at position 18 is Leu, Thr, Pro, His, lie or Cys;
  • Xaa at position 22 is Arg, Tyr, Ser, Thr or Ala; Xaa at position 24 is lie, Pro, Tyr or Leu;
  • Xaa at position 27 is Asp, or Gly;
  • Xaa at position 30 is Ala, lie, Leu or Gly;
  • Xaa at position 34 is Lys or Ser
  • Xaa at position 36 is Cys or Ser
  • Xaa at position 42 is Cys or Ser
  • Xaa at position 43 is His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu;
  • Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gin, or Thr;
  • Xaa at position 46 is Glu, Arg, Phe, Arg, lie or Ala;
  • Xaa at position 47 is Leu or Thr
  • Xaa at position 49 is Leu, Phe, Arg or Ser;
  • Xaa at position 50 is Leu, lie, His, Pro or Tyr;
  • Xaa at position 54 is Leu or His
  • Xaa at position 64 is Cys or Ser
  • Xaa at position 67 is Gin, Lys, Leu or Cys
  • Xaa at position 70 is Gin, Pro, Leu, Arg or Ser
  • Xaa at position 74 is Cys or Ser
  • Xaa at position 104 is Asp, Gly or Val
  • Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gin or Gly;
  • Xaa at position 115 is Thr, His, Leu or Ala; Xaa at position 120 is Gin, Gly, Arg, Lys or His
  • Xaa at position 123 is Glu, Arg, Phe or Thr
  • Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gin or Glu;
  • Xaa at position 146 is Arg or Gin
  • Xaa at position 147 is Arg or Gin; Xaa at position 156 is His, Gly or Ser;
  • Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly;
  • Xaa at position 162 is Glu, Leu, Gly or Trp;
  • Xaa at position 163 is Val, Gly, Arg or Ala;
  • Xaa at position 169 is Arg, Ser, Leu, Arg or Cys;
  • Xaa at position 170 is His, Arg or Ser;
  • N-termmus is . joined to the C-termmus directly or through a linker capable of joining the N-termmus to the C-termmus and having new C- and N- ermini at ammo acids;
  • the G-CSF receptor agonists of the present invention may contain ammo acid substitutions, deletions and/or insertions and may also have ammo acid deletions at either/or both the N- and C- termini.
  • the more preferred breakpoints at which new C-terminus and N-termmus can be made are; 38-39, 39-40, 40-41, 41-42, 48-49, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60- 61, 61-62, 62-63, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 96-97, 125-126, 126-127, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 136-137, 137- 138, 138-139, 139-140, 140-141 and 141-142.
  • the most preferred breakpoints at which new C-termmus and N-termmus can be made are; 38-39, 48-49, 96-97, 125- 126, 132-133 and 141-142.
  • linker (L) -joining the N-termmus to the C-terminus is a polypeptide selected from the group consisting of: GlyGlyGlySer (SEQ ID NO:2) ; GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:61) ; GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO: 62) ; SerGlyGlySerGlyGlySer (SEQ ID NO:63) ;
  • GluPheGlyAsnMet (SEQ ID NO: 64); GluPheGlyGlyAsnMet (SEQ ID NO:65); GluPheGlyGlyAsnGlyGlyAsnMet (SEQ ID NO: 66) ; and GlyGlySerAspMetAlaGly (SEQ ID NO: 67) .
  • the present invention also encompasses recombinant human G-CSF receptor agonists co-administered or sequentially with one or more additional colony stimulating factors (CSF) including, cytok es, lymphokmes, mterleukms, hematopoietic growth factors which include but are not limited to GM-CSF, c-mpl ligand (also known as TPO or MGDF) , M-CSF, erythropoietin (EPO) , IL-1, IL-4, IL-2, IL- 3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand (herein collectively referred to as "
  • co-administered mixtures may be characterized by having the usual activity of both of the peptides or the mixture may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of the G-CSF receptor agonists or the second colony stimulating factor alone.
  • the co- administration may also provide an enhanced effect on the activity or an activity different from that expected by the presence of the G-CSF ligand or the second colony stimulating factor.
  • the co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human G-CSF.
  • IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-administered with the polypeptides of the present invention.
  • in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before the expanded cells are infused into patients
  • Figure 1 schematically illustrates the sequence rearrangement of a protein.
  • the N-terminus (N) and the C- terminus (C) of the native protein are joined through a linker, or joined directly.
  • the protein is opened at a breakpoint creating a new N-terminus (new N) and a new C- terminus (new-C) resulting in a protein with a new linear amino acid sequence.
  • a rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C- terminus and opening of the protein at the breakpoint.
  • Figure 2 shows a schematic of Method I, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created.
  • the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C- terminus (a.a. 174) joined to the amino acid 11 (a.a. 1- 10 are deleted) through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
  • Figure 3 shows a schematic of Method II, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined without a linker and different N-terminus and C-terminus of the protein are created.
  • the sequence rearrangement results in a new gene encoding a protein with a new N- terminus created at amino acid 97 of the original protein, the original C-terminus (a.a. 174) joined to the original N- terminus and a new C-terminus created at amino acid 96 of the original sequence.
  • Figure 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created.
  • sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C- terminus (a.a. 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
  • Receptor agonists of the present invention may be useful in the treatment of diseases characterized by decreased levels of granulocytes of the hematopoietic system.
  • a G-CSF receptor agonist may be useful in the treatment or prevention of neutropenia.
  • Many drugs may cause bone marrow suppression or hematopoietic deficiencies.
  • examples of such drugs are AZT, DDI, alkylating agents and anti- metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti-convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics.
  • G-CSF receptor agonists may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.
  • Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis.
  • the present peptide may be useful in treating such hematopoietic deficiency.
  • Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel G-CSF receptor agonists.
  • These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention.
  • Appropriate vectors which can transform host cells capable of expressing the G- CSF receptor agonists include expression vectors comprising nucleotide sequences coding for the G-CSF receptor agonists joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.
  • Vectors incorporating modified sequences as described above are included in the present invention and are useful the production of the modified G-CSF receptor agonist polypeptides.
  • the vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.
  • a novel method for producing the novel family of human G-CSF receptor agonists involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of the novel G-CSF receptor agonist polypeptide.
  • suitable cells or cell lines may include various strains of bacteria such as E. coli , yeast, mammalian cells, or insect cells may be utilized as host cells in the method of the present invention.
  • compositions for treating the conditions referred to above.
  • Such compositions comprise a therapeutically effective amount of one or more of the G-CSF receptor agonists of the present invention in a mixture with a pharmaceutically acceptable carrier.
  • This composition can be administered either parenterally, intravenously or subcutaneously.
  • the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such a parenterally acceptable protein solution having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
  • a daily regimen may be in the range of 0.5 - 150 ⁇ g/kg of non- glycosylated G-CSF receptor agonists protein per kilogram of body weight. Dosages would be ad usted relative to the activity of a given receptor agonist and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day.
  • G-CSF receptor agonist may be adjusted higher or lower than the range of 0.5 - 150 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-adm ⁇ n ⁇ s ration with chemotherapeutic drugs and/or radiation; the use of glycosylated G-CSF receptor agonists; and various patient-related issues mentioned earlier n this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors.
  • a non-exclusive list of other appropriate hematopoietins, CSFs and mterleukms for simultaneous or serial co-admmistration with the polypeptides of the present invention includes GM-CSF, c-mpl ligand (also known as TPO or MGDF) , M-CSF, erythropoietin (EPO) , IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosmophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand (herein collectively referred to as "colony stimulating factors”), or combinations thereof.
  • the G-CSF receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in peripheral blood.
  • Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation.
  • Hematopoietic growth factors, including G- CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required.
  • the G-CSF receptor agonist of the present invention may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
  • the G-CSF receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors.
  • Colony stimulating factors CSFs
  • G- CSF Colony stimulating factors
  • the myeloid lineage which is comprised of monocytes (macrophages) , granulocytes (including neutrophils) and megakaryocytes, is critical m preventing infections and bleeding which can be life-threatening.
  • Neutropenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.
  • Bone marrow transplants have been used to treat his patient population.
  • problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow or other tissues, such as spleen or peripheral blood, is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells.
  • Stem cells and progenitor cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exists such that a greater number of multipotential hematopoietic progenitors will enhance hematopoietic recovery.
  • Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant.
  • Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors.
  • An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back into the patient afterwards.
  • autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection.
  • autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells.
  • the limited number of multipotential hematopoietic progenitors may be overcome by ex-vivo expansion of the multipotential hematopoietic progenitors.
  • stem cells can be specifically isolated based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
  • the fallowing patents contain further details on separating stem cells, CD34+ cells, culturing the cells with hematopoietic factors, the use of the cells for the treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.
  • 5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separating the stem cells from dedicated cells.
  • 5,199,942 iescribes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; (2) ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flt3 ligand, c-kit ligand, GM CSF, IL-1, GM-C ⁇ F/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
  • a growth factor selected from the group consisting of IL-3, flt3 ligand, c-kit ligand, GM CSF, IL-1, GM-C ⁇ F/IL-3 fusion protein and combinations thereof.
  • 5,240,856 relates to a cell separator that includes an apparatus for automatically controlling the cell separation process.
  • WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells .
  • WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.
  • WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokmes, for use n transplants.
  • WO 93/08268 describes a method for selectively expanding stem cells, comprising the steps of (a) separating CD34+ stem cells from other cells and (b) incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.
  • WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.
  • WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia.
  • WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.
  • WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.
  • WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.
  • WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.
  • stem cell refers to the multipotential hematopoietic cells as well as early myeloid progenitor and precursors cells which can be isolated from bone marrow, spleen or peripheral blood.
  • expansion refers to the proliferation and differentiation of the cells.
  • the present invention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells, (b) culturing the separated stem cells with a selective medium which contains a G-CSF receptor agonist and optionally a second colony stimulating factor, and (c) harvesting the cultured stems cells.
  • Stem cells as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc., may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics .
  • the phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population.
  • the CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors.
  • the CD34+ progenitors are heterogeneous, and may be divided into several sub-populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules.
  • the most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express CD90 (thy-1) .
  • Other surface antigens such HS CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors.
  • the separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers.
  • Various colony stimulating factors may be utilized in order to selectively expand cells.
  • Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6, IL-11, flt-3 ligand or combinations thereof.
  • the proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques (e.g. hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to incubation.
  • hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al . , Blood 73:1836-1841, 1989) .
  • no single factor has been shown to be as effective as the combination of multiple factors.
  • the present invention provides methods for ex vivo expansion that utilize novel G-CSF receptor agonists.
  • Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a G-CSF receptor agonist of the present invention.
  • a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a G-CSF receptor agonist of the present invention.
  • hematopoietic progenitor cells are good candidates for ex vivo gene transfection.
  • Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy.
  • Gene therapy Potential applications include; 1) the treatment of many congenital metabolic disorders and immunodeficiencies (Kay and Woo, Trends Genet . 10:253-257, 1994), 2) neurological disorders (Friedmann, Trends Genet . 10:210-214, 1994), 3) cancer (Culver and Blaese, Trends Genet . 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet . 10:139-144, 1994) .
  • Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opm . Genet . Dev. 3:102-109, 1993; Boris-Lawrie and Temm, Annal . New York Acad. Sci . 716:59-71, 1994; Miller, Current Top . Microbiol . Immunol .
  • Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA . 90:2122-2126, 1993; Curiel et al . , PNAS USA 88:8850-8854, 1991; Curiel, Annal . New York Acad. Sci .
  • the present invention provides an improvement to the existing methods of expanding hematopoietic cells, into which new genetic material has been introduced, in that it provides methods utilizing G-CSF receptor agonists that may have improved biological activity and/or physical properties .
  • the length of the ammo acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp & Woods, Mol .
  • linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:2) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.
  • Sequences of G-CSF receptor agonists capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence as described above.
  • Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below.
  • a novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. l_ ⁇ many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus.
  • those skilled in the art will recognize that selections of termini anywhere within the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.
  • Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti- parallel beta sheets, chain reversals and turns, and loops; Kabsch &_ Sander, Biopolymers 22: 2577-2637, 1983; the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann. .Rev. Biochem . 53:537-572; 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber & Mathews, Methods Enzymol . 154: 511-533, 1987) .
  • solvent exposure of residues is a site of post- translational attachment of carbohydrate which is necessarily on the surface of the protein.
  • methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops.
  • Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile & Salvatore, Eur. J. Biochem.
  • regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini.
  • those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region.
  • CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC 401 AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG 451 CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA 501 ACTGGGAATG GCCCCTGCCC TGCAGCCCTA ATAA (SEQ ID NO: 33)
  • CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA GTGAGAAAGA
  • Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala
  • Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala (SEQ ID NO:56)
  • E. col i strains such as DH50!TM (Life Technologies, Gaithersburg, MD) and TGI (Amersham Corp., Arlington Heights, IL) are used for transformation of ligation reactions and are the source of plasmid DNA for transfecting mammalian cells.
  • E. col strains such as MON105 and JM101, can be used for expressing the G-CSF receptor agonist of the present invention in the cytoplasm or pe ⁇ plasmic space.
  • MON105 ATCC#55204 F-, lamda-, IN(rrnD, rrE)l, rpoD+, rpoH358
  • DH5 ⁇ TM F-, ph ⁇ 80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recAl, endAl, hsdR17 (rk-,mk+) , phoA, supE441amda-, th ⁇ -1, gyrA96, relAl
  • TGI delta (lac-pro) , supE, th ⁇ -1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaM15)
  • DH5 ⁇ TM Subclonmg efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TGI and MON105 are rendered competent to take up DNA using a CaCl2 method.
  • LB medium 1% Bacto-tryptone, 0.5% Bacto-yeast extract, 150 mM NaCl
  • OD600 600 nanometers
  • Baush & Lomb Spectronic spectrophotometer Baush & Lomb Spectronic spectrophotometer
  • the cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl2 solution (50 mM CaCl2, 10 mM Tris- Cl, pH7.4) and are held at 4'C for 30 minutes.
  • the cells are again collected by centrifugation and resuspended in one-tenth culture volume of CaCl2 solution.
  • Ligated DNA is added to 0.2mL of these cells, and the samples are held at 4'C for 1 hour.
  • the samples are shifted fo 42'C for two minutes and lmL of LB is added prior to shaking the samples at 37 * C for one hour. Cells from these samples are spread on plates !
  • the plates are incubated overnight at 37 "C.
  • Single colonies are picked, grown in LB supplemented with appropriate antibiotic for 6-16 hours at 37 "C with shaking.
  • Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37°C while shaking.
  • 1 ul of cells are analyzed by PCR for the presence of a G-CSF gene.
  • the PCR is carried out using a combination of primers that anneal to the G-CSF gene and/or vector. After the PCR is complete, loading dye is added to the sample followed by electrophoresis as described earlier. gene has been ligated to the vector when a PCR product of the expected size is observed.
  • Method I Creation of genes with new N-terminus/C-terminus which contain a linker region.
  • Genes with new N-terminus/C-termmus which contain a linker region separating the original C-terminus and N- terminus can be made essentially following the method described in L. S. Mullins, et al J. Am . Chem. Soc . 116, 5529-5533 '1994) .
  • Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary ammo acid sequence of the protein. The steps are illustrated in Figure 2.
  • the primer set (new start) and
  • linker start is used to create and amplify, from the original gene sequence, the DNA fragment (“Fragment Start”) that contains the sequence encoding the new N-termmal portion of the new protein followed by the linker that connects the C- erminal and N-terminal ends of the original protein.
  • the primer set (“new stop” and “linker stop”) is used to create and ampHfy, from the original gene sequence, the DNA fragment (“Fragment Stop”) that encodes the same linker as used above, followed by the new C-termmal portion of the new protein.
  • the “new start” and “new stop” primers are designed to include the appropriate restriction enzyme recognition sites which allow cloning of the new gene into expression plasmids.
  • Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes.
  • a Perkin Elmer GeneAmp PCR Core Reagents kit is used.
  • a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl 2 - PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . "Fragment Start” and “Fragment Stop”, which have complementary sequence in the linker region and the coding sequence for the two amino acids on both sides of the linker, are joined together in a third PCR step to make the full-length gene encoding the new protein.
  • the DNA fragments "Fragment Start” and “Fragment Stop” are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined in equimolar quantities, heated at 70°C for ten minutes and slow cooled to allow annealing through their shared sequence in "linker start” and “linker stop”.
  • primers "new start” and “new stop” are added to the annealed fragments to create and amplify the full- length new N-terminus/C-terminus gene.
  • Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 60°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes .
  • a Perkin Elmer GeneAmp PCR Core Reagents kit is used.
  • a 100 ul reaction contains 100 pmole of each primer and approximately 0.5 ug of DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl 2 • PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
  • New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-terminus can be made using two steps of PCR amplification and a blunt end ligation.
  • the steps are illustrated in Figure 3.
  • the primer set (“new start” and "P-bl start”) is used to create and amplify, from the original gene sequence, the DNA fragment (“Fragment Start”) that contains the sequence encoding the new N- erminal portion of the new protein.
  • the primer set (“new stop” and "P-bl stop”) is used to create and amplify, from the original gene sequence, the DNA fragment (“Fragment Stop”) that contains the sequence encoding the new C-terminal portion of the new protein.
  • the “new start” and “new stop” primers are designed to include appropriate restriction sites which allow cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for 45 seconds and 72°C extension for 45 seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manuf cturer.
  • the "P-bl start” and “P-bl stop” primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start” and “Fragment Stop” to each other.
  • a 100 ul leaction contained 150 pmole of each primer and one ug of template DNA; and lx Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase.
  • PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) .
  • PCR reaction products are purified using a Wizard PCR Preps kit (Promega) .
  • the primers are designed to include appropriate restriction enzyme recognition sites which allow for the cloning of the new gene into expression vectors.
  • "Fragment Start” is designed to create a Ncol restriction site
  • “Fragment Stop” is designed to create a Hindlll restriction site.
  • Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega) . Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) .
  • Plasmid DNA is purified and sequence confirmed as below.
  • New N-terminus/C-terminus genes can be made based on the method described in R. A. Horlick, et al Protein Eng. 5:427-431 (1992) . Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA. The steps are illustrated in Figure 4.
  • PCR Polymerase chain reaction
  • the tandemly-duplicated template DNA is created by cloning and contains two copies of the gene separated by DNA sequence encoding a linker connecting the original C- and N- terminal ends of the two copies of the gene.
  • Specific primer sets are used to create and amplify a full-length new N terminus /C-terminus gene from the tandemly-duplicated template DNA. These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes.
  • a Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is used.
  • a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dAT?, 200 uM dTTP, 200 uM dCTP, 2.5 units A pliTaq DNA polymerase and 2 mM MgCl 2 .
  • PCR reactions are performed m a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) .
  • PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
  • Plasmid DNA can be isolated by a number of different methods and using commercially available kits known to those skilled in the art. A few such methods are shown herein. Plasmid DNA is isolated using the Promega WizardTM M niprep kit (Madison, WI) , the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) or Qiagen Plasmid Midi kit. These kits follow the same general procedure for plasmid DNA isolation. Briefly, ⁇ -11s are pelleted by centrifugation (5000 x g) , plasmid DNA released with sequential NaOH/acid treatment, and cellular debris is removed by centrifugation (10000 x g) .
  • the supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-bmding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells are inoculated into 50-100 mLs of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking.
  • the purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian, E. coli or other cells.
  • Purified plasmid DNA is resuspended in dH 2 0 and quantitated by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectromc 601 UV spectrometer.
  • DNA samples are sequenced using ABI PRISMTM DyeDeoxyTM terminator sequencing chemistry (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) kits (Part Number 401388 or 402078) according to the manufacturers suggested protocol usually modified by the addition of 5% DMSO to the sequencing mixture. Sequencing reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions.
  • Samples are purified to remove excess dye terminators with Centri-SepTM spin columns (Princeton Separations, Adelphia, NJ) and lyophilized. Fluorescent dye labeled sequencing reactions are resuspended n deionized formamide, and sequenced on denaturing 4.75% polyacrylamide- 8M urea gels using an ABI Model 373A automated DNA sequencer. Overlapping DNA sequence fragments are analyzed and assembled into master DNA contigs using Sequencher v2.1 DNA analysis software (Gene Codes Corporation, Ann Arbor, MI) .
  • the BHK-21 cell line can be obtained from the ATCC (Rockville, MD) .
  • the cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose) , supplemented to
  • BHK growth media 2mM (mM) L-glutamme and 10% fetal bovine serum (FBS) .
  • FBS fetal bovine serum
  • This formulation is designated BHK growth media.
  • Selective media is BHK growth media supplemented with 453 units/mL hygromycm B (Calbiochem, San Diego, CA) .
  • the BHK-21 cell line was previously stably transfected with the HSV transactivating protein VP16, which transactivates the IE110 promoter found on the plasmid pMON3359 (See Hippenmeyer et al., Bio /Technology, p .1037-1041, 1993) .
  • the VP16 protein drives expression of genes inserted behind the IE110 promoter.
  • BHK-21 cells expressing the transactivating protein VP ⁇ ⁇ are designated BHK-VP16.
  • the plasmid pMONlll ⁇ (See Highk et al . , Poul try Sci . , 70: 970-981, 1991) expresses the hygromyc resistance gene from the SV40 promoter.
  • a similar plasmid is available from ATCC, pSV2- hph.
  • BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X 10 5 cells per dish 24 hours prior to transfection.
  • Cells are transfected for 16 hours in 3 mL of "OPTIMEM”TM (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycm resistance plasmid, pMONlll ⁇ , and 80 ug of Gibco- BRL "LIPOFECTAMINE"TM per dish.
  • the media is subsequently aspirated and replaced with 3 mL of growth media.
  • media from each dish is collected and assayed for activity (transient conditioned media) .
  • the cells are removed from the dish by trypsm-EDTA, diluted 1:10 and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in tryps /EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluence, the conditioned media is re-assayed, and positive clones are expanded into growth media.
  • E. coli strain MON105 or JM101 harboring the plasmid of interest are grown at 37°C in M9 plus casammo acids medium with shaking in a air incubator Model G25 from New Brunswick Scientific (Edison, New Jersey) . Growth is monitored at OD600 until it reaches a value of 1, at which time nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a final concentration of 50 ⁇ g/mL. The cultures are then shaken at 37°C for three to four additional hours. A high degree of aeration is maintained throughout culture period in order to achieve maximal production of the desired gene product. The cells are examined under a light microsccpe for the presence of inclusion bodies (IB) .
  • IB inclusion bodies
  • One mL aliquots of the culture are removed for analysis of protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (see Maniatis et al . Molecular Cloning: A Laboratory Manual, 1982) .
  • the culture is centrifuged (5000 x g) to pellet the cells.
  • the cell pellet from a 330 mL E. col i culture is resuspended in 15 mL of sonication buffer (10 mM 2-amino-2- (hydroxymethyl) 1, 3-propanediol hydrochloride (Tris-HCl), pH 8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA)) .
  • sonication buffer 10 mM 2-amino-2- (hydroxymethyl) 1, 3-propanediol hydrochloride (Tris-HCl), pH 8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA)
  • Tris-HCl Tris-HCl
  • EDTA ethylenediaminetetraacetic acid
  • These resuspended cells are sonicated using the microtip probe of a Sonicator Cell Disruptor (Model W-375, Heat Systems- Ultrasonics, Inc., Farmingdale, New York) .
  • Extraction and refolding of proteins from inclusion body pellets Folli ing the final centrifugation step, the IB pellet is resuspended m 10 mL of 50 mM Tris-HCl, pH 9.5, 8 M urea and 5 mM dithiothreitol (DTT) and stirred at room temperature for approximately 45 minutes to allow for denaturation of the expressed protein.
  • DTT dithiothreitol
  • the extraction solution is transferred to a beaker containing 70 mL of 5mM Tris-HCl, pH 9.5 and 2.3 M urea and gently stirred while exposed to air at 4°C for 18 to 48 hours to allow the proteins to refold.
  • Refolding is monitored by analysis on a Vydac (Hesperia, Ca. ) C18 reversed phase high pressure liquid chromatography (RP-HPLC) column (0.46x25 cm) .
  • RP-HPLC reversed phase high pressure liquid chromatography
  • a linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid (TFA) is employed to monitor the refold. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute.
  • Denatured ,-rote ⁇ ns generally elute later in the gradient than the refolded proteins .
  • contaminating E. coli proteins are removed by acid precipitation.
  • the pH of the refold solution is titrated to between pH 5.0 and pH 5.2 using 15% (v/v) acetic acid (HOAc) . This solution is stirred at 4°C for 2 hours and then centrifuged for 20 minutes at 12,000 x g to pellet any insoluble protein.
  • HOAc acetic acid
  • the supernatant from the acid precipitation step is dialyzed using a Spectra/Por 3 membrane with a molecular weight cut off (MWCO) of 3,500 daltons.
  • MWCO molecular weight cut off
  • NaCl sodium chloride
  • the folded proteins can be affinity purified using affinity reagents such as mAbs or receptor subunits attached to a suitable matrix.
  • affinity reagents such as mAbs or receptor subunits attached to a suitable matrix.
  • purification can be accomplished using any of a variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic chromatography or reversed phase HPLC.
  • Protein Characterization The purified protein is analyzed by RP-HPLC, electrospray mass spectrometry, and SDS-PAGE. The protein quantitation is done by amino acid composition, RP-HPLC, and Bradford protein determination. In some cases tryptic peptide mapping is performed in conjunction with electrospray mass spectrometry to confirm the identity of the protein.
  • the factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) .
  • This cell line established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, B., et al . , Blood 70: 192, 1987; Valtieri,
  • AML 193 1.3 which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokme dependent AML 193 cells for growth factors for 24 hours. The cells are then replated at 1x105 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.
  • AML 193 1.3 cells are washed 6 times cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifugmg cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2 x 105 to 5 x 10 5 viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco ' s Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrm, lipids and 2-mercaptoethanol .
  • IMDM Iscove's modified Dulbecco ' s Medium
  • Bovine albumin (Boehrmger-Mannheim, Indianapolis, IN) is added at 500 ⁇ g/ ⁇ L; human transferrm (Boehrmger-Mannheim, Indianapolis, IN) is added at 100 ⁇ g/mL; soybean lipid (Boehrmger- Mannheim, Indianapolis, IN) is added at 50 ⁇ g/mL; and 2- mercaptoethanol (Sigma, St. Louis, MO) is added at 5 x 10 ⁇ 5 M.
  • Serial dilutions of G-CSF receptor agonist proteins are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 ⁇ l of medium containing G-CSF receptor agonist proteins once serial dilutions are completed.
  • Control wells contained tissue culture medium alone (negative control) .
  • AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 ⁇ l (2.5 x 104 cells) into each well. Tissue culture plates are incubated at 37°C with 5% C02 m humidified air for 3 days. On day 3, 0.5 ⁇ Ci
  • the G-CSF receptor agonist proteins were tested n a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions. Activity for each sample was determined by the concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample was determined from EC50 estimations for the sample and the standard as indicated above.
  • Cell lines such as BHK or the murine pro B cell line Baf/3, can be transfected with a colony stimulating factor receptor, ..uch as the human G-CSF receptor which the cell line does not have. These transfected cell lines can be used to determine the activity of the ligand of which the receptor has been transfected.
  • the new N-terminus/C-terminus gene in pMON3485 was created using Method I as described in Materials and Methods.
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:7) and L-ll start (SEQ ID NO:3) .
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in the plasmid, pMON13037 (WO 95/21254), using the primer set, 38 stop (SEQ ID NO:8) and L-ll stop (SEQ ID NO:4) .
  • the full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 39 start (SEQ ID NO:7) and 38 stop (SEQ ID NO:8) .
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and HindiII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI) .
  • the plasmid, pMON3934 (derivative of
  • PMON3359 was digested with restriction endonucleases Hindlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel-purified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD) . Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3485.
  • BHK cells were transfected with the plasmid, pMON3485, for protein expression and bioassay.
  • the new N-terminus/C-terminus gene in pMON3486 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in the plasmid, pMON13037, using the primer set, 97 start (SEQ ID NO:9) and L-ll start (SEQ ID NO:3) .
  • Fragment Stop was created and amplified from G-CSF Ser- 1 - 7 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:10) and L-ll stop (SEQ ID NO:4) .
  • the full-length new N terminus/C-termmus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 97 start (SEQ ID NO: 9) and 96 stop (SEQ ID NO:10) .
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hmdlll and gel-purified using a Magic DNA Clean-up System kit.
  • the plasmid, pMON3934 was digested with restriction endonucleases H dlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel- purified
  • the purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells. Transformant bacteria were selected on ampicillm-contammg plates. Plasmid DNA was isolated and sequenced to confirm the correct insert.
  • the resulting plasmi ⁇ was designated pMON3486.
  • BHK cells were transfected with the plasmid, pMON3486, for protein expression and bioassay.
  • the new N-terminus/C-terminus gene in pMON3487 was created using Method I as described in Materials and Methods.
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in the plasmid, pMON13037, using the primer set, 126 start (SEQ ID NO: 11) and L-ll start (SEQ ID NO:3) .
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:12) and L-ll stop (SEQ ID NO:4) .
  • the full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 126 start (SEQ ID NO:11) and 125 stop (SEQ ID NO:12) .
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hindlll and purified using a Magic DNA Clean-up System kit.
  • the plasmid, pMON3934 was digested with restriction endonucleases Hindlll and Ncol, resulting in an approxima ely 3800 base pair vector fragment, and gel- purified.
  • the purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E . col i strain DH5 ⁇ cells.
  • Plasmid DNA was isolated and sequenced to confirm the correct insert .
  • the resulting plasmid was designated
  • BHK cells were transfected with the plasmid, pMON3487, for protein expression and bioassay.
  • the new N-terminus/C-terminus gene in pMON3488 was created using Method I as described in Materials and Methods.
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in the plasmid, pMON13037, using the primer set, 133 start (SEQ ID NO:13) and L-ll start (SEQ ID NO:3) .
  • Fragment Stop was created and amplified from G-CSF Ser-*- 7 sequence in the plasmid, pMON13037 using the primer set, 132 stop (SEQ ID NO:14) and L-ll stop (SEQ ID NO:4) .
  • the full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 133 start (SEQ ID NO:13) and 132 stop (SEQ ID NO:14) .
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hindlll and purified using a Magic DNA Clean-up System kit.
  • the plasmid, pMON3934 was digested with restriction endonucleases Hindlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel- pu ⁇ fied.
  • the purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5ccells.
  • Transfo ⁇ ri t bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3488.
  • BHK cells were transfected with the plasmid, pMON3488, for protein expression and bioassay.
  • the new N-terminus/C-termmus gene in pMON3489 was created using Method I as described in Materials and Methods.
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence the plasmid, pMON13037, using the primer set, 142 start (SEQ ID NO:15) and L-ll start (SEQ ID NO:3) .
  • Fragment Stop was created and amplified from G-CSF Ser-'- 7 sequence in pMON13037 using the primer set, 141 stop
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hmdlll and purifiod using a Magic DNA Clean-up System kit.
  • the plasmid, pMON3934 was digested with restriction endonucleases H dlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel- purified.
  • the purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5ocells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert.
  • the resulting plasmid was designated PMON3489.
  • BHK cells were transfected with the plasmid, pMON3489, for protein expression and bioassay.
  • the new N-terminus/C-terminus gene in pMON3490 was created using Method II as described in Materials and Methods.
  • Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 39 start (SEQ ID NO:7) and P-bl start (SEQ ID NO:5) .
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMONl3037 using the primer set, 38 stop (SEQ ID NO:8) and P-bl stop (SEQ ID NO:6) .
  • Fragment Start was digested with restriction endonuclease Ncol
  • Fragment Stop was digested with restriction endonuclease Hindlll. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pMON3934.
  • Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated PMON3490. BHK cells were transfected with the plasmid, pMON3490, for protein expression and bioassay.
  • the new N-terminus/C-terminus gene in pMON3491 was created using Method II as described in Materials and Methods.
  • Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 97 start (SEQ ID NO: 9) and P-bl start (SEQ ID NO:5) .
  • Fragment Stop was created and amplified from G-CSF Ser ⁇ 7 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:10) and P-bl stop (SEQ ID NO:6) .
  • Fragment Start was digested with restriction endonuclease Ncol
  • Fragment Stop was digested with restriction endonuclease Hindlll.
  • the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pMON3934.
  • a portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert . The resulting plasmid was designated pMON3491. BHK cells were transfected with the plasmid, pMON3491, for protein expression and bioassay.
  • the plasmid, pMON3491 containing the gene sequence of (SEQ ID N0:31) encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON3492 was created using Method II as described in Materials and
  • Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 126 start (SEQ ID NO:ll) and P-bl start (SEQ ID NO:5) .
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:12) and P-bl stop (SEQ ID NO:6) .
  • Fragment Start was digested w.th restriction endonuclease Ncol, and Fragment Stop was digested with restriction endonuclease Hindlll.
  • the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pM0N3934.
  • a portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3492.
  • BHK cells were transfected with the plasmid, pMON3492, for protein expression and bioassay.
  • the new N-terminus/C-terminus gene in pMON3493 was created using Method II as described in Materials and Methods.
  • Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 133 start (SEQ ID NO:13) and P-bl start (SEQ ID NO:5) .
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO: 14) and P-bl stop (SEQ ID NO: 6) .
  • Fragment Start was digested with restriction endonuclease Ncol
  • Fragment Stop was digested with restriction endonuclease Hindlll.
  • the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hmdlll vector fragment of pMON3934.
  • a portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells.
  • Transformant bacteria were selected on ampicillm-contammg plates. Plasmid DNA was isolated and sequenced to confirm the correct insert . The resulting plasmid was designated pMON3493.
  • BHK cells were transfected with the plasmid, pMON3493, for prote * - i expression and bioassay.
  • Fragment Start was digested with restriction endonuclease Ncol
  • Fragment Stop was digested with restriction endonuclease Hmdlll.
  • the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pMON3934.
  • a portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert . The resulting plasmid was designated pMON3494. BHK cells were transfected with the plasmid, pMON3494, for protein expression and bioassay.
  • the genes encoding the G-CSF receptor agonists of Examples 1-10 were excised from the BHK vectors as a Ncol/Hindlll fragment and ligated with the ⁇ 3630 base pair Ncol/Hindlll vector fragment of pMON2341 (WO 94/12638) .
  • the resulting plasmids (Examples 11-20) are indicated in Table 4.
  • the plasmids were transformed into E. coli strain JM101 cells and expression of the G-CSF receptor agonist protein was evaluated.
  • the proteins expressed are the same as those expressed in the parental BHK expression vector except the proteins were immediately preceded by a Methionine-Alanine dipeptide and the Methionine is processed off by methionine aminopeptidase.
  • the complementary pair of synthetic oligomers, 141for.seq (SEQ ID NO:23) and 141rev.seq (SEQ ID NO:24) were annealed by heating 2ug of each synthetic oligomer in a 20ul reaction mixture containing 20mM Tris-HCl (7.5), lOmM MgCl 2 , and 50mM NaCl, at 80°C for 5 minutes, and allowing the mixture to slowly cool to ambient temperature (approximately 45 minutes) .
  • the oligomers create an Ncol site at the 5' end and a Nhel site at the 3' end.
  • the resulting plasmid was designated pMON25184 Plasmid, pMON25184 containing the gene sequence of (SEQ ID NO:38), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3454.
  • pMON25183 was constructed using an overlapping PCR primer method.
  • the synthetic oligomers, 132for.seq (SEQ ID NO:321 and 132rev.seq (SEQ ID NO:22), encode the Ncol and Nhel restriction recognition sequence, respectively.
  • Amplified DNA was generated by the DNA polymerase chain amplification method using the PCR Optimizer Kit
  • the reaction product was desalted using Centri-Sep spin columns (Princeton Separations) following the manufacturer's recommended protocol, digested with Ncol/Nhel, and gel purified from TAE-agarose gels using Gene Clean (Bio 101) and the DNA product was eluted in dH 2 ⁇
  • the purified PCR product was ligated with the - 4090 base pair Ncol/Nhel pMON3453 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in
  • Example 21 The resulting plasmid was designated pMON25183.
  • the resulting gene had 14 codon changes at the 5' end of the gene.
  • Plasmid, pMON25183 containing the gene sequence of (SEQ ID NO:37), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3453.
  • PMON25187 pM0N25187 was constructed using an overlapping PCR primer method.
  • Amplified DNA was generated by the DNA polymerase chain amplification method using the PCR Optimizer Kit (Invitrogen) .
  • the PCR reactions were performed using the manufacturer's recommended conditions, in 5X buffer B for seven cycles consisting of 94°C for 1', 65°C for 2', and
  • the resulting plasmid was designated pMON25187.
  • the resulting gene had 14 codon changes at the 5' end of the gene.
  • Plasmid, pMON25187 containing the gene sequence of (SEQ ID NO:41), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3458.
  • pMON25182 was constructed using the overlapping PCR primer approach described m Example 23.
  • the synthetic oligomer primers 125for.seq (SEQ ID NO:19) and 125rev.seq (SEQ ID NO:20) were used in the PCR reaction.
  • the PCR reaction conditions were identical to those used in Example 23 except the annealing temperature for the first seven cycles was 60°C.
  • the purified PCR product was ligated with - 4070 base pair Ncol/Nhel pMON3452 vector fragment.
  • pMON25186 was constructed using the overlapping PCR primer approach described in Example 23.
  • the synthetic oligomer primers 125for.seq (SEQ ID NO:19) and 125rev.seq (SEQ ID NO:20) were used in the PCR reaction.
  • the PCR reaction conditions were identical to those used m Example 23 except the annealing temperature for the first seven cycles was 60°C.
  • the purified PCR product was ligated with the - 4060 base pair Ncol/Nhel pMON3457 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21.
  • the resulting plasmid was designated pMON25186.
  • the resulting gene had 19 codon changes at the 5' end of the gene.
  • Plasmid, pMON25186 containing the gene sequence of (SEQ ID NO:40) DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3457
  • pMON25181 was constructed using PCR to amplify a DNA fragment from pMON3451 as the template using the oligomers 96for.seq (SEQ ID N0:17) and 96rev.seq (SEQ ID N0:18) .
  • the oligomer 96for.seq was designed to create six codon changes.
  • the PCR reaction conditions were the same as described in Example 25, except lOng of pMON3451 plasmid DNA was added.
  • the purified PCR product was ligated with the - 3980 base pair Ncol/Nhel pMON3451 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21.
  • the resulting plasmid was designated DMON25181.
  • pMON25185 was constructed using PCR to amplify a DNA fragment from pMON3451 as the template using the oligomers 96for.seq (SEQ ID NO:17) and 96rev.seq (SEQ ID NO:18) .
  • the oligomer 9697for.seq was designed to create six codon changes.
  • Tie PCR reaction conditions were the same as described in Example 25, except lOng of pMON3456 plasmid DNA was added.
  • the purified PCR product was ligated with the - 3970 base pair Ncol/Nhel pMON3456 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21.
  • the resulting plasmid was designated pMON25185.
  • Plasmid, pMON25185 containing the gene sequence of (SEQ ID NO: 39) DNA was retransformed into E. coli strain JMIOI cells for protein expression. The protein expressed is the same as that expressed from pMON3456.
  • the G-CSF amino acid substitution variants of the present invention were made using PCR mutagenesis techniques as described in WO 94/12639 and WO 94/12638. These and other variants (i.e. amino acid substitutions, insertions or deletions and N-terminal or C-terminal extensions) could also be made, by one skilled in the art, using a variety of other methods including synthetic gene assembly or site- directed mutagenesis (see Taylor et al . , Nucl . Acids Res . , 13:7864-8785, 1985; Kunkel et al. , Proc . Natl . Acad . Sci . USA, 82:488-492, 1985; Sambrook et al .
  • the plasmid DNA can be transfected into an appropriate mammalian cell, insect cell or bacterial strain such as E. coli for production.
  • G-CSF G-CSF
  • the G-CSF am o acid substitution variants were assayed m the Baf/3 cell l ne, transfected with the human G-CSF receptor, proliferation assay to determine their bioactivity relative to native G-CSF.
  • the G-CSF variants tested and their relative bioactivity are shown n Table 5. A "+” indicates that the activity was comparable to native G-CSF and "-" indicates that the activity was significantly decreased or not detected.
  • Examples 30-37 were made in a similar manner as described in Example 6 using the plasmid pMON13037 as the template and the oligonucleotide primers indicated in Table 6. The resulting gene and the designated plasmid pMON # and the protein encoded are indicated in Table 6.
  • the G-CSF receptor agonist genes in pMON3640, pMON3461, pMON3462, pMON3463, pMON3464, pMON3465, pMON3466 and pMON3467 were transferred to an E. coli expression vector, pMON2341, as an Ncol/Hindlll restriction fragment, resulting in the plasmids pMON3468, pMON3469, pMON3470, pMON3471, pMON3472, pMON3473, pMON3474 and pMON3498 respectively.
  • the plasmid, pMON3468 resulted in low expression levels in E. coli of the desired G-CSF receptor agonist.
  • the 5 ' end of the gene was redesigned to use codon selection that was AT rich to increase expression levels.
  • the oligonucleotides, Z4849AT.for (SEQ ID NO:84) and Z4849AT.rev (SEQ ID NO:85) were used to re-engineer the gene.
  • the resulting plasmid, pMON3499, containing the gene (SEQ ID NO: 94) encodes the G-CSF receptor agonist of (SEQ ID NO:103) .
  • the G-CSF receptor agonists were assayed in the Baf/3 cell line, transfected with the human G-CSF receptor, (Baf/3-G-CSF) proliferation assay to determine their bioactivity relative to native G-CSF.
  • the activity of the receptor agonists is shown in Table 7.
  • NAME G. D. Searle & Co.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Disclosed are G-CSF receptor agonists proteins, DNAs which encode the G-CSF hematopoietic receptor agonists proteins, methods of making the G-CSF hematopoietic receptor agonists proteins and methods of using the G-CSF hematopoietic receptor agonists proteins.

Description

NOVEL G-CSF RECEPTOR AGONISTS
The present application claims priority under 35 USC §119 (e) of United States provisional application Serial No. 60/004,382 filed October 05, 1995.
Field of the Invention The present invention relates to human G-CSF receptor agonists with activity on hematopoietic cell differentiation and expansion.
Background of the Invention
The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets) . The hematopoietic systems of the average male has been estimated to produce on the order of 4.5 x 1011 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight (De*; er et al . , BioEssays , 2*154-158, 1985) .
It is believed that small amounts of certain hematopoietic growth factors account for the differentiation of a small number of progenitor "stem cells" into the variety of blood cell lines, for the tremendous proliferation of those lines, and for the ultimate differentiation of mature blood cells from those lines. Because the hematopoietic growth factors are present in extremely small amounts, the detection and identification of these factors has relied upon an array of assays which as yet only distinguish among the different factors on the basis of stimulative effects on cultured cells under artificial conditions. U.S. Patent 4,999,291 discloses DNA and methods for making G-CSF the disclosure of which is incorporated herein by reference in it entirety.
U.S. Patent 4,810,643 relates to DNA and methods of making G-CSF and Cys to Ser substitution variants of G-CSF.
Kuga et al . ( Bi ochem. + Biophys . Res . Comm . 159:103- 111, 1988) made a series of G-CSF variants to partially define the structure-function relationship. Kuga et al . found that internal and C- erminal deletions abolished activity, while N-terminal deletions of up to 11 ammo acids and amino acid substitutions at positions 1, 2 and 3 were active.
Watanabe et al . ( Anal . Biochem . 195:38-44, 1991) made a variant to study G-CSF receptor binding in which amino acids 1 and 3 were changed to Tyr for radioiodination of the protein. Watanabe et al . found this T r1, Tyr^ G-CSF variant to be active.
WO 95/27732 describes, but does not show that the molecule has biological activity, a circularly permuted G- CSF ligand with a breakpoint at positions 68/69 creating a circularly permuted G-CSF ligand with a new N-termmus at the original position 69 of G-CSF and a new C-termmus at the original position 68 of G-CSF. WO 95/27732 also discloses circularly permuted GM-CΞF, IL-2 and IL-4.
Rearrangement of Protein Sequences
In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity and thereby provide organisms with a competitive advantage, especially since the basal mutation rate s low (Doolittle, Protein Sci ence 1:191-200, 1992) .
The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their ammo acid sequences (Cunningham, et al., Proc. Natl . Acad . Sci . U. S. A . 76:3218-3222, 1979; Teather & Erfle, J. Bacteriol . 172: 3837-3841, 1990; Schimming et al . , Eur. J. Biochem . 204: 13-19, 1992; Yamiuchi and Mmamikawa, FEBS Let t . 260:127-130, 1991: MacGregor et al . , FEBS Let t . 378:263-266, 1996) . The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton (J. Mol . Biol . 165:407-413, 1983) . A new Ν-termmus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of ammo acids as the original from the breakpoint until it reaches an ammo acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker) , to an ammo acid that is at or near the original Ν-termmus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the ammo acid that was Ν-termmal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain. This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg __ Creighton, J. Mol . Biol . 165:407-413, 1983; Li & Coffino, Mol . Cell . Biol . 13:2377-2383, 1993) . The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly α-helix (ιnterleukm-4; Kreitman et al . , Cytokine 7 :311-318, 1995), β-sheet (interleukin-1 ; Horlick et al . , Protein Eng. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate lsomerase; Luger et al . , Sci ence 243:206-210, 1989) . Broad categories of protein function are represented in these sequence reorganization studies :
Enzymes
T4 lysozyme Zhang et al . , Biochemistry 32:12311-12318 (1993) ; Zhang et al., Nat ure Struct . Biol . 1:434-438 (1995)
dihydrofolate Buchwalder et al. , Biochemistry reductase 31:1621-1630 (1994) ; Protasova et al., Prot. Eng. 7:1373-1377 (1995]
ribonuclease Tl Mullins et al . , J. Am . Chem . Soc . 116:5529-5533 (1994) ; Garrett et al . , Protein Sci ence 5:204-211 (1996)
Bacill us β-glucanse Hahn et al. , Proc. Natl . Acad. Sci .
U. S. A . 91:10417-10421 (1994)
aspartate Yang & Schachman, Proc . Natl . Acad. transcarbamoylase Sci . U. S.A . 90:11980-11984 (1993)
phosphoribosyl Luger et al . , Science 243:206-210 anthranilate (1989); Luger et al . , Prot. Eng. isomerase 3:249-258 (1990)
pepsin/pepsinogen Lin et al . , Protein Science 4:159-
166 (1995) glyceraldehyde-3- Vignais et al . , Protein Sci ence phosphate dehydro- 4:994-1000 (1995) genase
ornithine Li & Coffino, Mol . Cell . Biol decarboxylase 13:2377-2383 (1993)
yeast Ritco-Vonsovici et al . , Biochemistry phosphoglycerate 34:16543-16551 (1995) dehydrogenase
Enzyme Inhibitor
basic pancreatic Goldenberg & Creighton, J. Mol trypsin inhibitor Biol . 165:407-413 (1983)
Cyto ines
interleuki.i -lβ Horlick et al . , Protein Eng. 5:427- 431 (1992)
interleukin-4 Kreitman et al . , Cytokine 7:311- 318 (1995)
Tyrosine Kinase
Recognition Domain
α-spectrin SH3 Viguera, et al. , J. domain Mol . Biol . 247:670-681 (1995;
Transme brane Protein
omp A Koebnik & Kramer, J. Mol . Biol 250:617-626 (1995) Chimeric Protein mterleukιn-4— Kreitman et al . , Proc . Natl . Acad. Pseudomonas Sci . U. S . A . 91:6889-6893 (1994) . exotoxin fusion molecule
The results of these studies have been highly variable. In many cases substantially lower activity, solubility or thermodynamic stability were observed ( E. coli dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ormthme decarboxylase, omp A, yeast phosphoglycerate dehydrogenase) . In other cases, the sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus β-glucanase, mterleuk -lβ, αspectrm SH3 domain, peps ogen, ιnterleukm-4) . In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged ot-spectrm SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed mterleukιn-4— Pseudomonas exotoxin fusion molecule (Kreitman et al., Proc. Natl . Acad. Sci . U. S. A . 91:6889-6893, 1994; Kreitman et al . , Cancer _-.es. 55:3357-3363, 1995) .
The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type convert a subset of interactions that are long-range m the original sequence into short- range interactions in the new sequence, and vice versa. The fact that many of these sequence rearrangements are able to attain a conformation with at least some activity is persuasive evidence that protein folding occurs by multiple folding pathways (Viguera, et al . , J. Mol . Biol . 247:670- 681, 1995) . In the case of the SH3 domain of αspectrin, choosing new termini at locations that corresponded to β -hairpin turns resulted in proteins with slightly less stability, but which were nevertheless able to fold.
The positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N- terminus to the breakpoint is ca. 10 to 80% of the total sequence length) . The linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang & Schachman, Proc. Natl . Acad. Sci . U. S. A . 90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original -terminus. Flexible hydrophilic residues such as Gly and Ser are frequently used in the linkers. Viguera, et al. (J". Mol . Biol . 247:670-681, 1995) compared joining the original N- and C- termini with 3- or 4-residue linkers; the -residue linker was less thermodynamically stable. Protasova et al . { Protein Eng. 7:1373-1377, 1994) used 3- or 5-residue linkers in connecting the original N-termini of E. coli dihydrofolate reductase; only the 3-residue linker produced protein in good yield.
Summary of the Invention
The modified human G-CSF receptor agonists of the present invention can be represented by the Formula:
X1-(L)a-X2
wherein; a is 0 or 1;
X-- is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+1 through J; χ2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n; n is an integer ranging from 1 to J-l; and
L is a linker.
In the formula above the constituent amino acids residues of human G-CSF are numbered sequentially 1 through J from the amino to the carboxyl terminus. A pair of adjacent amino acids within this protein may be numbered n and n+1 respectively where n is an integer ranging from 1 to J-l. The r.sidue n+1 becomes the new N-terminus of the new G-CSF receptor agonist and the residue n becomes the new C- terminus of the new G-CSF receptor agonist.
The present invention relates to novel G-CSF receptor agonists of the following formula:
1 10
Xaa Xaa Xaa Gly Pro Ala Ser Ser Leu Pro Gin Ser Xaa 20
Leu Leu Xaa Xaa Xaa Glu Gin Val Xaa Lys Xaa Gin Gly Xaa Gly
30 40 Ala Xaa Leu Gin Glu Xaa Leu Xaa Ala Thr Tyr Lys Leu Xaa Xaa
50
Xaa Glu Xaa Xaa Val Xaa Xaa Gly His Ser Xaa Gly lie Pro Trp
60 70
Ala Pro Leu Ser Ser Xaa Pro Ser Xaa Ala Leu Xaa Leu Ala Gly
80 Xaa Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu
90 100
Leu Gin Ala Leu Glu Gly lie Ser Pro Glu Leu Gly Pro Thr Leu 110
Xaa Thr Leu Gin Xaa Asp Val Ala Asp Phe Ala Xaa Thr lie Trp
120 130
Gin Gin Met Glu Xaa Xaa Gly Met Ala Pro Ala Leu Gin Pro Thr
140
Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Xaa Gin Xaa Xaa Ala
150 160 Gly Gly Val Leu Val Ala Ser Xaa Leu Gin Xaa Phe Leu Xaa Xaa
170 Ser Tyr Arg Val Leu Xaa Xaa Leu Ala Gin Pro (SEQ ID NO:l)
wherein
Xaa at position 1 is Thr, Ser, Arg, Tyr or Gly;
Xaa at position 2 is Pro or Leu;
Xaa at position 3 is Leu, Arg, Tyr or Ser; Xaa at position 13 is Phe, Ser, His, Thr or Pro;
Xaa at position 16 is Lys, Pro, Ser, Thr or His;
Xaa at position 17 is Cys, Ser, Gly, Ala, lie, Tyr or Arg;
Xaa at position 18 is Leu, Thr, Pro, His, lie or Cys;
Xaa at position 22 is Arg, Tyr, Ser, Thr or Ala; Xaa at position 24 is lie, Pro, Tyr or Leu;
Xaa at position 27 is Asp, or Gly;
Xaa at position 30 is Ala, lie, Leu or Gly;
Xaa at position 34 is Lys or Ser;
Xaa at position 36 is Cys or Ser; Xaa at position 42 is Cys or Ser;
Xaa at position 43 is His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu;
Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gin, or Thr; Xaa at position 46 is Glu, Arg, Phe, Arg, lie or Ala;
Xaa at position 47 is Leu or Thr;
Xaa at position 49 is Leu, Phe, Arg or Ser; Xaa at position 50 is Leu, lie, His, Pro or Tyr;
Xaa at position 54 is Leu or His;
Xaa at position 64 is Cys or Ser;
Xaa at position 67 is Gin, Lys, Leu or Cys; Xaa at position 70 is Gin, Pro, Leu, Arg or Ser;
Xaa at position 74 is Cys or Ser;
Xaa at position 104 is Asp, Gly or Val;
Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gin or Gly;
Xaa at position 115 is Thr, His, Leu or Ala; Xaa at position 120 is Gin, Gly, Arg, Lys or His
Xaa at position 123 is Glu, Arg, Phe or Thr
Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gin or Glu;
Xaa at position 146 is Arg or Gin;
Xaa at position 147 is Arg or Gin; Xaa at position 156 is His, Gly or Ser;
Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly;
Xaa at position 162 is Glu, Leu, Gly or Trp;
Xaa at position 163 is Val, Gly, Arg or Ala;
Xaa at position 169 is Arg, Ser, Leu, Arg or Cys; Xaa at position 170 is His, Arg or Ser;
wherein optionally 1-11 ammo acids from the N-termmus and 1-5 from the C-termmus can be deleted; and
wherein the N-termmus is .joined to the C-termmus directly or through a linker capable of joining the N-termmus to the C-termmus and having new C- and N- ermini at ammo acids;
38-39 62-63 123-124
39-40 63-64 124-125
40-41 64-65 125-126
41-42 65-66 126-127
42-43 66-67 128-129 43-44 67-68 128-129
45-46 68-69 129-130
48-49 69-70 130-131
49-50 70-71 131-132
52-53 71-72 132-133 53-54 91-92 133-134
54-55 92-93 134-135
55-56 93-94 135-136
56-57 94-95 136-137
57-58 95-96 137-138 58-59 96-97 138-139
59-60 97-98 139-140
60-61 98-99 140-141
61-62 99-100 141-142 or 142-143. The G-CSF receptor agonists of the present invention may contain ammo acid substitutions, deletions and/or insertions and may also have ammo acid deletions at either/or both the N- and C- termini.
The more preferred breakpoints at which new C-terminus and N-termmus can be made are; 38-39, 39-40, 40-41, 41-42, 48-49, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60- 61, 61-62, 62-63, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 96-97, 125-126, 126-127, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 136-137, 137- 138, 138-139, 139-140, 140-141 and 141-142.
The most preferred breakpoints at which new C-termmus and N-termmus can be made are; 38-39, 48-49, 96-97, 125- 126, 132-133 and 141-142.
A preferred embodiment of the present invention the linker (L) -joining the N-termmus to the C-terminus is a polypeptide selected from the group consisting of: GlyGlyGlySer (SEQ ID NO:2) ; GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:61) ; GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO: 62) ; SerGlyGlySerGlyGlySer (SEQ ID NO:63) ;
GluPheGlyAsnMet (SEQ ID NO: 64); GluPheGlyGlyAsnMet (SEQ ID NO:65); GluPheGlyGlyAsnGlyGlyAsnMet (SEQ ID NO: 66) ; and GlyGlySerAspMetAlaGly (SEQ ID NO: 67) .
The present invention also encompasses recombinant human G-CSF receptor agonists co-administered or sequentially with one or more additional colony stimulating factors (CSF) including, cytok es, lymphokmes, mterleukms, hematopoietic growth factors which include but are not limited to GM-CSF, c-mpl ligand (also known as TPO or MGDF) , M-CSF, erythropoietin (EPO) , IL-1, IL-4, IL-2, IL- 3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand (herein collectively referred to as "colony stimulating factors" or "CSF") . These co-administered mixtures may be characterized by having the usual activity of both of the peptides or the mixture may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of the G-CSF receptor agonists or the second colony stimulating factor alone. The co- administration may also provide an enhanced effect on the activity or an activity different from that expected by the presence of the G-CSF ligand or the second colony stimulating factor. The co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human G-CSF. In addition to the list above, IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-administered with the polypeptides of the present invention.
In addition, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before the expanded cells are infused into patients
Brief Description of the Figures
Figure 1 schematically illustrates the sequence rearrangement of a protein. The N-terminus (N) and the C- terminus (C) of the native protein are joined through a linker, or joined directly. The protein is opened at a breakpoint creating a new N-terminus (new N) and a new C- terminus (new-C) resulting in a protein with a new linear amino acid sequence. A rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C- terminus and opening of the protein at the breakpoint.
Figure 2 shows a schematic of Method I, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C- terminus (a.a. 174) joined to the amino acid 11 (a.a. 1- 10 are deleted) through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
Figure 3 shows a schematic of Method II, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined without a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N- terminus created at amino acid 97 of the original protein, the original C-terminus (a.a. 174) joined to the original N- terminus and a new C-terminus created at amino acid 96 of the original sequence. Figure 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C- terminus (a.a. 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
Detailed Description of the Invention
Receptor agonists of the present invention may be useful in the treatment of diseases characterized by decreased levels of granulocytes of the hematopoietic system.
A G-CSF receptor agonist may be useful in the treatment or prevention of neutropenia. Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and anti- metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti-convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics. G-CSF receptor agonists may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.
Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis. The present peptide may be useful in treating such hematopoietic deficiency.
Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel G-CSF receptor agonists. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform host cells capable of expressing the G- CSF receptor agonists include expression vectors comprising nucleotide sequences coding for the G-CSF receptor agonists joined to transcriptional and translational regulatory sequences which are selected according to the host cells used. Vectors incorporating modified sequences as described above are included in the present invention and are useful the production of the modified G-CSF receptor agonist polypeptides. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells. As another aspect of the present invention, there is provided a novel method for producing the novel family of human G-CSF receptor agonists. The method of the present invention involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of the novel G-CSF receptor agonist polypeptide. Suitable cells or cell lines may include various strains of bacteria such as E. coli , yeast, mammalian cells, or insect cells may be utilized as host cells in the method of the present invention.
Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of one or more of the G-CSF receptor agonists of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously. When administered, the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art. The dosage regimen involved m a metnod for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.5 - 150 μg/kg of non- glycosylated G-CSF receptor agonists protein per kilogram of body weight. Dosages would be ad usted relative to the activity of a given receptor agonist and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day. In addition, there may exist specific circumstances where dosages of G-CSF receptor agonist would be adjusted higher or lower than the range of 0.5 - 150 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-admιnιs ration with chemotherapeutic drugs and/or radiation; the use of glycosylated G-CSF receptor agonists; and various patient-related issues mentioned earlier n this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietins, CSFs and mterleukms for simultaneous or serial co-admmistration with the polypeptides of the present invention includes GM-CSF, c-mpl ligand (also known as TPO or MGDF) , M-CSF, erythropoietin (EPO) , IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosmophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand (herein collectively referred to as "colony stimulating factors"), or combinations thereof. In addition to the list above, IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-admmistered with the polypeptides of the present invention.
The G-CSF receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in peripheral blood. Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation. Hematopoietic growth factors, including G- CSF and GM-CSF, have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required. The G-CSF receptor agonist of the present invention may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
The G-CSF receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors. Colony stimulating factors (CSFs), such as G- CSF, have been administered alone, co-administered with other CSFs, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and which is often the result of such treatment. However the period of severe neutropenia may not be totally eliminated. The myeloid lineage, which is comprised of monocytes (macrophages) , granulocytes (including neutrophils) and megakaryocytes, is critical m preventing infections and bleeding which can be life-threatening. Neutropenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.
Bone marrow transplants have been used to treat his patient population. However, several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow or other tissues, such as spleen or peripheral blood, is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells. Stem cells and progenitor cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exists such that a greater number of multipotential hematopoietic progenitors will enhance hematopoietic recovery. Therefore, the in vitro expansion of stem cells should enhance hematopoietic recovery and patient survival. Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant. However, Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors. An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back into the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells. The limited number of multipotential hematopoietic progenitors may be overcome by ex-vivo expansion of the multipotential hematopoietic progenitors. In addition, stem cells can be specifically isolated based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
The fallowing patents contain further details on separating stem cells, CD34+ cells, culturing the cells with hematopoietic factors, the use of the cells for the treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.
5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separating the stem cells from dedicated cells.
5,199,942 iescribes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; (2) ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flt3 ligand, c-kit ligand, GM CSF, IL-1, GM-CΞF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
5,240,856 relates to a cell separator that includes an apparatus for automatically controlling the cell separation process.
WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells .
WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.
WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokmes, for use n transplants.
WO 93/08268 describes a method for selectively expanding stem cells, comprising the steps of (a) separating CD34+ stem cells from other cells and (b) incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.
WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.
WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia.
WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.
WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.
WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.
WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.
The long term culturing of highly enriched CD34+ precursors of hematopoietic progenitor cells from human bone marrow in cultures containing IL-lα, IL-3, IL-6 or GM-CSF is discussed in Brandt et al ( J. Clin . Invest . 86:932-941, 1990) . One aspect of the present invention provides a method for selective ex-vivo expansion of stem cells. The term "stem cell" refers to the multipotential hematopoietic cells as well as early myeloid progenitor and precursors cells which can be isolated from bone marrow, spleen or peripheral blood. The term "expansion" refers to the proliferation and differentiation of the cells. The present invention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells, (b) culturing the separated stem cells with a selective medium which contains a G-CSF receptor agonist and optionally a second colony stimulating factor, and (c) harvesting the cultured stems cells. Stem cells, as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc., may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics . The phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population. The CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The CD34+ progenitors are heterogeneous, and may be divided into several sub-populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules. The most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express CD90 (thy-1) . Other surface antigens such HS CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors. The separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers. Various colony stimulating factors may be utilized in order to selectively expand cells.
Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6, IL-11, flt-3 ligand or combinations thereof. The proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques (e.g. hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to incubation.
Several methods for ex-vivo expansion of stem cells have been reported utilizing a number of selection methods and expansion using various colony stimulating factors including c-kit ligand (Brandt et al . , Blood 83:1507-1514, 1994; McKenna et al . , Blood 86:3413-3420, 1995), IL-3 (Brandt et al . , Blood 83:1507-1514, 1994; Sato et al . , Blood 82:3600-3609, 1993), G-CSF (Sato et al . , Blood 82:3600-3609, 1993), GM-CSF (Sato et al. , Blood 82:3600-3609, 1993), IL-1 (Muench et al . , Blood 81:3463-3473, 1993), IL-6 (Sato et al., Blood 82:3600-3609, 1993) , IL-11 (Lemoli et al. , Exp . Hem. 21:1668-1672, 1993; Sato et al . , Blood 82 :3600-3609, 1993), flt-3 ligand (McKenna et al . , Blood 86:3413 3420, 1995) and/or combinations thereof (Brandt et al . , Blood 83:1507 1514, 1994; Haylock et al . , Blood 80:1405-1412, 1992, Koller et al. , Biotechnology 11:358-363, 1993; Lemoli et al., Exp . Hem . 21:1668-1672, 1993), McKenna et al . , Blood 86:3413-3420, 1995; Muench et al . , Blood 81:3463-3473, 1993; Patchen et al . , Biotherapy 7:13-26, 1994; Sato et al . , Blood 82:3600-3609, 1993; Smith et al . , Exp . Hem. 21:870-877, 1993; Steen et al . , Stem Cells 12:214-224, 1994; Tsujino et al., Exp . Hem. 21:1379-1386, 1993) . Among the individual colony stimulating factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al . , Blood 73:1836-1841, 1989) . However, no single factor has been shown to be as effective as the combination of multiple factors. The present invention provides methods for ex vivo expansion that utilize novel G-CSF receptor agonists.
Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a G-CSF receptor agonist of the present invention.
Another projected clinical use of growth factors has been in the in vitro activation of hematopoietic progenitors and stem cells for gene therapy. Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body, hematopoietic progenitor cells are good candidates for ex vivo gene transfection. In order to have the gene of interest incorporated into the genome of the hematopoietic progenitor or stem cell one needs to stimulate cell division and DNA replication. Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy. Potential applications of gene therapy (review Crystal, Sci ence 270:404-410, 1995) include; 1) the treatment of many congenital metabolic disorders and immunodeficiencies (Kay and Woo, Trends Genet . 10:253-257, 1994), 2) neurological disorders (Friedmann, Trends Genet . 10:210-214, 1994), 3) cancer (Culver and Blaese, Trends Genet . 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet . 10:139-144, 1994) .
There are a variety of methods, known to those with skill in the art, for introducing genetic material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells. Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opm . Genet . Dev. 3:102-109, 1993; Boris-Lawrie and Temm, Annal . New York Acad. Sci . 716:59-71, 1994; Miller, Current Top . Microbiol . Immunol . 158:1-24, 1992) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629, 1988; Berkner, Current Top . Microbiol . Immunol . 158:39-66, 1992; Brody and Crystal, Annal . New York Acad. Sci . 716:90-103, 1994) . Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA . 90:2122-2126, 1993; Curiel et al . , PNAS USA 88:8850-8854, 1991; Curiel, Annal . New York Acad. Sci . 716:36-58, 1994) , electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al . , Annal . New York Acad . Sci . 716:23-35, 1994) .
The present invention provides an improvement to the existing methods of expanding hematopoietic cells, into which new genetic material has been introduced, in that it provides methods utilizing G-CSF receptor agonists that may have improved biological activity and/or physical properties .
Determination of the Linker
The length of the ammo acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp & Woods, Mol .
Immunol . 20: 483-489, 1983; Kyte & Doolittle, J. Mol . Biol . 157:105-132, 1982; solvent exposed surface area, Lee __ Richards, J. Mol . Biol . 55:379-400, 1971) and the ability to adopt the necessary conformation without deranging the configuration of the c-mpl receptor agonist
(conformationally flexible; Karplus & Schulz, Naturwissenschaften 72:212-213, (1985) . Assuming an average of translation of 2.0 to 3.8 A per residue, this would mean the length to test would be between 0 to 30 residues, with 0 to 15 residues being the preferred range. Exemplary of such an empirical series would be to construct linkers using a cassette sequence such as Gly-Gly-Gly-Ser (SEQ ID NO:2) repeated n times, where n is 1, 2, 3 or 4. Those skilled in the art will recognize that there are many such sequences that vary _.n length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short (cf., Sandhu, Cri tical Rev. Biotech . 12: 437-462, 1992); if they are too long, entropy effects will likely destabilize the three-dimensional fold, and may also make folding kmetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain.
Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number of possibilities that must be tested in an empirical selection of linkers. They will ΛISO recognize that it is sometimes the case that the positions of the ends of the polypeptide chain are ill- defined in structural models derived from x-ray diffraction or nuclear magnetic resonance spectroscopy data, and that when true, this situation will therefore need to be taken into account in order to properly estimate the length of the linker required. From those residues whose positions are well defined are selected two residues that are close in sequence to the chain ends, and the distance between their c-alpha carbons is used to calculate an approximate length for a linker between them. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8A per residue) are then selected. These linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:2) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.
Determination of the Amino and Carhπxvl Termini of G-CSF Receptor Agonists
Sequences of G-CSF receptor agonists capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence as described above. Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. l_ι many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, those skilled in the art will recognize that selections of termini anywhere within the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.
It is a central tenet of molecular biology that the primary amino acid sequence of a protein dictates folding to the three-dimensional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions . Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti- parallel beta sheets, chain reversals and turns, and loops; Kabsch &_ Sander, Biopolymers 22: 2577-2637, 1983; the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann. .Rev. Biochem . 53:537-572; 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber & Mathews, Methods Enzymol . 154: 511-533, 1987) . In some cases additional information is known about solvent exposure of residues; one example is a site of post- translational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available, or is not feasible to obtain, methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops. Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile & Salvatore, Eur. J. Biochem. 218:603-621, 1993) Thus using either the experimentally derived structural informaticn or predictive methods (e.g., Srinivisan & Rose Proteins : Struct . , Funct . & Geneti cs , 22: 81-99, 1995) the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The occurrence of sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so- called hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini. In contrast, those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region.
TAP E 1 Q IGONUCLEOTIPES
L-llstart . seq GCTCTGAGAG CCGCCAGAGC CGCCAGAGGG CTGCGCAAGG TGGCGTAGAA CGCG (SEQ ID NO:3)
L-llstop.seq CAGCCCTCTG GCGGCTCTGG CGGCTCTCAG AGCTTCCTGC TCAAGTCTTT AGAG (SEQ ID NO:4)
BlstartP. seq GGGCTGCGCA AGGTGGCG (SEQ ID NO:5) blstopP.seq ACACCATTGG GCCCTGCCAG C (SEQ ID NO:6) 39start .seq GATCGACCAT GGCTTACAAG CTGTGCCACC CC (SEQ ID NO:7)
38stop.Seq CGATCGAAGC TTATTAGGTG GCACACAGCT TCTCCT (SEQ ID NO:8)
97start .seq GATCGACCAT GGCTCCCGAG TTGGGTCCCA CC (SEQ ID NO: 9)
96stop.Seq CGATCGAAGC TTATTAGGAT ATCCCTTCCA GGGCCT (SEQ ID NO:10)
126start .seq GATCGACCAT GGCTATGGCC CCTGCCCTGC AG (SEQ ID NO:11)
125stop.Seq CGATCGAAGC TTATTATCCC AGTTCTTCCA TCTGCT (SEQ ID NO:12)
133start .seq GATCGACCAT GGCTACCCAG GGTGCCATGC CG (SEQ ID NO:13)
132stop.seq CGATCGAAGC TTATTAGGGC TGCAGGGCAG GGGCCA (SEQ ID NO:14)
142start .seq GATCGACCAT GGCTTCTGCT TTCCAGCGCC GG (SEQ ID NO:15)
141stop.Seq CGATCGAAGC TTATTAGGCG AAGGCCGGCA TGGCAC (SEQ ID NO:16)
96for.Seq ATATCCATGG CTCCGGAACT GGGTCCAACT CTG (SEQ ID NO:17)
96rev.Seq ACCTCCAGGA AGCTCTGCAG ATGG (SEQ ID NO: 18) 125for.seq TATATCCATG GCTATGGCTC CAGCTCTGCA ACCAACTCAA GGTGCAATGC CAGCATTTGC ATCTG (SEQ ID NO:19)
125rev.seq GATGGCTAGC AACCAGAACA CCACCTGCAC GACGTTGAAA AGCAGATGCA AATGCTGGCA TTG (SEQ ID NO:20)
132for.seq TATATCCATG GCTACTCAAG GTGCTATGCC AGCTTTTGCT TCTGCTTTTC AACGTCG (SEQ ID NO:21)
132rev.seq GCAGATGGCT AGCAACCAGA ACACCACCTG CACGACGTTG AAAAGCAGAA GCAAAAGC (SEQ ID NO:22)
141for.seq CATGGCTTCT GCTTTTCAAC GTCGTGCAGG TGGTGTTCTG GTTG (SEQ ID NO:23)
141rev.seq CTAGCAACCA GAACACCACC TGCACGACGT TGAAAAGCAG AAGC (SEQ ID NO:24)
49start .seq GATCGACCAT GGCTCTGCTC GGACACTCTC TG (SEQ ID NO:68)
48stop.seq CGATCGAAGC TTATTACACC AGCTCCTCGG GGTGGC (SEQ ID NO:69)
77start .seq GATCGACCAT GGCTCAACTC CATAGCGGCC TT (SEQ ID NO:70)
76stop.seq CGATCGAAGC TTATTAGCTC AAGCAGCCTG CCAGCT (SEQ ID NO:71)
82start .seq GATCGACCAT GGCTCTTTTC CTCTACCAGG GG (SEQ ID NO:72)
81stop.seq CGATCGAAGC TTATTAGCCG CTATGGAGTT GGCTCA (SEQ ID NO:73)
84start .seq GATCGACCAT GGCTCTCTAC CAGGGGCTCC TG (SEQ ID NO:74)
83stop.seq CGATCGAAGC TTATTAGAAA AGGCCGCTAT GGAGTT (SEQ ID NO:75)
91start .seq GATCGACCAT GGCTGCCCTG GAAGGGATAT CC (SEQ ID NO:76)
90stop.seq CGATCGAAGC TTATTACTGC AGGAGCCCCT GGTAGA (SEQ ID NO:77) 112start . seq GATCGACCAT GGCTGACTTT GCCACCACCA TC (SEQ ID NO: 78) lllstop.seq CGATCGAAGC TTATTAGGCG ACGTCCAGCT GCAGTG (SEQ ID NO: 79)
117start . seq GATCGACCAT GGCTATCTGG CAGCAGATGG AA (SEQ ID NO: 80)
116stop.seq CGATCGAAGC TTATTAGGTG GTGGCAAAGT CGGCGA (SEQ ID NO: 81)
119start .seq GATCGACCAT GGCTCAGCAG ATGGAAGAAC TG (SEQ ID NO: 82) llδstop.seq CGATCGAAGC TTATTACCAG ATGGTGGTGG CAAAGT (SEQ ID NO: 83)
Z4849at.for CATGGCTTTG TTAGGACATT CTTTAGGTAT TCCATGGGCT CCTCTGAGCT (SEQ ID NO:84!
Z4849at .rev CAGAGGAGCC CATGGAATAC CTAAAGAATG TCCTAACAAA GC (SEQ ID NO:85)
TAP E 2 DNA sequences
pM0N3485.Seq
1 ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC
51 TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC
101 AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG 151 GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT
201 GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC 251 AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC
301 ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT 351 TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC 401 ACCTTGCGCA GCCCTCTGGC GGCTCTGGCG GCTCTCAGAG CTTCCTGCTC
451 AAGTCTTTAG AGCAAGTGAG GAAGATCCAG GGCGATGGCG CAGCGCTCCA
501 GGAGAAGCTG TGTGCCACCT AATAA (SEQ ID NO: 25)
pMON3486.Seq
1 ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC 151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAG TGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC TCTGGCGGCT 251 CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGGAAG
301 ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA 351 GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC 401 CCTGGGCTCC CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC
451 TGCTTGAGCC AACTCCATAG CGGCCTTTTC CTCTACCAGG GGCTCCTGCA
501 GGCCCTGGAA GGGATATCCT AATAA (SEQ ID NO:26)
pMON3487.Seq
1 ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT
51 CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG 151 CCCTCTGGCG GCTCTGGCGG CTCTCAGAGC TTCCTGCTCA AGTCTTTAGA
201 GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT
251 GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC
301 TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT
351 GCAGCTGGCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC 401 AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC
451 TTGGACACAC TGCAGCTGGA CGTCGCCGAC TTTGCCACCA CCATCTGGCA
501 GCAGATGGAA GAACTGGGAT AATAA (SEQ ID NO:27)
pMON3488.Seq 1 ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG
51 GGCAGGAGGG GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC
151 TCTCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGGA AGATCCAGGG 201 CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC
251 ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
301 CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG 351 CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG 401 AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC 451 GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT
501 GGCCCCTGCC CTGCAGCCCT AATAA (SEQ ID NO:28)
pMON3489.Seq
1 ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC
101 AGCCCTCTGG CGGCTCTGGC GGCTCTCAGA GCTTCCTGCT CAAGTCTTTA
151 GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT 201 GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC
251 ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC
301 CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA
351 CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA
401 CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 451 CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG
501 TGCCATGCCG GCCTTCGCCT AATAA (SEQ ID NO:29)
PMON3490.seq
1 ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC
51 TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC
101 AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG
151 GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT 201 GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC
251 AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC
301 ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT 351 TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC 401 ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC 451 TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC
501 AGCGCTCCAG GAGAAGCTGT GTGCCACCTA ATAA (SEQ ID NO:30)
pMON3491.seq
1 ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT 201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG
251 GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
301 GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC 351 CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC
401 TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG
451 CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG
501 GCTCCTGCAG GCCCTGGAAG GGATATCCTA ATAA (SEQ ID NO: 31)
pMON3492.seq
1 ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT 51 CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG
151 CCCACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA
201 GTCTTTAGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG 251 AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG 301 CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG
351 CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT 401 TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 451 GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC 501 CATCTGGCAG CAGATGGAAG AACTGGGATA ATAA (SEQ ID NO: 32)
pMON3493.seq
1 ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG 51 GGCAG'3.-GGG GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCACACCATT GGGCCCTGCC
151 AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG TCTTTAGAGC AAGTGAGAAA
201 GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT GCCACCTACA
251 AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC 301 CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG
351 CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC 401 AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG 451 CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA 501 ACTGGGAATG GCCCCTGCCC TGCAGCCCTA ATAA (SEQ ID NO: 33)
pMON3494.seq
1 ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA 51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC
101 AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC
151 AAGTCTTTAG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA
201 GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC
251 TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 301 AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT
351 TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT
401 TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC
451 ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC
501 CACCCAGGGT GCCATGCCGG CCTTCGCCTA ATAA (SEQ ID NO: 34)
pMON25181.seq 1 ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC 151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG
251 GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA
301 GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC 351 CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC 401 TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG
451 CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG
501 GCTCCTGCAG GCCCTGGAAG GGATATCCTA A (SEQ ID NO:35)
pMON25182.seq
1 ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT
51 TGCATCTGCT TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG 151 CCCACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA
201 GTCTTTAGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG
251 AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG
301 CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG
351 CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT 401 TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
451 GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC
501 CATCTGGCAG CAGATGGAAG AACTGGGATA A (SEQ ID NO:36)
pMON25183.seq
1 ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG
51 TGCAGGTGGT GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCACACCATT GGGCCCTGCC 151 AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG TCTTTAGAGC AAGTGAGAAA
201 GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT GCCACCTACA
251 AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
301 CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG 351 CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC 401 AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG
451 CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA
501 ACTGGGAATG GCCCCTGCCC TGCAGCCCTA A (SEQ ID NO:37)
pMON25184.seq
1 ATGGCTTCTG CTTTTCAACG TCGTGCAGGT GGTGTTCTGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC
101 AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC 151 AAGTCTTTAG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA
201 GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC
251 TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 301 AGCC*GGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT
351 TTTCCrCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT
401 TGGGTCCC C CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC 451 ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC 501 CACCCAGGGT GCCATGCCGG CCTTCGCCTA A (SEQ ID NO: 38)
pMON25185.seq 1 ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC TCTGGCGGCT 251 CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGAAAG
301 ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA 351 GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC 401 CCTGGGCTCC CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC 451 TGCTTGAGCC AACTCCATAG CGGCCTTTTC CTCTACCAGG GGCTCCTGCA 501 GGCCCTGGAA GGGATATCCT AA (SEQ ID NO: -.9)
pMON25186.seq 1 ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT
51 TGCATCTGCT TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG
151 CCCTCTGGCG GCTCTGGCGG CTCTCAGAGC TTCCTGCTCA AGTCTTTAGA
201 GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT 251 GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC
301 TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT
351 GCAGCTGGCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC
401 AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC
451 TTGGACACAC TGCAGCTGGA CGTCGCCGAC TTTGCCACCA CCATCTGGCA 501 GCAGATGGAA GAACTGGGAT AA (SEQ ID NO: 40)
PMON25187.seq 1 ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG
51 TGCAGGTGGT GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC
151 TCTCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGAA AGATCCAGGG
201 CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC 251 ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
301 CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG
351 CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG
401 AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC
451 GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT 501 GGCCCCTGCC CTGCAGCCCT AA (SEQ ID NO: 41) pMON25188.seq
1 ATGGCTTCTG CTTTTCAACG TCGTGCAGGT GGTGTTCTGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC 101 AGCCCTCTGG CGGCTCTGGC GGCTCTCAGA GCTTCCTGCT CAAGTCTTTA
151 GAGCAAGTGA GAAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT
201 GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC 251 ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC
301 CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA 351 CCAGLΓ.GCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA
401 CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG
451 CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG
501 TGCCATGCCG GCCTTCGCCT AA (SEQ ID NO:42)
pMON3460.seq
1 ATGGCTCTGC TCGGACACTC TCTGGGCATC CCCTGGGCTC CCCTGAGCTC
51 CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA 101 GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC
151 CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT
201 TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC
251 TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC
301 CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT 351 GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG
401 CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA
451 AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA
501 CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA (SEQ ID NO:86)
pMON3461.seq l ATGGCΓCAAC TCCATAGCGG CCTTTTCCTC TACCAGGGGC TCCTGCAGGC
51 CCTGGAAGGG ATATCCCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC 101 TGGACGTCGC CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG
151 GGAATGGCCC CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC
201 CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC
251 AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC
301 ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC 351 TTTAGAGCAA GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA
401 AGCTGTGTGC CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC
451 GGACACTCTC TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA
501 GGCCCTGCAG CTGGCAGGCT GCTTGAGCTA ATAA (SEQ ID NO:87)
pMON3462.seq
1 ATGGCTCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC
51 CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT 101 TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT GGCCCCTGCC
151 CTGCAGCCCA CCCAGGGTGC CATGCCGGCC TTC._CCTCTG CTTTCCAGCG
201 CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC TTCCTGGAGG 251 TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT
301 GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG
351 AAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT
401 ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC 451 ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC TGCAGCTGGC 501 AGGCTGCTTG AGCCAACTCC ATAGCGGCTA ATAA (SEQ ID NO: 88)
pMON3463.seq
1 ATGGCTCTCT ACCAGGGGCT CCTGCAGGCC CTGGAAGGGA TATCCCCCGA
51 GTTGGGTCCC ACCTTGGACA CACTGCAGCT GGACGTCGCC GACTTTGCCA
101 CCACCATCTG GCAGCAGATG GAAGAACTGG GAATGGCCCC TGCCCTGCAG
151 CCCACCCAGG GTGCCATGCC GGCCTTCGCC TCTGCTTTCC AGCGCCGGGC 201 AGGAGGGGTC CTGGTTGCTA GCCATCTGCA GAGCTTCCTG GAGGTGTCGT
251 ACCGCGTTCT ACGCCACCTT GCGCAGCCCA CACCATTGGG CCCTGCCAGC
301 TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGAAAGAT
351 CCAGGGCGAT GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC
401 TGTGΓ.ΛCCC CGAGGAGCTG GTGCTGCTCG GACACTCTCT GGGCATCCCC 451 TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG GCCCTGCAGC TGGCAGGCTG
501 CTTGAGCCAA CTCCATAGCG GCCTTTTCTA ATAA (SEQ ID NO: 89)
pMON3464.seq
1 ATGGCTGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC
51 ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG
101 AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG
151 GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG 201 CCATCTGCAG AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG
251 CGCAGCCCAC ACCATTGGGC CCTGCCAGCT CCCTGCCCCA GAGCTTCCTG
301 CTCAAGTCTT TAGAGCAAGT GAGAAAGATC CAGGGCGATG GCGCAGCGCT
351 CCAGGAGAAG CTGTGTGCCA CCTACAAGCT GTGCCACCCC GAGGAGCTGG
401 TGCTGCTCGG ACACTCTCTG GGCATCCCCT GGGCTCCCCT GAGCTCCTGC 451 CCCAGCCAGG CCCTGCAGCT GGCAGGCTGC TTGAGCCAAC TCCATAGCGG
501 CCTTTTCCTC TACCAGGGGC TCCTGCAGTA ATAA (SEQ ID NO: 0)
pMON3465.seq
1 ATGGCTGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT
51 GGCCC 'TGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC TTCGCCTCTG
101 CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
151 TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC 201 ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG
251 AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG
301 TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA
351 CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC
401 TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC 451 CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC
501 CTTGGACACA CTGCAGCTGG ACGTCGCCTA ATAA (SEQ ID NO: 91) pMON 3466.seq
1 ATGGCTATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA 51 GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG
101 CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT GGAGGTGTCG
151 TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG GCCCTGCCAG
201 CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA GTGAGAAAGA
251 TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG 301 CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC
351 CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT
401 GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG GCTCCTGCAG
451 GCCCTGGAAG GGATATCCCC CGAGTTGGGT CCCACCTTGG ACACACTGCA
501 GCTGGACGTC GCCGACTTTG CCACCACCTA ATAA (SEQ ID NO: 92)
PMON3467.seq
1 ATGGCTCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC 51 CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG
101 GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC
151 GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT
201 GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG
251 GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC 301 CACCCCGAGG AGCTGGTGCT GCTCGGACAC TCTCTGGGCA TCCCCTGGGC
351 TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA GGCTGCTTGA
401 GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG
451 GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA
501 CGTCG CGAC TTTGCCACCA CCATCTGGTA ATAA (SEQ ID NO: 93)
pMON3499.seq
1 ATGGCTTTGT TAGGACATTC TTTAGGTATT CCATGGGCTC CTCTGAGCTC 51 CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA
101 GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC
151 CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT
201 TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC
251 TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC 301 CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT
351 GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG
401 CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA
451 AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA
501 CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA (SEQ ID NO: 94) TAELE 3
PROTEIN SEQUENCES pMON3485.Pep
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly lie Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala
Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe
Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe
Ala Thr Thr lie Trp Gin Gin Met Glu Glu L^u Gly Met Ala Pro
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser
Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr (SEQ ID NO:43)
pMON3486. Pep
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala
Asp Phe Ala Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu
Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala
Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu
Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser (SEQ ID NO:44)
pMON3487.Pep
Figure imgf000043_0001
pMON3488.Pep
Figure imgf000044_0001
pMON3489.Pep
Figure imgf000044_0002
pMON3490.Pep
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala
Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe
Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe
Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala
Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu
Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr (SEQ ID NO:48)
pMON3491.Pep
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala
Figure imgf000045_0001
pMON3492.Pep
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin
Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly
Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu
Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin
Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro
Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly (SEQ ID NO:50)
PMON3493.Pep Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu
Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu
His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu
Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gin Pro (SEQ ID NO:51)
pMON3494.Pep
Figure imgf000045_0002
Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala (SEQ ID NO: 52)
pMON25181.pep Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser
Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp
Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly
Leu Leu Gin Ala Leu Glu Gly He Ser (SEQ ID NO: 53)
pMON25182.pep
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly
Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu
Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro
Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly (SEQ ID NO:54)
pMON25183.pep
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro (SEQ ID NO: 55)
pMON25184.pep
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Set Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp
Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala
Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr
Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He
Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala (SEQ ID NO:56)
pMON25185.pep
Figure imgf000047_0001
pMON25186.pep
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu
Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala
Ala Leu Gin Glu Lys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu
Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser
Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala
Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly (SEQ ID NO:58) pMON25187 . pep
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly
Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val
Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly
Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gin Pro (SEQ ID NO:59)
pMON25188.pep
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu
Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala
Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu
Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin
Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin
Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala (SEQ ID NO:60) pMON3460.Pep
Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu
His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu
Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu
Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Al i Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val
Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu
Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu
Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He
Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val (SEQ ID NO: 95)
pMON3461.Pep Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met
Figure imgf000049_0001
3462. Pep
Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser
Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp
Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Tr.r Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly (SEQ ID NO: 97)
3463.Pep Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe (SEQ ID NO: 98)
3464. Pep
Figure imgf000049_0002
Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly
Leu Phe Leu Tyr Gin Gly Leu Leu Gin (SEQ ID NO: 99)
Figure imgf000050_0001
3466.Pep
He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr (SEQ ID NO:101)
3467.Pep
Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr
Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala
Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly
Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu
Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu
Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His
Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly
He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp
Figure imgf000051_0001
Materials ar.H Methods
Recombinant DNA methods
Unless noted otherwise, all specialty chemicals were obtained from Sigma Co., (St. Louis, MO) . Restriction endonuclea-es and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN) .
Transformation of E. col i s ains
E. col i strains, such as DH50!™ (Life Technologies, Gaithersburg, MD) and TGI (Amersham Corp., Arlington Heights, IL) are used for transformation of ligation reactions and are the source of plasmid DNA for transfecting mammalian cells. E. col strains, such as MON105 and JM101, can be used for expressing the G-CSF receptor agonist of the present invention in the cytoplasm or peπplasmic space.
MON105 ATCC#55204: F-, lamda-, IN(rrnD, rrE)l, rpoD+, rpoH358
DH5α™: F-, phι80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recAl, endAl, hsdR17 (rk-,mk+) , phoA, supE441amda-, thι-1, gyrA96, relAl
TGI: delta (lac-pro) , supE, thι-1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaM15)
DH5α™ Subclonmg efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TGI and MON105 are rendered competent to take up DNA using a CaCl2 method. Typically, 20 to 50 L of cells -re grown in LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 150 mM NaCl) to a density of approximately 1.0 optical density unit at 600 nanometers (OD600) as measured by a Baush & Lomb Spectronic spectrophotometer (Rochester, NY) . The cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl2 solution (50 mM CaCl2, 10 mM Tris- Cl, pH7.4) and are held at 4'C for 30 minutes. The cells are again collected by centrifugation and resuspended in one-tenth culture volume of CaCl2 solution. Ligated DNA is added to 0.2mL of these cells, and the samples are held at 4'C for 1 hour. The samples are shifted fo 42'C for two minutes and lmL of LB is added prior to shaking the samples at 37*C for one hour. Cells from these samples are spread on plates ! B medium plus 1.5% Bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycin-resistant transformants . The plates are incubated overnight at 37 "C. Single colonies are picked, grown in LB supplemented with appropriate antibiotic for 6-16 hours at 37 "C with shaking. Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37°C while shaking. Before harvesting the cultures, 1 ul of cells are analyzed by PCR for the presence of a G-CSF gene. The PCR is carried out using a combination of primers that anneal to the G-CSF gene and/or vector. After the PCR is complete, loading dye is added to the sample followed by electrophoresis as described earlier. gene has been ligated to the vector when a PCR product of the expected size is observed.
Methods for creation of genes with new N-terminus/C-terminus
Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region. Genes with new N-terminus/C-termmus which contain a linker region separating the original C-terminus and N- terminus can be made essentially following the method described in L. S. Mullins, et al J. Am . Chem. Soc . 116, 5529-5533 '1994) . Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary ammo acid sequence of the protein. The steps are illustrated in Figure 2.
In the first step, the primer set ("new start" and
"linker start") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Start") that contains the sequence encoding the new N-termmal portion of the new protein followed by the linker that connects the C- erminal and N-terminal ends of the original protein. In the second step, the primer set ("new stop" and "linker stop") is used to create and ampHfy, from the original gene sequence, the DNA fragment ("Fragment Stop") that encodes the same linker as used above, followed by the new C-termmal portion of the new protein. The "new start" and "new stop" primers are designed to include the appropriate restriction enzyme recognition sites which allow cloning of the new gene into expression plasmids. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2- PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . "Fragment Start" and "Fragment Stop", which have complementary sequence in the linker region and the coding sequence for the two amino acids on both sides of the linker, are joined together in a third PCR step to make the full-length gene encoding the new protein. The DNA fragments "Fragment Start" and "Fragment Stop" are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined in equimolar quantities, heated at 70°C for ten minutes and slow cooled to allow annealing through their shared sequence in "linker start" and "linker stop". In the third PCR step, primers "new start" and "new stop" are added to the annealed fragments to create and amplify the full- length new N-terminus/C-terminus gene. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 60°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes . A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and approximately 0.5 ug of DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2 • PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
Method II. Creation of genes with new N-terminus/C-terminus without a linker region.
New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-terminus can be made using two steps of PCR amplification and a blunt end ligation. The steps are illustrated in Figure 3. In the first step, the primer set ("new start" and "P-bl start") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Start") that contains the sequence encoding the new N- erminal portion of the new protein. In the second step, the primer set ("new stop" and "P-bl stop") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Stop") that contains the sequence encoding the new C-terminal portion of the new protein. The "new start" and "new stop" primers are designed to include appropriate restriction sites which allow cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for 45 seconds and 72°C extension for 45 seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manuf cturer. The "P-bl start" and "P-bl stop" primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start" and "Fragment Stop" to each other. A 100 ul leaction contained 150 pmole of each primer and one ug of template DNA; and lx Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase. PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . PCR reaction products are purified using a Wizard PCR Preps kit (Promega) .
The primers are designed to include appropriate restriction enzyme recognition sites which allow for the cloning of the new gene into expression vectors. Typically "Fragment Start" is designed to create a Ncol restriction site , and "Fragment Stop" is designed to create a Hindlll restriction site. Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega) . Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined with and annealed to the ends of the ~ 3800 base pair Ncol/Hindlll vector fragment of pMON3934 by heating at 50°C for ten minutes and allowed to slow cool. The three fragments are ligated together using T4 DNA ligase (Boehringer Mannheim) . The result is a plasmid containing the full-length new N- terminus/C-terminus gene. A portion of the ligation reaction is used to transform E. coli strain DH5αcells (Life
Technologies, Gaithersburg, MD) . Plasmid DNA is purified and sequence confirmed as below.
Method III. Creation of new N-terminus/C-terminus genes by tandem-duplication method
New N-terminus/C-terminus genes can be made based on the method described in R. A. Horlick, et al Protein Eng. 5:427-431 (1992) . Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA. The steps are illustrated in Figure 4.
The tandemly-duplicated template DNA is created by cloning and contains two copies of the gene separated by DNA sequence encoding a linker connecting the original C- and N- terminal ends of the two copies of the gene. Specific primer sets are used to create and amplify a full-length new N terminus /C-terminus gene from the tandemly-duplicated template DNA. These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is used. A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dAT?, 200 uM dTTP, 200 uM dCTP, 2.5 units A pliTaq DNA polymerase and 2 mM MgCl2. PCR reactions are performed m a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
DNA isolation and characterization
Plasmid DNA can be isolated by a number of different methods and using commercially available kits known to those skilled in the art. A few such methods are shown herein. Plasmid DNA is isolated using the Promega Wizard™ M niprep kit (Madison, WI) , the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) or Qiagen Plasmid Midi kit. These kits follow the same general procedure for plasmid DNA isolation. Briefly, ι -11s are pelleted by centrifugation (5000 x g) , plasmid DNA released with sequential NaOH/acid treatment, and cellular debris is removed by centrifugation (10000 x g) . The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-bmding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells are inoculated into 50-100 mLs of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking. The purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian, E. coli or other cells.
Sequence confirmation.
Purified plasmid DNA is resuspended in dH20 and quantitated by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectromc 601 UV spectrometer. DNA samples are sequenced using ABI PRISM™ DyeDeoxy™ terminator sequencing chemistry (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) kits (Part Number 401388 or 402078) according to the manufacturers suggested protocol usually modified by the addition of 5% DMSO to the sequencing mixture. Sequencing reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions. Samples are purified to remove excess dye terminators with Centri-Sep™ spin columns (Princeton Separations, Adelphia, NJ) and lyophilized. Fluorescent dye labeled sequencing reactions are resuspended n deionized formamide, and sequenced on denaturing 4.75% polyacrylamide- 8M urea gels using an ABI Model 373A automated DNA sequencer. Overlapping DNA sequence fragments are analyzed and assembled into master DNA contigs using Sequencher v2.1 DNA analysis software (Gene Codes Corporation, Ann Arbor, MI) .
Expression of G-CSF receptor agonists in mammalian cells
Mammalian Cell Transfection/Production of Conditioned Media
The BHK-21 cell line can be obtained from the ATCC (Rockville, MD) . The cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose) , supplemented to
2mM (mM) L-glutamme and 10% fetal bovine serum (FBS) . This formulation is designated BHK growth media. Selective media is BHK growth media supplemented with 453 units/mL hygromycm B (Calbiochem, San Diego, CA) . The BHK-21 cell line was previously stably transfected with the HSV transactivating protein VP16, which transactivates the IE110 promoter found on the plasmid pMON3359 (See Hippenmeyer et al., Bio /Technology, p .1037-1041, 1993) . The VP16 protein drives expression of genes inserted behind the IE110 promoter. BHK-21 cells expressing the transactivating protein VP ■ ό are designated BHK-VP16. The plasmid pMONlllδ (See Highk et al . , Poul try Sci . , 70: 970-981, 1991) expresses the hygromyc resistance gene from the SV40 promoter. A similar plasmid is available from ATCC, pSV2- hph.
BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X 105 cells per dish 24 hours prior to transfection. Cells are transfected for 16 hours in 3 mL of "OPTIMEM"™ (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycm resistance plasmid, pMONlllδ, and 80 ug of Gibco- BRL "LIPOFECTAMINE"™ per dish. The media is subsequently aspirated and replaced with 3 mL of growth media. At 48 hours post-transfection, media from each dish is collected and assayed for activity (transient conditioned media) . The cells are removed from the dish by trypsm-EDTA, diluted 1:10 and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in tryps /EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluence, the conditioned media is re-assayed, and positive clones are expanded into growth media.
Expression of G-CSF receptor agonists n E. coli
E. coli strain MON105 or JM101 harboring the plasmid of interest are grown at 37°C in M9 plus casammo acids medium with shaking in a air incubator Model G25 from New Brunswick Scientific (Edison, New Jersey) . Growth is monitored at OD600 until it reaches a value of 1, at which time nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a final concentration of 50 μg/mL. The cultures are then shaken at 37°C for three to four additional hours. A high degree of aeration is maintained throughout culture period in order to achieve maximal production of the desired gene product. The cells are examined under a light microsccpe for the presence of inclusion bodies (IB) . One mL aliquots of the culture are removed for analysis of protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (see Maniatis et al . Molecular Cloning: A Laboratory Manual, 1982) . The culture is centrifuged (5000 x g) to pellet the cells.
Inclusion Body preparation. Extraction. Refolding. Dialysis. DEAE Chromatography. and Characterization of the G-CSF receptor agonists which accumulate as inclusion bodies in .£____. coli .
Isolation of Inclusion Bodies:
The cell pellet from a 330 mL E. col i culture is resuspended in 15 mL of sonication buffer (10 mM 2-amino-2- (hydroxymethyl) 1, 3-propanediol hydrochloride (Tris-HCl), pH 8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA)) . These resuspended cells are sonicated using the microtip probe of a Sonicator Cell Disruptor (Model W-375, Heat Systems- Ultrasonics, Inc., Farmingdale, New York) . Three rounds of sonication in sonication buffer followed by centrifugation are employed to disrupt the cells and wash the inclusion bodies (IB) . The first round of sonication is a 3 minute burst followed by a 1 minute burst, and the final two rounds of sonication are for 1 minute each.
Extraction and refolding of proteins from inclusion body pellets : Folli ing the final centrifugation step, the IB pellet is resuspended m 10 mL of 50 mM Tris-HCl, pH 9.5, 8 M urea and 5 mM dithiothreitol (DTT) and stirred at room temperature for approximately 45 minutes to allow for denaturation of the expressed protein.
The extraction solution is transferred to a beaker containing 70 mL of 5mM Tris-HCl, pH 9.5 and 2.3 M urea and gently stirred while exposed to air at 4°C for 18 to 48 hours to allow the proteins to refold. Refolding is monitored by analysis on a Vydac (Hesperia, Ca. ) C18 reversed phase high pressure liquid chromatography (RP-HPLC) column (0.46x25 cm) . A linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid (TFA) , is employed to monitor the refold. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute.
Denatured ,-roteιns generally elute later in the gradient than the refolded proteins .
Purification:
Following the refold, contaminating E. coli proteins are removed by acid precipitation. The pH of the refold solution is titrated to between pH 5.0 and pH 5.2 using 15% (v/v) acetic acid (HOAc) . This solution is stirred at 4°C for 2 hours and then centrifuged for 20 minutes at 12,000 x g to pellet any insoluble protein.
The supernatant from the acid precipitation step is dialyzed using a Spectra/Por 3 membrane with a molecular weight cut off (MWCO) of 3,500 daltons. The dialysis is against 2 changes of 4 liters (a 50-fold excess) of lOmM
Tris-HCl, pH 8.0 for a total of 18 hours. Dialysis lowers the sample conductivity and removes urea prior to DEAE chromatography. The sample is then centrifuged (20 minutes at 12,000 x g) to pellet any insoluble protein following dialysis. A Bio-Rad Bio-Scale DEAE2 column (7 x 52 mm) is used for ion exchange chromatography. The column is equilibrated in a buffer containing lOmM Tris-HCl, pH 8.0. The protein is eluted using a 0-to-500 mM sodium chloride (NaCl) gradient, in equilibration buffer, over 45 column volumes . A flow rate of 1 mL per minute is used throughout the run. Column fractions (2 mL per fraction) are collected across the gradient and analyzed by RP HPLC on a Vydac (Hesperia, Ca.) C18 column (0.46 x 25 cm) . A linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is employed. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute. Pooled fractions are then dialyzed against 2 changes of 4 liters (50-to-500-fold excess) of 10 mM ammonium acetate (NH4Ac), pH 4.0 for a total of 18 hours. Dialysis is performed using a
Spectra/Por 3 membrane with a MWCO of 3,500 daltons. Finally, the sample is sterile filtered using a 0.22μm syringe filter (μStar LB syringe filter, Costar, Cambridge, Ma.), and stored at 4°C. In some cases the folded proteins can be affinity purified using affinity reagents such as mAbs or receptor subunits attached to a suitable matrix. Alternatively, (or in addition) purification can be accomplished using any of a variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic chromatography or reversed phase HPLC.
These and other protein purification methods are described in detail in Methods in Enzymology, Volume 182 'Guide to Protein Purification" edited by Murray Deutscher, Academic Press, San Diego, CA (1990) .
Protein Characterization: The purified protein is analyzed by RP-HPLC, electrospray mass spectrometry, and SDS-PAGE. The protein quantitation is done by amino acid composition, RP-HPLC, and Bradford protein determination. In some cases tryptic peptide mapping is performed in conjunction with electrospray mass spectrometry to confirm the identity of the protein.
AML Proliferation Assay The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) . This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, B., et al . , Blood 70: 192, 1987; Valtieri,
M., et al., J. Immunol . 138:4042, 1987) . The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al . , J. Immunol . 139: 348, 1987) . A cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokme dependent AML 193 cells for growth factors for 24 hours. The cells are then replated at 1x105 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.
AML 193 1.3 cells are washed 6 times cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifugmg cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2 x 105 to 5 x 105 viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco ' s Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrm, lipids and 2-mercaptoethanol . Bovine albumin (Boehrmger-Mannheim, Indianapolis, IN) is added at 500 μg/πιL; human transferrm (Boehrmger-Mannheim, Indianapolis, IN) is added at 100 μg/mL; soybean lipid (Boehrmger- Mannheim, Indianapolis, IN) is added at 50 μg/mL; and 2- mercaptoethanol (Sigma, St. Louis, MO) is added at 5 x 10~ 5 M. Serial dilutions of G-CSF receptor agonist proteins are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 μl of medium containing G-CSF receptor agonist proteins once serial dilutions are completed. Control wells contained tissue culture medium alone (negative control) . AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 μl (2.5 x 104 cells) into each well. Tissue culture plates are incubated at 37°C with 5% C02 m humidified air for 3 days. On day 3, 0.5 μCi
Figure imgf000065_0001
(2 Ci/rriM, New England Nuclear, Boston, MA) is added in 50 μl of tissue culture medium. Cultures are incubated at 37°C with 5% C02 in humidified air for 18-24 hours. Cellular DNA is harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, MD) using a TOMTEC cell harvester (TOMTEC,
Orange, CT) which utilized a water wash cycle followed by a 70% ethanol wash cycle. Filter mats are allowed to air dry and then placed into sample bags to which scintillation fluid (Scmtiverse II, Fisher Scientific, St. Louis, MO or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, MD) is added. Beta emissions of samples from individual tissue culture wells are counted in a LKB BetaPlate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD) and data is expressed as counts per minute of
Figure imgf000065_0002
incorporated into cells from each tissue culture well. Activity of each G-CSF receptor agonist proteins preparation is quantitated by measuring cell proliferation ( H-thymιdιne incorporation) induced by graded concentrations of G-CSF receptor agonist. Typically, concentration ranges from 0.05 pM - 105 pM are quantitated in these assays. Activity is determined by measuring the dose of G-CSF receptor agonist protein which provides 50% of maximal proliferation (EC50 = 0.5 x (maximum average counts per minute of
Figure imgf000066_0001
incorporated per well among triplicate cultures of all concentrations of G-CSF receptor agonists tested - background proliferation measured by 3H-thymιdιne incorporation observed in triplicate cultures lacking any factor) . This EC50 value is also equivalent to 1 unit of bioactivity. Every assay is performed with native mterleukm-3 and G-CSF as reference standards so that relative activity levels could be assigned.
Typically, the G-CSF receptor agonist proteins were tested n a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions. Activity for each sample was determined by the concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample was determined from EC50 estimations for the sample and the standard as indicated above.
Other n vitro cell based prolif rati n assays
Other in vitro cell based proliferation assays, known to those skilled in the art, may also be useful to determine the activity of the G-CSF receptor agonists in a similar manner as described in the AML 193.1.3 cell proliferation assay. Transfected cell lines:
Cell lines, such as BHK or the murine pro B cell line Baf/3, can be transfected with a colony stimulating factor receptor, ..uch as the human G-CSF receptor which the cell line does not have. These transfected cell lines can be used to determine the activity of the ligand of which the receptor has been transfected.
EXAMPLE 1
Construction of PMON348
The new N-terminus/C-terminus gene in pMON3485 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:7) and L-ll start (SEQ ID NO:3) . Fragment Stop was created and amplified from G-CSF Ser17 sequence in the plasmid, pMON13037 (WO 95/21254), using the primer set, 38 stop (SEQ ID NO:8) and L-ll stop (SEQ ID NO:4) . The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 39 start (SEQ ID NO:7) and 38 stop (SEQ ID NO:8) .
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and HindiII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI) . The plasmid, pMON3934 (derivative of
PMON3359) , was digested with restriction endonucleases Hindlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel-purified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD) . Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3485.
BHK cells were transfected with the plasmid, pMON3485, for protein expression and bioassay.
The plasmid, pMON3485 containing the gene sequence of (SEQ ID NO:25), encodes the following amino acid sequence:
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr (SEQ ID NO:43)
EXAMPLE 2
Construction of PMON3486
The new N-terminus/C-terminus gene in pMON3486 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in the plasmid, pMON13037, using the primer set, 97 start (SEQ ID NO:9) and L-ll start (SEQ ID NO:3) . Fragment Stop was created and amplified from G-CSF Ser-1-7 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:10) and L-ll stop (SEQ ID NO:4) . The full-length new N terminus/C-termmus G-CSF Ser17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 97 start (SEQ ID NO: 9) and 96 stop (SEQ ID NO:10) .
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hmdlll and gel-purified using a Magic DNA Clean-up System kit. The plasmid, pMON3934, was digested with restriction endonucleases H dlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel- purified The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5α cells. Transformant bacteria were selected on ampicillm-contammg plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmiα was designated pMON3486.
BHK cells were transfected with the plasmid, pMON3486, for protein expression and bioassay.
The plasmid, pMON3486 containing the gene sequence of (SEQ ID NO:26), encodes the following ammo acid sequence:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu
Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala
Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser (SEQ ID NO:44)
EXAMPLE 3
Construction of PMON3487
The new N-terminus/C-terminus gene in pMON3487 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in the plasmid, pMON13037, using the primer set, 126 start (SEQ ID NO: 11) and L-ll start (SEQ ID NO:3) . Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:12) and L-ll stop (SEQ ID NO:4) . The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 126 start (SEQ ID NO:11) and 125 stop (SEQ ID NO:12) . The resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hindlll and purified using a Magic DNA Clean-up System kit. The plasmid, pMON3934, was digested with restriction endonucleases Hindlll and Ncol, resulting in an approxima ely 3800 base pair vector fragment, and gel- purified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E . col i strain DH5α cells.
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert . The resulting plasmid was designated
PMON3487.
BHK cells were transfected with the plasmid, pMON3487, for protein expression and bioassay. The plasmid, pMON3487 containing the gene sequence of (SEQ ID NO:27), encodes the following amino acid sequence:
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu
Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala
Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala
Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys
Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu
Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp
Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly (SEQ ID NO:45)
EXAMPLE 4
Construction of PMON3488
The new N-terminus/C-terminus gene in pMON3488 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in the plasmid, pMON13037, using the primer set, 133 start (SEQ ID NO:13) and L-ll start (SEQ ID NO:3) . Fragment Stop was created and amplified from G-CSF Ser-*-7 sequence in the plasmid, pMON13037 using the primer set, 132 stop (SEQ ID NO:14) and L-ll stop (SEQ ID NO:4) . The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 133 start (SEQ ID NO:13) and 132 stop (SEQ ID NO:14) .
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hindlll and purified using a Magic DNA Clean-up System kit. The plasmid, pMON3934, was digested with restriction endonucleases Hindlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel- puπfied. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5ccells.
Transfoπri t bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3488.
BHK cells were transfected with the plasmid, pMON3488, for protein expression and bioassay.
The plasmid, pMON3488 containing the gene sequence of (SEQ ID NO:28), encodes the following ammo acid sequence:
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro (SEQ ID NO:46)
EXAMPLE 5
Construction of PMON3489
The new N-terminus/C-termmus gene in pMON3489 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence the plasmid, pMON13037, using the primer set, 142 start (SEQ ID NO:15) and L-ll start (SEQ ID NO:3) . Fragment Stop was created and amplified from G-CSF Ser-'-7 sequence in pMON13037 using the primer set, 141 stop
(SEQ ID NO:16) and L-ll stop (SEQ ID NO:4) . The full-length new N termmus/C-termmus G-CSF Ser17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 142 start (SEQ ID NO:15) and 141 stop (SEQ ID NO:16) .
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases Ncol and Hmdlll and purifiod using a Magic DNA Clean-up System kit. The plasmid, pMON3934, was digested with restriction endonucleases H dlll and Ncol, resulting in an approximately 3800 base pair vector fragment, and gel- purified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5ocells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated PMON3489.
BHK cells were transfected with the plasmid, pMON3489, for protein expression and bioassay.
The plasmid, pMON3489 containing the gene sequence of (SEQ ID NO:29), encodes the following ammo acid sequence:
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu
Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala
Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu
Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin
Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin
Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly
Ala Met Pro Ala Phe Ala (SEQ ID NO:47)
EXAMPLE 6
Construction of PMON3490
The new N-terminus/C-terminus gene in pMON3490 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 39 start (SEQ ID NO:7) and P-bl start (SEQ ID NO:5) . Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMONl3037 using the primer set, 38 stop (SEQ ID NO:8) and P-bl stop (SEQ ID NO:6) . Fragment Start was digested with restriction endonuclease Ncol, and Fragment Stop was digested with restriction endonuclease Hindlll. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pMON3934.
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated PMON3490. BHK cells were transfected with the plasmid, pMON3490, for protein expression and bioassay.
The plasmid, pMON3490 containing the gene sequence of (SEQ ID NO:30), encodes the following amino acid sequence:
Figure imgf000075_0001
EXAMPLE 7
Construction of PMON3491
The new N-terminus/C-terminus gene in pMON3491 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 97 start (SEQ ID NO: 9) and P-bl start (SEQ ID NO:5) . Fragment Stop was created and amplified from G-CSF Ser^7 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:10) and P-bl stop (SEQ ID NO:6) . Fragment Start was digested with restriction endonuclease Ncol, and Fragment Stop was digested with restriction endonuclease Hindlll. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pMON3934. A portion of the ligation reaction was used to transform E. coli strain DH5α cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert . The resulting plasmid was designated pMON3491. BHK cells were transfected with the plasmid, pMON3491, for protein expression and bioassay.
The plasmid, pMON3491 containing the gene sequence of (SEQ ID N0:31) , encodes the following amino acid sequence:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser
Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp
Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala
Gly Cys Le I Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser (SEQ ID NO:49)
EXAMPLE 8
Construction of PMON3492
The new N-terminus/C-terminus gene in pMON3492 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 126 start (SEQ ID NO:ll) and P-bl start (SEQ ID NO:5) . Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:12) and P-bl stop (SEQ ID NO:6) . Fragment Start was digested w.th restriction endonuclease Ncol, and Fragment Stop was digested with restriction endonuclease Hindlll. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pM0N3934. A portion of the ligation reaction was used to transform E. coli strain DH5α cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3492.
BHK cells were transfected with the plasmid, pMON3492, for protein expression and bioassay.
The plasmid, pMON3492 containing the gene sequence of
(SEQ ID NO:32), encodes the following amino acid sequence:
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe
Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin
Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly
Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu
Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin
Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro
Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr
He Trp Gin Gin Met Glu Glu Leu Gly (SEQ ID NO:50)
EXAMPLE 9
Construction of PMON3493
The new N-terminus/C-terminus gene in pMON3493 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 133 start (SEQ ID NO:13) and P-bl start (SEQ ID NO:5) . Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO: 14) and P-bl stop (SEQ ID NO: 6) . Fragment Start was digested with restriction endonuclease Ncol, and Fragment Stop was digested with restriction endonuclease Hindlll. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hmdlll vector fragment of pMON3934. A portion of the ligation reaction was used to transform E. coli strain DH5α cells. Transformant bacteria were selected on ampicillm-contammg plates. Plasmid DNA was isolated and sequenced to confirm the correct insert . The resulting plasmid was designated pMON3493.
BHK cells were transfected with the plasmid, pMON3493, for prote*- i expression and bioassay.
The plasmid, pMON3493 containing the gene sequence of (SEQ ID NO:33), encodes the following ammo acid sequence:
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu
His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu
Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Al Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu
Leu Gly Met Ala Pro Ala Leu Gin Pro (SEQ ID NO:51)
EXAMPLE 10
Construction of PMON3494 The new N-termmus/C-termmus gene in pMON3494 was created using Method II as described m Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, u3ing the primer set, 142 start (SEQ ID N0:15) and P-bl start (SEQ ID N0:5) . Fragment Stop was created and amplified from G-CSF Ser---7 sequence 1 ' pMON13037 using the primer set, 141 stop (SEQ ID NO:16) and P-bl stop (SEQ ID NO:6) . Fragment Start was digested with restriction endonuclease Ncol, and Fragment Stop was digested with restriction endonuclease Hmdlll. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair Ncol-Hindlll vector fragment of pMON3934. A portion of the ligation reaction was used to transform E. coli strain DH5α cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert . The resulting plasmid was designated pMON3494. BHK cells were transfected with the plasmid, pMON3494, for protein expression and bioassay.
The pxasmid, pMON3494 containing the gene sequence of
(SEQ ID NO:34) , encodes the following amino acid sequence:
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser
Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala
Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly
Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala (SEQ ID NO: 52)
Examples 11-20
The genes encoding the G-CSF receptor agonists of Examples 1-10 were excised from the BHK vectors as a Ncol/Hindlll fragment and ligated with the ~ 3630 base pair Ncol/Hindlll vector fragment of pMON2341 (WO 94/12638) . The resulting plasmids (Examples 11-20) are indicated in Table 4. The plasmids were transformed into E. coli strain JM101 cells and expression of the G-CSF receptor agonist protein was evaluated. The proteins expressed are the same as those expressed in the parental BHK expression vector except the proteins were immediately preceded by a Methionine-Alanine dipeptide and the Methionine is processed off by methionine aminopeptidase. Overnight growths of cells (20 Klett units) were inoculated in lOmL of minimal M9 medium supplemented with vitamin Bl and trace minerals and incubated with shaking at 37°C until initial Klett readings of -120 units were obtained. At 120 Klett units 50uL of lOmg/ L nalidixic acid was added. Four hours post-induction, a 1ml aliquot was removed for protein expression analysis by SDS-PAGE. Cells were also examined using light microscopy for the presence of inclusion bodies. Only pMON3450 and pMON3455 had significant expression levels of the G-CSF receptor agonist protein. In an effort to improve expression levels of G-CSF receptor agonists, the 5' end of the genes were re- engineered to incorporate AT-rich codons and E. coli preferred codons between the unique Ncol and Nhel restriction endonuclease recognition sites (Examples 21-28) .
Figure imgf000081_0001
Example 21
Construction of PMON25184
The complementary pair of synthetic oligomers, 141for.seq (SEQ ID NO:23) and 141rev.seq (SEQ ID NO:24) , (Midland Certified Reagent Co., Midland TX) were annealed by heating 2ug of each synthetic oligomer in a 20ul reaction mixture containing 20mM Tris-HCl (7.5), lOmM MgCl2 , and 50mM NaCl, at 80°C for 5 minutes, and allowing the mixture to slowly cool to ambient temperature (approximately 45 minutes) . When properly annealed the oligomers create an Ncol site at the 5' end and a Nhel site at the 3' end. Approximately 15 ng of the annealed oligomer pair was ligated with the gel-purified - 4120 base pair Ncol/Nhel vector frajment of pMON3454 (-molar ratio of 10:1) . The resulting gene, had seven codon changes at the 5' end of the gene. The ligation reaction was used to transform E. coli strain DH5α and the desired codon changes were confirmed by
DNA sequence analysis. The resulting plasmid was designated pMON25184 Plasmid, pMON25184 containing the gene sequence of (SEQ ID NO:38), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3454.
Example 22
Construction of PMON25188
The complementary pair of synthetic oligomers,
141for.seq (SEQ ID NO:23) and 141rev.seq (SEQ ID NO:24), (Midland Certified Reagent Co., Midland TX) were annealed by heating 2ug of each synthetic oligomer in a 20ul reaction mixture containing 20mM Tris-HCl (7.5), lOmM MgCl2 , and 50mM NaCl, at 80°C for 5 minutes, and allowing the mixture to slowly cool to ambient temperature (approximately 45 minutes) . When properly annealed the oligomers create an Ncol site at the 5' end and a Nhel site at the 3' end. Approximately 15ng of the annealed oligomer pair was ligated with the ~ 4110 base pair Ncol/Nhel gel-purified pMON3459 (-molar ratio of 10:1) . The ligation mixture was used to transform E. coli strain DH5α and the desired codon changes were confirmed by DNA sequence analysis. The resulting plasmid was designated pMON25188. The resulting gene, had seven codon changes at the 5' end of the gene. Plasmid,
PMON25188 containing the gene sequence of (SEQ ID NO:42), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3459. Example 23
Construction of PMON25183
pMON25183 was constructed using an overlapping PCR primer method. The synthetic oligomers, 132for.seq (SEQ ID NO:321 and 132rev.seq (SEQ ID NO:22), encode the Ncol and Nhel restriction recognition sequence, respectively. Amplified DNA was generated by the DNA polymerase chain amplification method using the PCR Optimizer Kit
(Invitrogen) . The PCR reactions were performed using the manufacturer's recommended conditions using 5X buffer B (300mM Tris-HCl pH8.5, 75 mM (NH4)2S04, lOmM MgCl2) for seven cycles consisting of 94°C for 1', 65°C for 2', and 72°C for 2', followed by 20 cycles of 94°C for 1', and 72°C for 3', and a final cycle of 7 minutes at 72°C using a Perkin Elmer Model 480 DNA thermal cycler (Perkin Elmer) . The reaction product was desalted using Centri-Sep spin columns (Princeton Separations) following the manufacturer's recommended protocol, digested with Ncol/Nhel, and gel purified from TAE-agarose gels using Gene Clean (Bio 101) and the DNA product was eluted in dH2θ The purified PCR product was ligated with the - 4090 base pair Ncol/Nhel pMON3453 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in
Example 21. The resulting plasmid was designated pMON25183. The resulting gene, had 14 codon changes at the 5' end of the gene. Plasmid, pMON25183 containing the gene sequence of (SEQ ID NO:37), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3453.
Exampl 24
Construction of PMON25187 pM0N25187 was constructed using an overlapping PCR primer method. The synthetic oligomers, 132for.seq (SEQ ID N0:21) and 132rev.seq (SEQ ID N0:22), encode the Ncol and Nhel restriction recognition sequence, respectively.
Amplified DNA was generated by the DNA polymerase chain amplification method using the PCR Optimizer Kit (Invitrogen) . The PCR reactions were performed using the manufacturer's recommended conditions, in 5X buffer B for seven cycles consisting of 94°C for 1', 65°C for 2', and
72°C for 2', followed by 20 cycles of 94°C for 1', and 72°C for 3', and a final cycle of 7 minutes at 72°C using a Perkin Elmer Model 480 DNA thermal cycler (Perkin Elmer) . The reaction product was desalted using Centri-Sep spin columns (P mceton Separations) following the manufacturer's recommended protocol, digested with Ncol/Nhel, and gel purified from TAE-agarose gels using Gene Clean (Bio 101) and the DNA product was eluted in dH20. The purified PCR product was ligated with the - 4080 base pair Ncol/Nhel pMON3458 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25187. The resulting gene, had 14 codon changes at the 5' end of the gene. Plasmid, pMON25187 containing the gene sequence of (SEQ ID NO:41), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3458.
E ample 25
Construction of PMON25182
pMON25182 was constructed using the overlapping PCR primer approach described m Example 23. The synthetic oligomer primers 125for.seq (SEQ ID NO:19) and 125rev.seq (SEQ ID NO:20) were used in the PCR reaction. The PCR reaction conditions were identical to those used in Example 23 except the annealing temperature for the first seven cycles was 60°C. The purified PCR product was ligated with - 4070 base pair Ncol/Nhel pMON3452 vector fragment.
Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25182. The resulting gene, had 19 codon changes at the 5' end of the gene. Plasmid, pMON25182 containing the gene sequence of (SEQ ID NO:36), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3452.
Example 26
Construction of PMON25186
pMON25186 was constructed using the overlapping PCR primer approach described in Example 23. The synthetic oligomer primers 125for.seq (SEQ ID NO:19) and 125rev.seq (SEQ ID NO:20) were used in the PCR reaction. The PCR reaction conditions were identical to those used m Example 23 except the annealing temperature for the first seven cycles was 60°C. The purified PCR product was ligated with the - 4060 base pair Ncol/Nhel pMON3457 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25186. The resulting gene, had 19 codon changes at the 5' end of the gene. Plasmid, pMON25186 containing the gene sequence of (SEQ ID NO:40), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3457. Examples 27
Construction of p_.ON2._181
pMON25181 was constructed using PCR to amplify a DNA fragment from pMON3451 as the template using the oligomers 96for.seq (SEQ ID N0:17) and 96rev.seq (SEQ ID N0:18) . The oligomer 96for.seq was designed to create six codon changes. The PCR reaction conditions were the same as described in Example 25, except lOng of pMON3451 plasmid DNA was added. The purified PCR product was ligated with the - 3980 base pair Ncol/Nhel pMON3451 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated DMON25181. The resulting gene, had 6 codon changes at the 5' end of the gene. Plasmid, pMON25181 containing the gene sequence of (SEQ ID NO:35), DNA was retransformed into E. coli strain JMIOI cells for protein expression. The protein expressed is the same as that expressed from pMON3451.
Exam les 28
Construction of PMON25185
pMON25185 was constructed using PCR to amplify a DNA fragment from pMON3451 as the template using the oligomers 96for.seq (SEQ ID NO:17) and 96rev.seq (SEQ ID NO:18) . The oligomer 9697for.seq was designed to create six codon changes. Tie PCR reaction conditions were the same as described in Example 25, except lOng of pMON3456 plasmid DNA was added. The purified PCR product was ligated with the - 3970 base pair Ncol/Nhel pMON3456 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25185. The resulting gene, had 6 codon changes at the 5' end of the gene. Plasmid, pMON25185 containing the gene sequence of (SEQ ID NO: 39) , DNA was retransformed into E. coli strain JMIOI cells for protein expression. The protein expressed is the same as that expressed from pMON3456.
EXAMPLE 29
The G-CSF amino acid substitution variants of the present invention were made using PCR mutagenesis techniques as described in WO 94/12639 and WO 94/12638. These and other variants (i.e. amino acid substitutions, insertions or deletions and N-terminal or C-terminal extensions) could also be made, by one skilled in the art, using a variety of other methods including synthetic gene assembly or site- directed mutagenesis (see Taylor et al . , Nucl . Acids Res . , 13:7864-8785, 1985; Kunkel et al. , Proc . Natl . Acad . Sci . USA, 82:488-492, 1985; Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, WO 94/12639 and WO 94/12638) . These substitutions can be made one at a time or in combination with other amino acid substitutions, and/or deletions, and/or insertions and/or extensions. After sequence verification of the changes, the plasmid DNA can be transfected into an appropriate mammalian cell, insect cell or bacterial strain such as E. coli for production. Known variants of G-CSF, which are active, include substitutions at positions 1 (Thr to Ser, Arg or Gly, 2 (Pro to Leu), 3 (Leu to Arg or Ser) and 17 (Cys to Ser) and deletions of amino acids 1-11 (Kuga et al . Biochemicla and Biophysi cal Research Comm. 159:103-111, 1989) . It is understood that these G-CSF amino acid substitution variants could serve as the template sequence for the rearrangement of the amino acid sequence as described in the other examples. Bioactivity determination of G-CSF am o acid substitution variants .
The G-CSF am o acid substitution variants were assayed m the Baf/3 cell l ne, transfected with the human G-CSF receptor, proliferation assay to determine their bioactivity relative to native G-CSF. The G-CSF variants tested and their relative bioactivity are shown n Table 5. A "+" indicates that the activity was comparable to native G-CSF and "-" indicates that the activity was significantly decreased or not detected.
TABLE 5
CELL PROLIFERATION ACTIVITY OF G-CSF VARIANTS IN BAF/3 CELL LINE TRANSFECTED WITH THE HUMAN G-CSF RECEPTOR
Figure imgf000089_0001
TABLE 5 con .
Figure imgf000090_0001
44 Pro Gin 44 Pro Trp 44 Pro Gly 44 Pro Thr 46 Glu Ala 46 Glu Arg 46 Glu Phe 46 Glu He 47 Leu Thr 49 Leu Phe 49 Leu Arg 49 Leu Ser TABLE 5 cont
Figure imgf000091_0001
TABLE 5 cont
Figure imgf000092_0001
159 Ser Gly 162 Glu Gly 162 Glu Trp 162 Glu Leu 163 Val Arg 163 Val Ala 163 Val Gly 165 Tyr Cys not determined 169 Ser Leu 169 Ser Cys 169 Ser Arg 170 His Arg 170 His Ser EXAMLPLE 3 0 - 37
Examples 30-37 were made in a similar manner as described in Example 6 using the plasmid pMON13037 as the template and the oligonucleotide primers indicated in Table 6. The resulting gene and the designated plasmid pMON # and the protein encoded are indicated in Table 6.
TABLE 6
Figure imgf000094_0001
The G-CSF receptor agonist genes in pMON3640, pMON3461, pMON3462, pMON3463, pMON3464, pMON3465, pMON3466 and pMON3467 were transferred to an E. coli expression vector, pMON2341, as an Ncol/Hindlll restriction fragment, resulting in the plasmids pMON3468, pMON3469, pMON3470, pMON3471, pMON3472, pMON3473, pMON3474 and pMON3498 respectively.
EXAMPLE 38
The plasmid, pMON3468, resulted in low expression levels in E. coli of the desired G-CSF receptor agonist. The 5 ' end of the gene was redesigned to use codon selection that was AT rich to increase expression levels. The oligonucleotides, Z4849AT.for (SEQ ID NO:84) and Z4849AT.rev (SEQ ID NO:85), were used to re-engineer the gene. The resulting plasmid, pMON3499, containing the gene (SEQ ID NO: 94) encodes the G-CSF receptor agonist of (SEQ ID NO:103) .
EXAMPLE 39
The G-CSF receptor agonists were assayed in the Baf/3 cell line, transfected with the human G-CSF receptor, (Baf/3-G-CSF) proliferation assay to determine their bioactivity relative to native G-CSF. The activity of the receptor agonists is shown in Table 7.
TABLE 7 G-CSF receptor agonist activity in Baf/3-G-CSF cell proliferation assay
Figure imgf000096_0001
Additional techniques for the construction of the variant genes, recombinant protein expression , protein purification, protein characterization, biological activity determination can be found m WO 94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO 95/20977, WO 95/21254 which are hereby incorporated by reference in their entirety.
All references, patents or applications cited herein are incorporated by reference in their entirety as if written herein.
Various other examples will be apparent to the person skilled the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.
SEQUENCE LISTING
I) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: G. D. Searle & Co.
(B) STREET: P. 0. Box 5110
(C) CITY: Chicago
(D) STATE: Illinois
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP) : 60680
(G) TELEPHONE: (708)470-6501 (H) TELEFAX: (708)470-6881
(A) NAME: Monsanto Company
(B) STREET: 800 North Lindbergh Boulevard
(C) CITY: St. Louis
(D) STATE: Missouri
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP) : 63167
(G) TELEPHONE: (314)647-3131 (H) TELEFAX: (314)694-5435
(ii) TITLE OF INVENTION: G-CSF Receptor Agonists
(iii) NUMBER OF SEQUENCES: 103
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: US 2907 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/004,382
(B) FILING DATE: 05-OCT-1995
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 1
(D) OTHER INFORMATIO :/note= "Xaa at position 1 is Thr, Ser, Arg, Tyr or Gly;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATIONS
(D) OTHER INFORMATION: /note- "Xaa at position 2 is Pro or Leu; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATIONS
(D) OTHER INFORMATION: /note= "Xaa at position 3 is Leu, Arg, Tyr or Ser,-"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13
(D) OTHER INFORMATION:/note= "Xaa at position 13 is Phe, Ser, His, Thr or Pro;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 16
(D) OTHER INFORMATION:/note= "Xaa at position 16 is Lys, Pro, Ser, thr or His;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 17
(D) OTHER INFORMATION: /note= "Xaa at position 17 is Cys, Ser, Gly, Ala, He, Tyr or Arg;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 18
(D) OTHER INFORMATION: /note= "Xaa at position 18 is Leu, Thr, Pro, His, He or Cys;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:22
(D) OTHER INFORMATIO :/note= "Xaa at position 22 is Arg, Tyr, Ser, Thr or Ala;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:24
(D) OTHER INFORMATION: /note- "Xaa at position 24 is He, Pro, Tyr or Leu; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site ( B ) LOCATION : 27
(D) OTHER INFORMATION : /note- " Xaa at position 27 is Asp , or Gly; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:30
(D) OTHER INFORMATION: /note= "Xaa at position 30 is Ala, He, Leu or Gly; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:34
(D) OTHER INFORMATION: /note= "Xaa at position 34 is Lys or Ser; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:36
(D) OTHER INFORMATION: /note- "Xaa at position 36 is Cys or Ser; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:42
(D) OTHER INFORMATION:/note= "Xaa at position 42 is Cys or Ser; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:43
(D) OTHER INFORMATION: /note- "Xaa at position 43 is His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 44
(D) OTHER INFORMATION:/note= "Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gin, or Thr;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:46
(D) OTHER INFORMATION: /note= "Xaa at position 46 is Glu, Arg, Phe, Arg, He or Ala;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:47
(D) OTHER INFORMATION: /note- "Xaa at position 47 is Leu or Thr; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site ( B ) LOCATION : 49
( D) OTHER INFORMATION : /note= " Xaa at position 49 i s Leu , Phe , Arg or Ser ; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 50
(D) OTHER INFORMATION: /note= "Xaa at position 50 is Leu, He, His, Pro or Tyr;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:54
(D) OTHER INFORMATION: /note= "Xaa at position 54 is Leu or His; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 64
(D) OTHER INFORMATION: /note= "Xaa at position 64 is Cys or Ser; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:67
(D) OTHER INFORMATIO :/note= "Xaa at position 67 is Gin, Lys, Leu or Cys; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:70
(D) OTHER INFORMATION: /note= "Xaa at position 70 is Gin, Pro, Leu, Arg or Ser; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:74
(D) OTHER INFORMATION: /note= "Xaa at position 74 is Cys or Ser,- "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:104
(D) OTHER INFORMATION: /note- "Xaa at position 104 is Asp, Gly or Val; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:108
(D) OTHER INFORMATION:/note= "Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gin or Gly;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:115
(D) OTHER INFORMATION: /no e= "Xaa at position 115 is Thr, His, Leu or Ala,-"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:120
(D) OTHER INFORMATION: /note= "Xaa at position 120 is Gin, Gly, Arg, Lys or His"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:123
(D) OTHER INFORMATION: /note= "Xaa at position 123 is Glu, Arg, Phe or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:144
(D) OTHER INFORMATION: /note= "Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gin or Glu;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:146
(D) OTHER INFORMATION: /note- "Xaa at positionl46 is Arg or Gin;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:147
(D) OTHER INFORMATION: /note= "Xaa ap position 147 is Arg or Gin;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:156
(D) OTHER INFORMATION: /note= "Xaa at position 156 is His, Gly or Ser; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:159
(D) OTHER INFORMATION: /note- "Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly;"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:162
(D) OTHER INFORMATION:/note= "Xaa at position 162 is Glu, Leu, Gly or Trp; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 163
(D) OTHER INFORMATION: /note= "Xaa at position 163 is Val, Gly, Arg or Ala; "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 169
(D) OTHER INFORMATION:/note= "Xaa at position 169 is Arg, Ser, Leu, Arg or Cys,-"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 170
(D) OTHER INFORMATION:/note= "Xaa at position 170 is His, Arg or Ser; "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Xaa Xaa Gly Pro Ala Ser Ser Leu Pro Gin Ser Xaa Leu Leu Xaa 1 5 10 15
Xaa Xaa Glu Gin Val Xaa Lys Xaa Gin Gly Xaa Gly Ala Xaa Leu Gin 20 25 30
Glu Xaa Leu Xaa Ala Thr Tyr Lys Leu Xaa Xaa Xaa Glu Xaa Xaa Val 35 40 45
Xaa Xaa Gly His Ser Xaa Gly He Pro Trp Ala Pro Leu Ser Ser Xaa 50 55 60
Pro Ser Xaa Ala Leu Xaa Leu Ala Gly Xaa Leu Ser Gin Leu His Ser 65 70 75 80
Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 85 90 95
Pro Glu Leu Gly Pro Thr Leu Xaa Thr Leu Gin Xaa Asp Val Ala Asp 100 105 110
Phe Ala Xaa Thr He Trp Gin Gin Met Glu Xaa Xaa Gly Met Ala Pro 115 120 125
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Xaa 130 135 140
Gin Xaa Xaa Ala Gly Gly Val Leu Val Ala Ser Xaa Leu Gin Xaa Phe 145 150 155 160
Leu Xaa Xaa Ser Tyr Arg Val Leu Xaa Xaa Leu Ala Gin Pro 165 170
(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Gly Gly Ser
1
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single <D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GCTCTGAGAG CCGCCAGAGC CGCCAGAGGG CTGCGCAAGG TGGCGTAGAA CGCG 54
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CAGCCCTCTG GCGGCTCTGG CGGCTCTCAG AGCTTCCTGC TCAAGTCTTT AGAG 54 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GGGCTGCGCA AGGTGGCG 18
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ACACCATTGG GCCCTGCCAG C 21
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GATCGACCAT GGCTTACAAG CTGTGCCACC CC 32
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CGATCGAAGC TTATTAGGTG GCACACAGCT TCTCCT 36
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GATCGACCAT GGCTCCCGAG TTGGGTCCCA CC 32
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CGATCGAAGC TTATTAGGAT ATCCCTTCCA GGGCCT 36
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GATCGACCAT GGCTATGGCC CCTGCCCTGC AG 32
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CGATCGAAGC TTATTATCCC AGTTCTTCCA TCTGCT 36
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GATCGACCAT GGCTACCCAG GGTGCCATGC CG 32
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CGATCGAAGC TTATTAGGGC TGCAGGGCAG GGGCCA 36
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GATCGACCAT GGCTTCTGCT TTCCAGCGCC GG 32
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
CGATCGAAGC TTATTAGGCG AAGGCCGGCA TGGCAC 36
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
ATATCCATGG CTCCGGAACT GGGTCCAACT CTG 33
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
ACCTCCAGGA AGCTCTGCAG ATGG 24
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 65 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
TATATCCATG GCTATGGCTC CAGCTCTGCA ACCAACTCAA GGTGCAATGC CAGCATTTGC 60
ATCTG 65
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
GATGGCTAGC AACCAGAACA CCACCTGCAC GACGTTGAAA AGCAGATGCA AATGCTGGCA 60
TTG 63
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TATATCCATG GCTACTCAAG GTGCTATGCC AGCTTTTGCT TCTGCTTTTC AACGTCG 57
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GCAGATGGCT AGCAACCAGA ACACCACCTG CACGACGTTG AAAAGCAGAA GCAAAAGC 58
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CATGGCTTCT GCTTTTCAAC GTCGTGCAGG TGGTGTTCTG GTTG 44
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CTAGCAACCA GAACACCACC TGCACGACGT TGAAAAGCAG AAGC 44
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC 60
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC 120
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC 180
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC 240
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC 300
ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT 360
CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCTCTGGC 420
GGCTCTGGCG GCTCTCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG GAAGATCCAG 480
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT AATAA 525
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS: ; 1 1
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC 60
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG 120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT 180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC 240
TCTGGCGGCT CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGGAAG 300
ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA GCTGTGCCAC 360
CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC CCTGAGCTCC 420
TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG CGGCCTTTTC 480
CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCT AATAA 525
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT CGCCTCTGCT 60
TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG 120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCTCTGGCG GCTCTGGCGG CTCTCAGAGC 180
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG 240
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC 300
TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA 360
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG 420
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC 480
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAT AATAA 525
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG GGCAGGAGGG 60
GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC 120
CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC TCTCAGAGCT TCCTGCTCAA GTCTTTAGAG 180 CAAGTGAGGA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC 240
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT 300
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT 360
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 420
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG 480
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCT AATAA 525
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 525 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC 60
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTCTGG CGGCTCTGGC 120
GGCTCTCAGA GCTTCCTGCT CAAGTCTTTA GAGCAAGTGA GGAAGATCCA GGGCGATGGC 180
GCAGCGCTCC AGGAGAAGCT GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAGCTGGTG 240
CTGCTCGGAC ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC 300
CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA CCAGGGGCTC 360
CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC ACTGCAGCTG 420 GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG AATGGCCCCT 480
GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT AATAA 525
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC 60
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC 120
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC 180
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC 240
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC 300
ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT 360
CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA 420
TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA 480
AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC 60
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG 120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT 180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC 240
ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA 300
GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG 360
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC 420
CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC 480
GGCCTTTTCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (Synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT CGCCTCTGCT 60
TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG 120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCACACCAT TGGGCCCTGC CAGCTCCCTG 180
CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA 240
GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG 300
CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG 360
CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG 420
CAGGCCCTGG AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC 480
GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGATA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG GGCAGGAGGG 60
GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC 120
CTTGCGCAGC CCACACCATT GGGCCCTGCC AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG 180 TCTTTAGAGC AAGTGAGAAA GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT 240
GCCACCTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC 300
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC 360
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC 420
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC 480
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC 60
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT 120
GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG 180
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG 240
GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 300
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC 360
CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA 420 CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA 480
ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC 60
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG 120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT 180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC 240
ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA 300
GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG 360
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC 420
CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC 480
GGCCTTTTCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCTA A 531
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT TGCATCTGCT 60
TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG 120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCACACCAT TGGGCCCTGC CAGCTCCCTG 180
CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA 240
GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG 300
CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG 360
CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG 420
CAGGCCCTGG AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC 480
GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGATA A 531
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG TGCAGGTGGT 60
GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC 120
CTTGCGCAGC CCACACCATT GGGCCCTGCC AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG 180
TCTTTAGAGC AAGTGAGAAA GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT 240
GCCACCTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC 300
CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC 360
CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC 420
CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC 480
ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCTA A 531
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
ATGGCTTCTG CTTTTCAACG TCGTGCAGGT GGTGTTCTGG TTGCTAGCCA TCTGCAGAGC 60
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT 120
GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG 180 GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG 240
GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 300
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC 360
CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA 420
CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA 480
ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTA A 531
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC 60
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG 120
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT 180
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC 240
TCTGGCGGCT CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGAAAG 300
ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA GCTGTGCCAC 360
CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC CCTGAGCTCC 420 TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG CGGCCTTTTC 480
CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCT AA 522
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT TGCATCTGCT 60
TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG 120
TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCTCTGGCG GCTCTGGCGG CTCTCAGAGC 180
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG 240
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC 300
TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA 360
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG 420
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC 480
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAT AA 522
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG TGCAGGTGGT 60
GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC 120
CTTGCGCAGC CCTCTGGCGG CTCTGGCGGC TCTCAGAGCT TCCTGCTCAA GTCTTTAGAG 180
CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC 240
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT 300
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT 360
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 420
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG 480
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCT AA 522
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: ATGGCTTCTG CTTTTCAACG TCGTGCAGGT GGTGTTCTGG TTGCTAGCCA TCTGCAGAGC 60
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTCTGG CGGCTCTGGC 120
GGCTCTCAGA GCTTCCTGCT CAAGTCTTTA GAGCAAGTGA GAAAGATCCA GGGCGATGGC 180
GCAGCGCTCC AGGAGAAGCT GTGTGCCACC TACAAGCTGT GCCACCCCGA GGAGCTGGTG 240
CTGCTCGGAC ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC 300
CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA CCAGGGGCTC 360
CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC ACTGCAGCTG 420
GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG AATGGCCCCT 480
GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT AA 522
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu 1 5 10 15
Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin 20 25 30
Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin 35 40 45
Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr 50 55 60 Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp 65 70 75 80
Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin 85 90 95
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly 100 105 110
Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg 115 120 125
Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin 130 135 140
Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp
145 150 155 160
Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr 165 170
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp
1 5 10 15
Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 20 25 30
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 35 40 45
Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe
50 55 60
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly 65 70 75 80
Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val 85 90 95 Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala 100 105 110
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
115 120 125
Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu 130 135 140
Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr 145 150 155 160
Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 165 170
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala 1 5 10 15
Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser 20 25 30
Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly 35 40 45
Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin 50 55 60
Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu 65 70 75 80
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro 85 90 95
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly 100 105 110
Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu
115 120 125 Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr 130 135 140
Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met 145 150 155 160
Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 165 170
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
TGGAATAAAA AAGAGAGAAG GAAAAGGATA GAAGAAGGGG GGGGAAGGGA GAAAAGGCAA 60
TTCGGAGGTA ACGAAGAAGC GGTGGGAAGG GGTATGAAAA AAATTTGGTG GGTAAAAG 118
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 1 5 10 15
Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin
20 25 30
Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu 35 40 45 Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 50 55 60
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 65 70 75 80
Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 85 90 95
Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu 100 105 110
Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu 115 120 125
Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala 130 135 140
Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu 145 150 155 160
Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala 165 170
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu 1 5 10 15
Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin 20 25 30
Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin 35 40 45
Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr 50 55 60
Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp 65 70 75 80 Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin 85 90 95
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly 100 105 110
Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg 115 120 125
Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser 130 135 140
Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He 145 150 155 160
Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr 165 170
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp
1 5 10 15
Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 20 25 30
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 35 40 45
Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 50 55 60
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro 65 70 75 80
Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu 85 90 95
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 100 105 110 Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 115 120 125
Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 130 135 140
Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu 145 150 155 160
Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 165 170
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala
1 5 10 15
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 20 25 30
Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin 35 40 45
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 50 55 60
Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu 65 70 75 80
Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 85 90 95
Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser 100 105 110
Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 115 120 125
Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He 130 135 140 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 145 150 155 160
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly 165 170
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala 1 5 10 15
Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser 20 25 30
Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala 35 40 45
Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg 50 55 60
Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr 65 70 75 80
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu 85 90 95
Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin 100 105 110
Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin 115 120 125
Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr 130 135 140
Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp 145 150 155 160
Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 165 170 (2) INFORMATION FOR SEQ ID NO: 52:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH. 174 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS- single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
1 5 10 15
Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin 20 25 30
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 35 40 45
Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu 50 55 60
Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80
Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser 85 90 95
Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 100 105 110
Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He 115 120 125
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 130 135 140
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala 145 150 155 160
Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala 165 170
(2) INFORMATION FOR SEQ ID NO: 53:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 1 5 10 15
Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 20 25 30
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 35 40 45
Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 50 55 60
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro 65 70 75 80
Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu 85 90 95
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 100 105 110
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu
115 120 125
Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 130 135 140
Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu 145 150 155 160
Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 165 170
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala 1 5 10 15
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 20 25 30
Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin 35 40 45
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 50 55 60
Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu 65 70 75 80
Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 85 90 95
Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser 100 105 110
Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 115 120 125
Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He 130 135 140
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 145 150 155 160
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly 165 170
(2) INFORMATION FOR SEQ ID NO: 55:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala 1 5 10 15 Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser 20 25 30
Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala 35 40 45
Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg 50 55 60
Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr 65 70 75 80
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu 85 90 95
Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin 100 105 110
Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin 115 120 125
Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr 130 135 140
Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp 145 150 155 160
Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 165 170
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 1 5 10 15
Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin 20 25 30
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 35 40 45 Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu 50 55 60
Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80
Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser 85 90 95
Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 100 105 110
Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He 115 120 125
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 130 135 140
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala 145 150 155 160
Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala 165 170
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp
1 5 10 15
Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 20 25 30
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 35 40 45
Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 50 55 60
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly 65 70 75 80 Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val 85 90 95
Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala 100 105 110
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser 115 120 125
Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu 130 135 140
Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr 145 150 155 160
Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 165 170
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 169 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala 1 5 10 15
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 20 25 30
Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin 35 40 45
Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu 50 55 60
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 65 70 75 80
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His 85 90 95
Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala 100 105 110 Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu 115 120 125
Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly 130 135 140
Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr 145 150 155 160
He Trp Gin Gin Met Glu Glu Leu Gly 165
(2) INFORMATION FOR SEQ ID NO: 59:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 am o acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala 1 5 10 15
Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser 20 25 30
Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly 35 40 45
Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin 50 55 60
Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu 65 70 75 80
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro 85 90 95
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly 100 105 110
Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu 115 120 125
Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr 130 135 140 Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met 145 150 155 160
Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 165 170
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 1 5 10 15
Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin 20 25 30
Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu 35 40 45
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 50 55 60
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 65 70 75 80
Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 85 90 95
Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu 100 105 110
Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu 115 120 125
Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala 130 135 140
Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu 145 150 155 160
Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala 165 170 (2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Gly Gly Gly Ser Gly Gly Gly Ser 1 5
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Ser Gly Gly Ser Gly Gly Ser
1 5 (2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Glu Phe Gly Asn Met 1 5
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Glu Phe Gly Gly Asn Met
1 5
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Glu Phe Gly Gly Asn Gly Gly Asn Met
1 5 (2) INFORMATION FOR SEQ ID NO: 67:
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH. 7 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Gly Gly Ser Asp Met Ala Gly 1 5
(2) INFORMATION FOR SEQ ID NO: 68:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 68:
GATCGACCAT GGCTCTGCTC GGACACTCTC TG 32
(2) INFORMATION FOR SEQ ID NO: 69:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION. /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: CGATCGAAGC TTATTACACC AGCTCCTCGG GGTGGC 36
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
GATCGACCAT GGCTCAACTC CATAGCGGCC TT 32
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
CGATCGAAGC TTATTAGCTC AAGCAGCCTG CCAGCT 36
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72.
GATCGACCAT GGCTCTTTTC CTCTACCAGG GG 32
(2) INFORMATION FOR SEQ ID NO: 73:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
CGATCGAAGC TTATTAGCCG CTATGGAGTT GGCTCA 36
(2) INFORMATION FOR SEQ ID NO: 74:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
GATCGACCAT GGCTCTCTAC CAGGGGCTCC TG 32
(2) INFORMATION FOR SEQ ID NO: 75:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
CGATCGAAGC TTATTAGAAA AGGCCGCTAT GGAGTT 36
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
GATCGACCAT GGCTGCCCTG GAAGGGATAT CC 32
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
CGATCGAAGC TTATTACTGC AGGAGCCCCT GGTAGA 36
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
GATCGACCAT GGCTGACTTT GCCACCACCA TC 32
(2) INFORMATION FOR SEQ ID NO: 79:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
CGATCGAAGC TTATTAGGCG ACGTCCAGCT GCAGTG 36
(2) INFORMATION FOR SEQ ID NO: 80:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
GATCGACCAT GGCTATCTGG CAGCAGATGG AA 32
(2) INFORMATION FOR SEQ ID NO: 81:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
CGATCGAAGC TTATTAGGTG GTGGCAAAGT CGGCGA 36
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
GATCGACCAT GGCTCAGCAG ATGGAAGAAC TG 32
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
CGATCGAAGC TTATTACCAG ATGGTGGTGG CAAAGT 36
(2) INFORMATION FOR SEQ ID NO: 84: (l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
CATGGCTTTG TTAGGACATT CTTTAGGTAT TCCATGGGCT CCTCTGAGCT 50
(2) INFORMATION FOR SEQ ID NO. 85:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85.
CAGAGGAGCC CATGGAATAC CTAAAGAATG TCCTAACAAA 40
(2) INFORMATION FOR SEQ ID NO: 86:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: ATGGCTCTGC TCGGACACTC TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC 60
CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG 120
GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG 180
CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG 240
GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC 300
CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC 360
GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC 420
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG 480
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
ATGGCTCAAC TCCATAGCGG CCTTTTCCTC TACCAGGGGC TCCTGCAGGC CCTGGAAGGG 60
ATATCCCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC 120
ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG 180
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT 240 AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC 300
ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA 360
GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG 420
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC 480
CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
ATGGCTCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 60
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG 120
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC 180
TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC 240
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT 300
GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG 360
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG 420 GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 480
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
ATGGCTCTCT ACCAGGGGCT CCTGCAGGCC CTGGAAGGGA TATCCCCCGA GTTGGGTCCC 60
ACCTTGGACA CACTGCAGCT GGACGTCGCC GACTTTGCCA CCACCATCTG GCAGCAGATG 120
GAAGAACTGG GAATGGCCCC TGCCCTGCAG CCCACCCAGG GTGCCATGCC GGCCTTCGCC 180
TCTGCTTTCC AGCGCCGGGC AGGAGGGGTC CTGGTTGCTA GCCATCTGCA GAGCTTCCTG 240
GAGGTGTCGT ACCGCGTTCT ACGCCACCTT GCGCAGCCCA CACCATTGGG CCCTGCCAGC 300
TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGAAAGAT CCAGGGCGAT 360
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 420
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 480
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION. /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
ATGGCTGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC ACTGCAGCTG 60
GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG AATGGCCCCT 120
GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA 180
GGAGGGGTCC TGGTTGCTAG CCATCTGCAG AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA 240
CGCCACCTTG CGCAGCCCAC ACCATTGGGC CCTGCCAGCT CCCTGCCCCA GAGCTTCCTG 300
CTCAAGTCTT TAGAGCAAGT GAGAAAGATC CAGGGCGATG GCGCAGCGCT CCAGGAGAAG 360
CTGTGTGCCA CCTACAAGCT GTGCCACCCC GAGGAGCTGG TGCTGCTCGG ACACTCTCTG 420
GGCATCCCCT GGGCTCCCCT GAGCTCCTGC CCCAGCCAGG CCCTGCAGCT GGCAGGCTGC 480
TTGAGCCAAC TCCATAGCGG CCTTTTCCTC TACCAGGGGC TCCTGCAGTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 91:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: ATGGCTGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT GGCCCCTGCC 60
CTGCAGCCCA CCCAGGGTGC CATGCCGGCC TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA 120
GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC 180
CACCTTGCGC AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC 240
AAGTCTTTAG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG 300
TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC 360
ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG 420
AGCCAACTCC ATAGCGGCCT TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA 480
TCCCCCGAGT TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
ATGGCTATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG 60
GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT 120
AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC 180
ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA 240 GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG 300
CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC 360
CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC 420
GGCCTTTTCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCCC CGAGTTGGGT 480
CCCACCTTGG ACACACTGCA GCTGGACGTC GCCGACTTTG CCACCACCTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
ATGGCTCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC 60
ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT 120
CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA 180
TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA 240
AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC 300
CACCCCGAGG AGCTGGTGCT GCTCGGACAC TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC 360
TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT 420 TTCCTCTACC AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC 480
TTGGACACAC TGCAGCTGGA CGTCGCCGAC TTTGCCACCA CCATCTGGTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 534 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
ATGGCTTTGT TAGGACATTC TTTAGGTATT CCATGGGCTC CTCTGAGCTC CTGCCCCAGC 60
CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG 120
GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG 180
CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG 240
GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC 300
CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC 360
GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC 420
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG 480
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA 534
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys 1 5 10 15
Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser 20 25 30
Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 35 40 45
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 50 55 60
Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 65 70 75 80
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 85 90 95
Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 100 105 110
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro 115 120 125
Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu 130 135 140
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 145 150 155 160
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 165 170
(2) INFORMATION FOR SEQ ID NO: 96:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
Figure imgf000158_0001
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu 1 5 10 15
Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu 20 25 30
Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu 35 40 45
Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe 50 55 60
Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His 65 70 75 80
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala 85 90 95
Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu 100 105 110
Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala 115 120 125
Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu 130 135 140
Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser 145 150 155 160
Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser 165 170
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro
1 5 10 15 Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe 20 25 30
Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala 35 40 45
Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin 50 55 60
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu 65 70 75 80
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu 85 90 95
Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu 100 105 110
Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu 115 120 125
Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly 130 135 140
His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin 145 150 155 160
Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly 165 170
(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE : protein
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 98 :
Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu
1 5 10 15
Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr 20 25 30
Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin 35 40 45 Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg 50 55 60
Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val 65 70 75 80
Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro 85 90 95
Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val 100 105 110
Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala 115 120 125
Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
130 135 140
Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu 145 150 155 ' 160
Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe 165 170
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
1 5 10 15
Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu 20 25 30
Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro 35 40 45
Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala 50 55 60
Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His 65 70 75 80 Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser 85 90 95
Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly 100 105 110
Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro 115 120 125
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro 130 135 140
Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser 145 150 155 160
Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin 165 170
(2) INFORMATION FOR SEQ ID NO: 100:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala 1 5 10 15
Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala 20 25 30
Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser 35 40 45
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr 50 55 60
Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser 65 70 75 80
Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu 85 90 95
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu 100 105 110 Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro 115 120 125
Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly 130 135 140
Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro 145 150 155 160
Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 165 170
(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 1 5 10 15
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala 20 25 30
Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser 35 40 45
Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala 50 55 60
Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg
65 70 75 80
Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr 85 90 95
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu 100 105 110
Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin 115 120 125
Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin 130 135 140 Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr 145 150 155 160
Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr 165 170
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin 1 5 10 15
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly 20 25 30
Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg 35 40 45
Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser 50 55 60
Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He 65 70 75 80
Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys 85 90 95
Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He 100 105 110
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala 115 120 125
Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu 130 135 140
Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp 145 150 155 160
Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp 165 170 (2) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys
1 5 10 15
Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser 20 25 30
Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 35 40 45
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 50 55 60
Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 65 70 75 80
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 85 90 95
Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 100 105 110
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro 115 120 125
Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu 130 135 140
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 145 150 155 160
Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 165 170

Claims

WHAT IS CLAIMED IS:
1. A human G-CSF receptor agonist polypeptide, comprising a modified G-CSF ammo acid sequence of the Formula:
1 10
Xaa Xaa Xaa Gly Pro Ala Ser Ser Leu Pro Gin Ser Xaa
20 Leu Leu Xaa Xaa Xaa Glu Gin Val Xaa Lys Xaa Gin Gly Xaa Gly
30 40 Ala Xaa Leu Gin Glu Xaa Leu Xaa Ala Thr Tyr Lys Leu Xaa Xaa
50
Xaa Glu Xaa Xaa Val Xaa Xaa Gly His Ser Xaa Gly He Pro Trp 60 70
Ala Pro Leu Ser Ser Xaa Pro Ser Xaa Ala Leu Xaa Leu Ala Gly
80
Xaa Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu
90 100
Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu
110 Xaa Thr Leu Gin Xaa Asp Val Ala Asp Phe Ala Xaa Thr He Trp
120 130
Gin Gin Met Glu Xaa Xaa Gly Met Ala Pro Ala Leu Gin Pro Thr 140
Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Xaa Gin Xaa Xaa Ala
150 160
Gly Gly Val Leu Val Ala Ser Xaa Leu Gin Xaa Phe Leu Xaa Xaa
170
Ser Tyr Arg Val Leu Xaa Xaa Leu Ala Gin Pro (SEQ ID NO:l)
wherein
Xaa at position 1 is Thr, Ser, Arg, Tyr or Gly;
Xaa at position 2 is Pro or Leu;
Xaa at position 3 is Leu, Arg, Tyr or Ser;
Xaa at position 13 is Phe, Ser, His, Thr or Pro; Xaa at position 16 is Lys, Pro, Ser, Thr or His; Xaa at position 17 is Cys, Ser, Gly, Ala, He, Tyr or Arg;
Xaa at position 18 is Leu, Thr, Pro, His, He or Cys;
Xaa at position 22 is Arg, Tyr, Ser, Thr or Ala;
Xaa at position 24 is He, Pro, Tyr or Leu; Xaa at position 27 is Asp, or Gly;
Xaa at position 30 is Ala, He, Leu or Gly;
Xaa at position 34 is Lys or Ser;
Xaa at position 36 is Cys or Ser;
Xaa at position 42 is Cys or Ser; Xaa at position 43 is His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu;
Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gin, or Thr;
Xaa at position 46 is Glu, Arg, Phe, Arg, He or Ala; Xaa at position 47 is Leu or Thr;
Xaa at position 49 is Leu, Phe, Arg or Ser;
Xaa at position 50 is Leu, He, His, Pro or Tyr;
Xaa at position 54 is Leu or His;
Xaa at position 64 is Cys or Ser; Xaa at position 67 is Gin, Lys, Leu or Cys;
Xaa at position 70 is Gin, Pro, Leu, Arg or Ser;
Xaa at position 74 is Cys or Ser;
Xaa at position 104 is Asp, Gly or Val;
Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gin or Gly; Xaa at position 115 is Thr, His, Leu or Ala;
Xaa at position 120 is Gin, Gly, Arg, Lys or His
Xaa at position 123 is Glu, Arg, Phe or Thr
Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gin or Glu;
Xaa at position 146 is Arg or Gin; Xaa at position 147 is Arg or Gin;
Xaa at position 156 is His, Gly or Ser;
Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly;
Xaa at position 162 is Glu, Leu, Gly or Trp;
Xaa at position 163 is Val, Gly, Arg or Ala; Xaa at position 169 is Arg, Ser, Leu, Arg or Cys;
Xaa at position 170 is His, Arg or Ser;
wherein optionally 1-11 amino acids from the N-terminus and 1-5 from the C-terminus can be deleted;
wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-terminus at amino acids;
38 -39 62 - 63 123-124 39 -40 63 - 64 124-125 40-41 64- 65 125-126 41-42 65 - 66 126-127 42-43 66-67 128-129
43-44 67-68 128-129
45-46 68-69 129-130
48-49 69-70 130-131 49-50 70-71 131-132
52-53 71-72 132-133
53-54 91-92 133-134
54-55 92-93 134-135
55-56 93-94 135-136 56-57 94-95 136-137
57-58 95-96 137-138
58-59 96-97 138-139
59-60 97-98 139-140
60-61 98-99 140-141 61-62 99-100 141-142 or 142-143; and
said G-CSF receptor agonist polypeptide may optionally be immediately preceded by (me hionine--*-) , (alanine----) or (methionine . alanine"-*) .
2. The G-CSF receptor agonist polypeptide, as recited in claim 1, wherein said linker is selected from the group consisting of;
GlyGlyGlySer (SEQ ID NO:2);
GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:61);
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:62);
ΞerGlyGlySerGlyGlySer (SEQ ID NO: 63); GluPheGlyAsnMet (SEQ ID NO:64);
GluPheGlyGlyAsnMet (SEQ ID NO:65);
GluPheGlyGlyAsnGlyGlyAsnMet (SEQ ID NO: 66); and
GlyGlySerAspMetAlaGly (SEQ ID NO: 67).
3. The G-CSF receptor agonist polypeptide of claim
1, selected from the group consisting of;
Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala
Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe
Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu
Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe
Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr (SEQ ID N0:48);
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met
Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly Asp
Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala
Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser (SEQ ID NO:49);
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe
Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His
Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin
Figure imgf000169_0001
Leu Lys Ser Leu Glu Gin Val Arg Lys He Gin Gly
Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu
Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu
Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin
Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro
Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr
He Trp Gin Gin Met Glu Glu Leu Gly (SEQ ID NO:50);
Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu
Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser
Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu
Gly He Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro (SEQ ID NO: 51); and
Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
Figure imgf000170_0001
4. A nucleic acid molecule comprising a DNA sequence encoding the G-CSF receptor agonist polypeptide of claim 1.
5. A nucleic acid molecule comprising a DNA sequence encoding the G-CSF receptor agonist polypeptide of claim 2.
6. A nucleic acid molecule comprising a DNA sequence encoding the G-CSF receptor agonist polypeptide of claim 3.
7. A nucleic acid molecule of claim 6 selected from group consisting of;
1 ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC
51 TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC
101 AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG
151 GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT 201 GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC
251 AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC
301 ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT
351 TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC
401 ACCTT CGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC 451 TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC
501 AGCGCTCCAG GAGAAGCTGT GTGCCACCTA ATAA (SEQ ID NO: 30) ;
1 ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC 51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT
201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG
251 GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA 301 GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC 351 CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC
401 TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG
451 CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG
501 GCTCCTGCAG GCCCTGGAAG GGATATCCTA ATAA (SEQ ID NO: 31) ;
1 ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT
51 CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG 151 CCCACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA
201 GTCTTTAGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG
251 AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG
301 CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG
351 CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT 401 TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
451 GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC
501 CATCTGGCAG CAGATGGAAG AACTGGGATA ATAA (SEQ ID NO: 32) ;
1 ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG
51 GGCAGGAGGG GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCACACCATT GGGCCCTGCC
151 AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG TCTTTAGAGC AAGTGAGAAA
201 GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT GCCACCTACA 251 AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
301 CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG
351 CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC
401 AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG
451 CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA 501 ACTGGGAATG GCCCCTGCCC TGCAGCCCTA ATAA (SEQ ID NO:33) ;
Figure imgf000171_0001
351 CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC
401 TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG
451 CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG
501 GCTCCTGCAG GCCCTGGAAG GGATATCCTA A (SEQ ID NO: 35) ;
1 ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT
51 TGCATCTGCT TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC
101 TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG 151 CCCACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA
201 GTCTTTAGAG CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG
251 AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG
301 CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG
351 CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT 401 TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
451 GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC
501 CATCTGGCAG CAGATGGAAG AACTGGGATA A (SEQ ID NO: 36) ;
1 ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG
51 TGCAGGTGGT GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT
101 CGTACCGCGT TCTACGCCAC CTTGCGCAGC CCACACCATT GGGCCCTGCC
151 AGCTCCCTGC CCCAGAGCTT CCTGCTCAAG TCTTTAGAGC AAGTGAGAAA
201 GATCCAGGGC GATGGCGCAG CGCTCCAGGA GAAGCTGTGT GCCACCTACA 251 AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC
301 CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG
351 CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC
401 AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG
451 CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA 501 ACTGGGAATG GCCCCTGCCC TGCAGCCCTA A (SEQ ID NO: 37) ;
1 ATGGCTTCTG CTTTTCAACG TCGTGCAGGT GGTGTTCTGG TTGCTAGCCA
51 TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC 101 AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC
151 AAGTCTTTAG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA
201 GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC
251 TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC
301 AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT 351 TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT
401 TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC
451 ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC
501 CACCCAGGGT GCCATGCCGG CCTTCGCCTA A (SEQ ID NO:38);
1 ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC
51 CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC
101 CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC
151 CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT 201 GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC TCTGGCGGCT
251 CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA AGTGAGAAAG
301 ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA 351 GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC
401 CCTGGGCTCC CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC
451 TGCTTGAGCC AACTCCATAG CGGCCTTTTC CTCTACCAGG GGCTCCTGCA
501 GGCCCTGGAA GGGATATCCT AA (SEQ ID NO:39);
Figure imgf000173_0001
8. A method of producing a G-CSF receptor agonist polypeptide comprising: growing under suitable nutrient conditions, a host cell transformed or transfected with a replicable vector comprising said nucleic acid molecule of claim 4, 5, 6 or 7 in a manner allowing expression of said G-CSF receptor agonist polypeptide and recovering said G-CSF receptor agonist polypeptide.
9. A composition comprising; a G-CSF receptor agonist polypeptide according to claim 1, 2, or 3; and a pharmaceutically acceptable carrier.
10. A composition comprising; a G-CSF receptor agonist polypeptide according to claim 1, 2, or 3; a colony stimulating factor; and a pharmaceutically acceptable carrier.
11. A composition comprising; a G-CSF receptor agonist polypeptide according to claim 1, 2, or 3; a colony stimulating factor selected from the group consisting of:
GM-CSF, c-mpl ligand, M-CSF, erythropoietin, IL-1, IL-4, IL- 2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor; and
a pharmaceutically acceptable carrier.
12. A method of stimulating the production of hematopoietic cells in a patient comprising the step of; administering said G-CSF receptor agonist polypeptide of claim 1, 2, or 3 to said patent.
13. A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering said composition of claim 9, 10 or 11 to said patient.
14. A method for selective ex vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells; (b) culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, or 3 ; and
(c) harvesting said cultured cells.
15. A method for selective ex vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells; (b) culturing said separated stem cells with a selected culture medium comprising the composition of claim 9, 10 or 11; and
(c) harvesting said cultured cells.
16. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells; (b) separating stem cells from other cells; (c) culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, or 3;
(d) harvesting said cultured cells; and
(e) transplanting said cultured cells into said patient.
17. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells; (b) separating stem cells from other cells; (c) culturing said separated stem cells with a selected culture medium comprising the composition of claim 9;
(d) harvesting said cultured cells; and
(e) transplanting said cultured cells into said patient.
18. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells; (b) separating stem cells from other cells; (c) culturing said separated stem cells with a selected culture medium comprising the composition of claim 10;
(d) harvesting said cultured cells; and
(e) transplanting said cultured cells into said patient.
19. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells; (b) separating stem cells from other cells; (c) culturing said separated stem cells with a selected culture medium comprising the composition of claim
11;
(d) harvesting said cultured cells; and
(e) transplanting said cultured cells into said patient.
20. A method of human gene therapy, comprising the steps of;
(a) removing stem cells from a patient;
(b) separating said stem cells from other cells; (c) culturing said separated stem cells with a selected culture medium comprising the hematopoietic protein of claim 1, 2, or 3 ;
(d) introducing DNA into said cultured cells;
(e) harvesting said transduced cells; and (f) transplanting said transduced cells into said patient .
21. A method of human gene therapy, comprising the steps of; (a) removing stem cells from a patient; (b) separating said stem cells from other cells;
(c) culturing said separated stem cells with a selected media comprising the composition of claim 9; (d) introducing DNA into said cultured cells;
(e) harvesting said transduced cells; and
(f) transplanting said transduced cells into said patient.
22. A method of human gene therapy, comprising the steps of;
(a) removing stem cells from a patient;
(b) separating said stem cells from other cells;
(c) culturing said separated stem cells with a selected media comprising the composition of claim 10;
(d) introducing DNA into said cultured cells;
(e) harvesting said transduced cells; and
(f) transplanting said transduced cells into said patient.
23. A method of human gene therapy, comprising the steps of;
(a) removing stem cells from a patient; (b) separating said stem cells from other cells;
(c) culturing said separated stem cells with a selected media comprising the composition of claim 11;
(d) introducing DNA into said cultured cells; (e) harvesting said transduced cells; and
(f) transplanting said transduced cells into said patient.
24. A method of claim 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 wherein said stem cells are isolated from peripheral blood.
PCT/US1996/015935 1995-10-05 1996-10-04 Novel g-csf receptor agonists WO1997012977A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU73900/96A AU717733B2 (en) 1995-10-05 1996-10-04 Novel G-CSF receptor agonists
JP51445997A JP2002515729A (en) 1995-10-05 1996-10-04 Novel G-CSF receptor agonist
EP96936191A EP0859843A1 (en) 1995-10-05 1996-10-04 Novel g-csf receptor agonists
US08/833,167 US6100070A (en) 1995-10-05 1997-04-04 G-CSF receptor agonists
US09/344,837 US6358505B1 (en) 1995-10-05 1999-06-25 G-CSF receptor agonists

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US483295P 1995-10-05 1995-10-05
US60/004,382 1995-10-05

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US08/833,167 Continuation-In-Part US6100070A (en) 1995-10-05 1997-04-04 G-CSF receptor agonists

Publications (1)

Publication Number Publication Date
WO1997012977A1 true WO1997012977A1 (en) 1997-04-10

Family

ID=21712745

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/015935 WO1997012977A1 (en) 1995-10-05 1996-10-04 Novel g-csf receptor agonists

Country Status (4)

Country Link
EP (1) EP0859843A1 (en)
AU (1) AU717733B2 (en)
CA (1) CA2234042A1 (en)
WO (1) WO1997012977A1 (en)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018905A1 (en) * 1998-09-25 2000-04-06 G.D. Searle & Co. Method of producing permuteins by scanning permutagenesis
DE19860801A1 (en) * 1998-12-30 2000-07-06 Rhein Biotech Proz & Prod Gmbh Recombinant growth factor with the biological activity of a G-CSF (Granulocyte Colony Stimulating Factor)
EP1073683A1 (en) * 1998-04-30 2001-02-07 Tanox, Inc. G-csf receptor agonist antibodies and screening method therefor
US6261550B1 (en) 1993-01-28 2001-07-17 Amgen Inc. G-CSF hybrid molecules and pharmaceutical compositions
WO2002028896A1 (en) * 2000-09-18 2002-04-11 Isis Innovation Limited Analogues of human granulocite colony stimulating factor (g-csf)
US6555660B2 (en) 2000-01-10 2003-04-29 Maxygen Holdings Ltd. G-CSF conjugates
US6646110B2 (en) 2000-01-10 2003-11-11 Maxygen Holdings Ltd. G-CSF polypeptides and conjugates
JP2004508044A (en) * 2000-09-08 2004-03-18 マサチューセッツ　インスティテュート　オブ　テクノロジー G-CSF analog compositions and methods
US6831158B2 (en) 2000-01-10 2004-12-14 Maxygen Holdings Ltd. G-CSF conjugates
WO2006067170A1 (en) * 2004-12-23 2006-06-29 Laboratoires Serono S.A. G-csf polypeptides and uses thereof
US7655766B2 (en) 2005-06-01 2010-02-02 Carsten Germansen Compositions comprising positional isomers of PEGylated G-CSF
US9867777B2 (en) 2010-01-19 2018-01-16 Hanmi Science Co., Ltd. Liquid formulations for long-acting G-CSF conjugate

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0299782A2 (en) * 1987-07-17 1989-01-18 Schering Biotech Corporation Expression vectors for the production of human Granulocyte-Macrophage Colony Stimulation Factor in a mammalian cell host(18.05.92)
EP0396158A1 (en) * 1985-08-23 1990-11-07 Kirin-Amgen, Inc. Production of pluripotent granulocyte colony-stimulating factor
WO1995027732A2 (en) * 1994-04-08 1995-10-19 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Circularly permuted ligands and circularly permuted chimeric molecules

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0396158A1 (en) * 1985-08-23 1990-11-07 Kirin-Amgen, Inc. Production of pluripotent granulocyte colony-stimulating factor
EP0299782A2 (en) * 1987-07-17 1989-01-18 Schering Biotech Corporation Expression vectors for the production of human Granulocyte-Macrophage Colony Stimulation Factor in a mammalian cell host(18.05.92)
WO1995027732A2 (en) * 1994-04-08 1995-10-19 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Circularly permuted ligands and circularly permuted chimeric molecules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEISHA S. MULLINS ET AL.: "Transposition of protein sequences: Circular permutation of Ribonuclease T1", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 116, no. 13, 29 June 1994 (1994-06-29), DC US, pages 5529 - 5533, XP002024483 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6261550B1 (en) 1993-01-28 2001-07-17 Amgen Inc. G-CSF hybrid molecules and pharmaceutical compositions
US7381804B2 (en) 1993-01-28 2008-06-03 Amgen Inc. G-CSF analog compositions and methods
US6632426B2 (en) 1993-01-28 2003-10-14 Amgen Inc. G-CSF analog compositions and methods
US8058398B2 (en) 1993-01-28 2011-11-15 Amgen Inc. Modified G-CSF polypeptide
KR100479790B1 (en) * 1998-04-30 2005-03-30 타녹스 인코퍼레이티드 G-CSF receptor agonist antibodies and screening method therefor
EP1073683A1 (en) * 1998-04-30 2001-02-07 Tanox, Inc. G-csf receptor agonist antibodies and screening method therefor
EP1073683A4 (en) * 1998-04-30 2001-08-22 Tanox Inc G-csf receptor agonist antibodies and screening method therefor
WO2000018905A1 (en) * 1998-09-25 2000-04-06 G.D. Searle & Co. Method of producing permuteins by scanning permutagenesis
DE19860801A1 (en) * 1998-12-30 2000-07-06 Rhein Biotech Proz & Prod Gmbh Recombinant growth factor with the biological activity of a G-CSF (Granulocyte Colony Stimulating Factor)
US6555660B2 (en) 2000-01-10 2003-04-29 Maxygen Holdings Ltd. G-CSF conjugates
US6831158B2 (en) 2000-01-10 2004-12-14 Maxygen Holdings Ltd. G-CSF conjugates
US6646110B2 (en) 2000-01-10 2003-11-11 Maxygen Holdings Ltd. G-CSF polypeptides and conjugates
JP2004508044A (en) * 2000-09-08 2004-03-18 マサチューセッツ　インスティテュート　オブ　テクノロジー G-CSF analog compositions and methods
JP4799803B2 (en) * 2000-09-08 2011-10-26 マサチューセッツ インスティテュート オブ テクノロジー G-CSF analog compositions and methods
WO2002028896A1 (en) * 2000-09-18 2002-04-11 Isis Innovation Limited Analogues of human granulocite colony stimulating factor (g-csf)
US7790174B2 (en) 2004-12-23 2010-09-07 Merck Serono Sa G-CSF polypeptides and uses thereof
US7863016B2 (en) 2004-12-23 2011-01-04 Merck Serono S.A. G-CSF polypeptides and uses thereof
US7863018B2 (en) 2004-12-23 2011-01-04 Merck Serono Sa G-CSF polypeptides and uses thereof
WO2006067170A1 (en) * 2004-12-23 2006-06-29 Laboratoires Serono S.A. G-csf polypeptides and uses thereof
US7655766B2 (en) 2005-06-01 2010-02-02 Carsten Germansen Compositions comprising positional isomers of PEGylated G-CSF
US9867777B2 (en) 2010-01-19 2018-01-16 Hanmi Science Co., Ltd. Liquid formulations for long-acting G-CSF conjugate

Also Published As

Publication number Publication date
AU717733B2 (en) 2000-03-30
AU7390096A (en) 1997-04-28
CA2234042A1 (en) 1997-04-10
EP0859843A1 (en) 1998-08-26

Similar Documents

Publication Publication Date Title
AU734594B2 (en) Circularly permuted polypeptides as novel stem cell factor receptor agonists
US6660257B1 (en) Circular permuteins of flt3 ligand
US6730303B1 (en) Fused G-CSF and IL-3 proteins and uses thereof
AU717733B2 (en) Novel G-CSF receptor agonists
AU705083B2 (en) Multi-functional hematopoietic receptor agonists
US6358505B1 (en) G-CSF receptor agonists
AU725547B2 (en) Multi-functional chimeric hematopoietic receptor agonists
WO1997012985A9 (en) Multi-functional hematopoietic receptor agonists
US6967092B1 (en) Multi-functional chimeric hematopoietic receptor agonists
WO1997012978A1 (en) NOVEL c-mpl RECEPTOR AGONISTS
AU703627B2 (en) Interleuken-3 (IL-3) receptor agonists
MXPA99003875A (en) NOVEL flt-3 RECEPTOR AGONISTS
MXPA99003877A (en) Multi-functional chimeric hematopoietic receptor agonists

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1996936191

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2234042

Country of ref document: CA

Ref country code: CA

Ref document number: 2234042

Kind code of ref document: A

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1996936191

Country of ref document: EP