WO1996023815A1 - Ob gene product antibodies - Google Patents

Ob gene product antibodies Download PDF

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Publication number
WO1996023815A1
WO1996023815A1 PCT/US1996/000957 US9600957W WO9623815A1 WO 1996023815 A1 WO1996023815 A1 WO 1996023815A1 US 9600957 W US9600957 W US 9600957W WO 9623815 A1 WO9623815 A1 WO 9623815A1
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WO
WIPO (PCT)
Prior art keywords
xaa
leu
gin
ser
glu
Prior art date
Application number
PCT/US1996/000957
Other languages
French (fr)
Inventor
William F. Heath, Jr.
Joseph V. Manetta
James E. Shields
Original Assignee
Eli Lilly And Company
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 Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to AU47665/96A priority Critical patent/AU4766596A/en
Publication of WO1996023815A1 publication Critical patent/WO1996023815A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors

Definitions

  • the invention belongs to the general field of immunology as applied to biopharmaceutical research and development.
  • the invention includes both monoclonal and polyclonal antibodies that specifically bind fragments of the human ob gene product as well as certain analogs of the protein fragments.
  • Obesity is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the U.S. population and 27% of the Canadian population are over weight. Kuczmarski, Amer. J. of Clin. Nut. 55: 495S - 502S (1992); Reeder ec al . , Can. Med. Ass. J.. 21- 226-233 (1992). Upper body obesity carries the highest risk factor known for Type II Diabetes and is a significant risk factor for cardiovascular disease and cancer as well. Recent cost estimates for medical complications associated with obesity are $150 billion world wide. The problem has now become so serious that the Surgeon General has begun a national initiative to combat obesity in America.
  • Hypertension, dyslipidemia, and insulin resistance are the primary pathologies associated with obesity. Many studies have demonstrated that weight reduction through diet and exercise dramatically improves these serious medical conditions. Unfortunately, obese individuals generally fail to significantly reduce their body mass through diet and exercise and have a near 95% failure rate. This failure may be due to genetically inherited factors that contribute to increased appetite, preference for high calorie foods, reduced physical activity, reduced lipolytic metabolism, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition. Therefore, new pharmacological agents that can reverse obesity in spite of genetic predisposition are needed. that can reverse obesity in spite of genetic predisposition are needed.
  • the ob lob mouse model of obesity and diabetes is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome.
  • Zhang and co-workers published the positional cloning of the mouse gene linked to this condition.
  • This report discloses the mouse cDNA sequence encoding a 167 amino acid protein that is expressed exclusively in adipose tissue and compares the mouse ob gene product to its human homolog.
  • the report also discloses a point mutation resulting in the conversion of an Arg codon to a stop codon at position 105.
  • This mutant gene is postulated to expresses a truncated protein that lacks the biological function of the complete intact protein.
  • Physiologist have long postulated that excess fat cells laid down through overeating signals the brain that the body is obese which, in turn, causes the body to eat less and burn more fuel.
  • Parabiotic experiments support a "feedback" model and suggest that a circulating peptide hormone may regulate the size of the body's fat depot.
  • the newly disclosed ob gene product mentioned above is now believed to be such a hormone.
  • the present specification discloses novel ob gene product fragments and analogs of such fragments that have the same biological activity as the full protein with varying potencies.
  • the antibodies of the present invention can be used to purify the ob gene product fragments herein disclosed.
  • the claimed antibodies may also be used as a means for diagnosing whether obese patients are expressing the ob gene product.
  • the present invention solves various problems associated with purifying potentially valuable pharmaceutical peptides and with identifying individuals who require therapy using such peptides.
  • the present invention encompasses monoclonal or polyclonal antibodies that specifically bind to proteins and peptides set forth in SEQ ID NOS: 1-31.
  • the invention further includes a method for isolating the aforementioned proteins and peptides comprising immobilizing the antibody onto a surface and contacting the immobilized antibody with a mixture containing one of the proteins or peptides then separating the immobilized protein from the mixture and recovering the protein by removing it from the immobilized antibody complex.
  • Val Leu Gin lie Ala Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu
  • Xaa at position 4 is Gin or Glu; Xaa at position 7 is Gin or Glu; Xaa at position 22 is Gin, Asn, or Asp; Xaa at position 28 is Gin or Glu; Xaa at position 34 is Gin or Glu; Xaa at position 54 is Met, methionine sulfoxide, Leu,
  • Xaa at position 56 is Gin or Glu
  • Xaa at position 62 is Gin or Glu
  • Xaa at position 63 is Gin or Glu
  • Xaa at position 68 is Met, methionine sulfoxide, Leu
  • Xaa at position 72 is Gin, Asn, or Asp
  • Xaa at position 75 is Gin or Glu
  • Xaa at position 78 is Gin, Asn, or Asp
  • Xaa at position 82 is Gin, Asn, or Asp
  • Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or
  • Xaa at position 108 is Asp or Glu; Xaa at position 130 is Gin or Glu; Xaa at position 134 is Gin or Glu; Xaa at position 136 is Met, methionine sulfoxide, Leu,
  • Xaa at position 138 is Gin, Trp, Tyr, Phe, He, Val, or
  • Xaa at position 139 is Gin or Glu
  • Xaa at position 2 is Gin or Glu
  • Xaa at position 5 is Gin or Glu
  • Xaa at position 20 is Asn, Asp or Gin;
  • Xaa at position 26 is Gin or Glu
  • Xaa at position 32 is Gin or Glu
  • Xaa at position 52 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 54 is Gin or Glu
  • Xaa at position 60 is Gin or Glu
  • Xaa at position 61 is Gin or Glu
  • Xaa at position 66 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 70 is Asn, Asp or Gin;
  • Xaa at position 73 is Gin or Glu
  • Xaa at position 76 is Asn, Asp or Gin;
  • Xaa at position 80 is Asn, Asp or Gin
  • Xaa at position 98 is Trp or Gin
  • Xaa at position 128 is Gin or Glu
  • Xaa at position 132 is Gin or Glu
  • Xaa at position 134 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 136 is Trp or Gin; and Xaa at position 137 is Gin or Glu.
  • Xaa at position 17 is Asn, Asp or Gin;
  • Xaa at position 23 is Gin or Glu
  • Xaa at position 29 is Gin or Glu
  • Xaa at position 49 is He, Leu, Met or methionine sulfoxide;
  • Xaa at position 51 is Gin or Glu;
  • Xaa at position 57 is Gin or Glu
  • Xaa at position 58 is Gin or Glu
  • Xaa at position 63 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 67 is Asn, Asp or Gin; Xaa at position 70 is Gin or Glu;
  • Xaa at position 73 is Asn, Asp or Gin;
  • Xaa at position 77 is Asn, Asp or Gin;
  • Xaa at position 95 is Trp or Gin
  • Xaa at position 125 is Gin or Glu
  • Xaa at position 129 is Gin or Glu
  • Xaa at position 131 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 133 is Trp or Gin
  • Xaa at position 134 is Gin or Glu.
  • Xaa at position 7 is Asn, Asp or Gin
  • Xaa at position 13 is Gin or Glu
  • Xaa at position 19 is Gin or Glu
  • Xaa at position 39 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 41 is Gin or Glu
  • Xaa at position 47 is Gin or Glu
  • Xaa at position 48 is Gin or Glu
  • Xaa at position 53 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 57 is Asn, Asp or Gin;
  • Xaa at position 60 is Gin or Glu
  • Xaa at position 63 is Asn, Asp or Gin; ; Xaa at position 67 is Asn, Asp or Gin;
  • Xaa at position 85 is Trp or Gin
  • Xaa at position 115 is Gin or Glu
  • Xaa at position 119 is Gin or Glu
  • Xaa at position 121 is He, Leu, Met or methionine sulfoxide; Xaa at position 123 is Trp or Gin; and Xaa at position 124 is Gin or Glu.
  • Xaa at position 2 is Asn, Asp or Gin;
  • Xaa at position 8 is Gin or Glu
  • Xaa at position 14 is Gin or Glu
  • Xaa at position 34 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 36 is Gin or Glu
  • Xaa at position 42 is Gin or Glu
  • Xaa at position 43 is Gin or Glu
  • Xaa at position 48 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 52 is Asn, Asp or Gin;
  • Xaa at position 55 is Gin or Glu
  • Xaa at position 58 is Asn, Asp or Gin;
  • Xaa at position 62 is Asn, Asp or Gin;
  • Xaa at position 80 is Trp or Gin
  • Xaa at position 110 is Gin or Glu
  • Xaa at position 114 is Gin or Glu
  • Xaa at position 116 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 118 is Trp or Gin
  • Xaa at position 119 is Gin or Glu.
  • Xaa at position 1 is Gin or Glu
  • Xaa at position 21 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 23 is Gin or Glu
  • Xaa at position 29 is Gin or Glu
  • Xaa at position 30 is Gin or Glu
  • Xaa at position 35 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 39 is Asn, Asp or Gin;
  • Xaa at position 42 is Gin or Glu
  • Xaa at position 45 is Asn, Asp or Gin
  • Xaa at position 49 is Asn, Asp or Gin
  • Xaa at position 67 is Trp or Gin
  • Xaa at position 97 is Gin or Glu
  • Xaa at position 101 is Gin or Glu
  • Xaa at position 103 is He, Leu, Met or methionine sulfoxide; Xaa at position 105 is Trp or Gin; Xaa at position 106 is Gin or Glu.
  • Xaa at position 19 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 21 is Gin or Glu
  • Xaa at position 27 is Gin or Glu
  • Xaa at position 28 is Gin or Glu
  • Xaa at position 33 is He, Leu, Met or methionine sulfoxide; ;
  • Xaa at position 37 is Asn, Asp or Gin;
  • Xaa at position 40 is Gin or Glu
  • Xaa at position 43 is Asn, Asp or Gin;
  • Xaa at position 47 is Asn, Asp or Gin;
  • Xaa at position 65 is Trp or Gin
  • Xaa at position 95 is Gin or Glu
  • Xaa at position 99 is Gin or Glu
  • Xaa at position 101 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 103 is Trp or Gin
  • Xaa at position 104 is Gin or Glu.
  • Xaa at position 13 is lie, Leu, Met or methionine sulfoxide
  • Xaa at position 15 is Gin or Glu
  • Xaa at position 21 is Gin or Glu
  • Xaa at position 22 is Gin or Glu
  • Xaa at position 27 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 31 is Asn, Asp or Gin;
  • Xaa at position 34 is Gin or Glu
  • Xaa at position 37 is Asn, Asp or Gin;
  • Xaa at position 41 is Asn, Asp or Gin;
  • Xaa at position 59 is Trp or Gin
  • Xaa at position 89 is Gin or Glu
  • Xaa at position 93 is Gin or Glu
  • Xaa at position 95 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 97 is Trp or Gin
  • Xaa at position 98 is Gin or Glu.
  • Xaa at position 1 is He, Leu, Met, methionine sulfoxide or absent;
  • Xaa at position 3 is Gin or Glu
  • Xaa at position 9 is Gin or Glu
  • Xaa at position 10 is Gin or Glu
  • Xaa at position 15 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 19 is Asn, Asp, or Gin;
  • Xaa at position 22 is Gin or Glu
  • Xaa at position 25 is Asn, Asp, or Gin
  • Xaa at position 29 is Asn, Asp, or Gin;
  • Xaa at position 47 is Trp or Gin
  • Xaa at position 77 is Gin or Glu
  • Xaa at position 81 is Gin or Glu
  • Xaa at position 83 is- He, Leu, Met or methionine sulfoxide
  • Xaa at position 85 is Trp or Gin
  • Xaa at position 86 is Gin or Glu.
  • Xaa at position 1 is Gin or Glu
  • Xaa at position 2 is Gin or Glu
  • Xaa at position 7 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 11 is Asn, Asp, or Gin;
  • Xaa at position 14 is Gin or Glu
  • Xaa at position 17 is Asn, Asp, or Gin
  • Xaa at position 21 is Asn, Asp, or Gin
  • Xaa at position 39 is Trp or Gin
  • Xaa at position 69 is Gin or Glu
  • Xaa at position 73 is Gin or Glu
  • Xaa at position 75 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 77 is Trp or Gin
  • Xaa at position 78 is Gin or Glu.
  • Xaa at position 1 is Asn, Asp or Gin
  • Xaa at position 4 is Gin or Glu
  • Xaa at position 7 is Asn, Asp or Gin;
  • Xaa at position 11 is Asn, Asp or Gin
  • Xaa at position 29 is Trp or Gin
  • Xaa at position 59 is Gin or Glu
  • Xaa at position 63 is Gin or Glu
  • Xaa at position 65 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 67 is Trp or Gin
  • Xaa at position 68 is Gin or Glu.
  • Xaa at position 16 is Trp or Gin; Xaa at position 46 is Gin or Glu;
  • Xaa at position 50 is Gin or Glu
  • Xaa at position 52 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 54 is Trp or Gin
  • Xaa at position 55 is Gin or Glu.
  • Xaa at position 8 is Trp or Gin; Xaa at position 38 is Gin or Glu; Xaa at position 42 is Gin or Glu;
  • Xaa at position 44 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 46 is Trp or Gin
  • Xaa at position 47 is Gin or Glu.
  • Xaa at position 11 is Asn, Asp, or Gin
  • Xaa at position 17 is Gin or Glu
  • Xaa at position 23 is Gin or Glu
  • Xaa at position 43 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 45 is Gin or Glu
  • Xaa at position 51 is Gin or Glu
  • Xaa at position 52 is Gin or Glu
  • Xaa at position 57 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 61 is Asn, Asp, or Gin;
  • Xaa at position 64 is Gin or Glu
  • Xaa at position 67 is Asn, Asp, or Gin
  • Xaa at position 71 is Asn, Asp, or Gin
  • Xaa at position 89 is Trp or Gin
  • Xaa at position 119 is Gin or Glu
  • Xaa at position 123 is Gin or Glu
  • Xaa at position 125 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 127 is Trp or Gin
  • Xaa at position 128 is Gin or Glu.
  • Xaa at position 3 is Gin or Glu
  • Xaa at position 6 is Asn, Asp or Gin;
  • Xaa at position 10 is Asn, Asp or Gin;
  • Xaa at position 28 is Trp or Gin
  • Xaa at position 58 is Gin or Glu
  • Xaa at position 62 is Gin or Glu
  • Xaa at position 64 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 66 is Trp or Gin
  • Xaa at position 67 is Gin or Glu.
  • Xaa at position 17 is Trp or Gin
  • Xaa at position 47 is Gin or Glu
  • Xaa at position 51 is Gin or Glu
  • Xaa at position 53 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 55 is Trp or Gin
  • Xaa at position 56 is Gin or Glu.
  • Xaa at position 6 is Trp or Gin
  • Xaa at position 36 is Gin or Glu
  • Xaa at position 40 is Gin or Glu
  • Xaa at position 42 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 44 is Trp or Gin
  • Xaa at position 45 is Gin or Glu.
  • Xaa at position 4 is Gin or Glu
  • Xaa at position 22 is Gin, Asn, or Asp;
  • Xaa at position 28 is Gin or Glu
  • Xaa at position 34 is Gin or Glu
  • Xaa at position 54 is Met, methionine sulfoxide, Leu,
  • Xaa at position 68 is Met, methionine sulfoxide, Leu,
  • Xaa at position 72 is Gin, Asn, or Asp;
  • Xaa at position 75 is Gin or Glu
  • Xaa at position 78 is Gin, Asn, or Asp
  • Xaa at position 82 is Gin, Asn, or Asp;
  • Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or Leu.
  • Xaa at position 8 is Asp or Glu
  • Xaa at position 30 is Gin or Glu
  • Xaa at position 34 is Gin or Glu
  • Xaa at position 36 is Met, methionine sulfoxide, Leu,
  • Xaa at position 38 is Gin, Trp, Tyr, Phe, He, Val, or
  • Xaa at position 39 is Gin or Glu.
  • Xaa at position 4 is Gin or Glu
  • Xaa at position 7 is Gin or Glu
  • Xaa at position 22 is Asn, Asp or Gin;
  • Xaa at position 28 is Gin or Glu
  • Xaa at position 34 is Gin or Glu
  • Xaa at position 54 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 56 is Gin or Glu
  • Xaa at position 62 is Gin or Glu
  • Xaa at position 63 is Gin or Glu
  • Xaa at position 68 is He, Leu, Met or methionine sulfoxide
  • Xaa at position 72 is Asn, Asp or Gin;
  • Xaa at position 75 is Gin or Glu
  • Xaa at position 78 is Asn, Asp or Gin
  • Xaa at position 82 is Asn, Asp or Gin;
  • Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or Leu.
  • Xaa at position 11 is Gin or Glu
  • Xaa at position 15 is Gin or Glu
  • Xaa at position 17 is Met or methionine sulfoxide, Leu, He, Val, Ala, or Gly;
  • Xaa at position 19 is Trp or Gin
  • Xaa at position 20 is Gin or Glu.
  • Xaa at position 4 is Gin or Glu
  • Xaa at position 7 is Gin or Glu
  • Xaa at position 22 is Asn, Asp or Gin;
  • Xaa at position 28 is Gin or Glu
  • Xaa at position 34 is Gin or Glu
  • Xaa at position 54 is Met, methionine sulfoxide, Leu,
  • Xaa at position 56 is Gin or Glu
  • Xaa at position 62 is Gin or Glu
  • Xaa at position 63 is Gin or Glu
  • Xaa at position 68 is Met, methionine sulfoxide, Leu,
  • Xaa at position 72 is Asn, Asp or Gin;
  • Xaa at position 75 is Gin or Glu
  • Xaa at position 78 is Asn, Asp or Gin;
  • Xaa at position 82 is Asn, Asp or Gin;
  • Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or
  • Xaa at position 2 is Gin or Glu
  • Xaa at position 6 is Gin or Glu
  • Xaa at position 8 is Met, methionine sulfoxide, Leu,
  • Xaa at position 10 is Trp or Gin
  • Xaa at position 11 is Gin or Glu. 1
  • Xaa at position 4 is Gin or Glu
  • Xaa at position 7 is Gin or Glu
  • Xaa at position 22 is Asn, Asp or Gin
  • Xaa at position 28 is Gin or Glu
  • Xaa at position 34 is Gin or Glu
  • Xaa at position 54 is Met, methionine sulfoxide, Leu,
  • Xaa at position 56 is Gin or Glu
  • Xaa at position 62 is Gin or Glu
  • Xaa at position 63 is Gin or Glu
  • Xaa at position 68 is Met, methionine sulfoxide, Leu,
  • Xaa at position 72 is Asn, Asp or Gin
  • Xaa at position 75 is Gin or Glu
  • Xaa at position 78 is Asn, Asp or Gin
  • Xaa at position 82 is Asn, Asp or Gin
  • Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or Leu;
  • Xaa at position 130 is Gin or Glu
  • Xaa at position 134 is Gin or Glu
  • Xaa at position 136 is Met, methionine sulfoxide, Leu,
  • Xaa at position 138 is Trp or Gin.
  • antibody includes antibody fragments which retain their specific binding capacity. Antibodies that specifically bind to SEQ ID NOS: 1-6 are preferred.
  • the protein or peptide must be first be either purified, synthesized or recombinantly expressed. Relatively crude antigenic preparations of the protein or peptide may be used for immunization purposes. However, highly purified protein is required to determine accurately if hybridomas are producing the sought after monoclonal antibodies (mabs) or to assay the antibody titers of immune serum.
  • the peptide fragments herein disclosed can be prepared via chemical synthesis. Also, recombinant DNA techniques may be used to express these proteins.
  • SEQUENCE LISTING may be synthesized by solid-phase methodology utilizing a 430A peptide synthesizer (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) and synthesis cycles supplied by PE-Applied Biosystems. Boc amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical supply houses. Sequential Boc chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides. For the production of C-terminal acids, the corresponding PAM resin is used. Asn, Gin, and Arg are coupled using preformed hydroxy benzotriazole esters. The following side chain protecting groups may be used: Arg, Tosyl Asp, cyclohexyl
  • Boc deprotection may be accomplished with trifluoroacetic acid in methylene chloride. Following completion of the synthesis the peptides may be deprotected and cleaved from the resin with anhydrous hydrogen fluoride (HF) containing 10% meta-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at -5°C to 5°C, preferably on ice for 60 minutes. After removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and lyophilized.
  • HF hydrous hydrogen fluoride
  • LISTING may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the ordinarily skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed, all of which encode any one given peptide set forth in the SEQUENCE LISTING. The methodology of synthetic gene construction is well known in the art. See Brown, et al . (1979) Methods in Enzymology, Academic Press, N.Y. , Vol. 68, pgs. 109-151. A O 9623815
  • DNA sequences can be designed based on the amino acid sequences herein disclosed. Once designed, the sequence itself may be generated using conventional DNA synthesizing apparatus such as the Model 380A or 380B DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404).
  • Model 380A or 380B DNA synthesizers PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404.
  • restriction endonucleases are engineered into either end of the coding region to facilitate isolation from, and integration into, known amplification and expression vectors.
  • the particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The choice of restriction sites are chosen so as to properly orient and position the coding sequence with control sequences to achieve expression of the protein of interest.
  • the promoter-operator region of the synthetic gene is placed in the same sequential orientation with respect to the ATG start codon of the synthetic gene.
  • circular DNA plasmids are useful as vectors in recombinant DNA procedures for .securing high levels of expression of exogenous genes. Having constructed an expression vector for any given polypeptide set forth in the SEQUENCE LISTING, the next step is to place the vector into a suitable cell and thereby construct a recombinant host cell useful for expressing the polypeptide. Techniques for transforming cells with recombinant DNA vectors are well known in the art and may be found in such general references as Maniatis, et al . supra . Host cells made be constructed from either eukaryotic or prokaryotic cells.
  • Prokaryotic host cells generally produce the protein at higher rates and are easier to culture. Proteins which are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Such protein aggregates typically must be solubilized, denatured and refolded using techniques well known in the art. See Kreuger, et al . (1990) in Protein Folding, Gierasch and King, eds. , pgs 136-142, American 9623815
  • antibodies specific for the peptide may be raised by conventional methods that are well known in the art. Repeated injections into a host of choice over a period of weeks or months generally elicits an immune response and results in significant serum titers. Preferred hosts are mammalian species and more highly preferred species are rabbits, goats, sheep and mice. The most preferred species is rabbit. Blood drawn from such immunized animals may be processed by established methods to obtain antiserum (polyclonal antibodies) reactive with the chosen immunogen. The antiserum may then be affinity purified by adsorption to the immunogen according to techniques known in the art.
  • Affinity purified antiserum may be further purified by isolating the immunoglobulin fraction within the antiserum using procedures known in the art.
  • the resulting material will be a heterogeneous population of immunoglobulins that specifically bind to the immunogen.
  • antibodies of interest may also be generated by preparing a semi-synthetic immunogen consisting of a peptide fragment bound to an immunogenic carrier.
  • suitable immunogenic carriers such as bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin are also well known in the art as are techniques for coupling the two proteins.
  • Mab ⁇ of the present invention are readily prepared using isolated peptide fragments (immunogens) set forth in the SEQUENCE LISTING. Methods for producing mabs have been practiced for over 15 years and are well known to those of ordinary skill in the art. Repeated intraperitoneal or subcutaneous injections of immunogen in adjuvant will elicit an immune response in most animals. Hyperimmunized B-lymphocytes are removed from the animal and fused with a suitable fusion partner cell line capable of being cultured indefinitely.
  • Preferred animals whose B-lymphocytes may be hyperimmunized and used in the production of mabs are mammals. More preferred animals are rats and mice and most preferred is the BALB/c mouse strain. Numerous mammalian cell lines are suitable fusion partners for the production of hybridomas. Many such lines are commercially available from the ATCC and commercial suppliers. Preferred fusion partner cell lines are derived from mouse myelomas and the HL-1' Friendly myeloma-653 cell line (Ventrex, Portland, ME) is most preferred.
  • the resulting hybridomas are cultured in a selective growth medium for one to two weeks.
  • Two well known selection systems are available for eliminating unfused myeloma cells, or fusions between myeloma cells, from the mixed hybridoma culture. The choice of selection system depends on the strain of mouse immunized and myeloma fusion partner used.
  • the AAT selection system described by Taggart and Samloff, Science 219. 1228 (1982), may be used; however, the HAT
  • Spent growth medium is then screened for immunospecific mab secretion.
  • Enzyme linked immunosorbant assay (ELISA) procedures are best suited for this purpose; though, radioimmune assays adapted for large volume screening are also acceptable. Multiple screens designed to consecutively pare down the considerable number of irrelevant or less desired cultures should be performed. Cultures that secrete mabs reactive with the immunogen should be screened for cross- reactivity with other proteins such as the immunogenic carrier.
  • Mabs that preferentially bind to the immunogen of interest may be isotyped using commercially available assays. Binding constants may also be determined by well known methods such a Scatchard analysis. Those skilled in the art will then be able to choose a particular antibody in light of these characteristics based on the intended use for the mab.
  • Hybridoma cultures which secrete the sought-after mabs should be sub-cloned several times to establish monoclonality and stability.
  • Well known methods for sub-cloning eukaryotic, non-adherent cell cultures include limiting dilution, soft agarose and fluorescence activated cell sorting techniques the latter of which is exemplified in U.S. Patent No. 4,264,341. After each sub-cloning, the resultant cultures must be re- assayed for antibody secretion and isotype to ensure that a stable antibody-secreting culture has been established. Immunoaffinitv purification ⁇
  • the claimed antibodies are essential reagents for preparing an immunoa finity surface necessary to practice the isolation method of the invention.
  • the immunoaffinity surface is formed by immobilizing a claimed antibody to a substance so that at least some of the antibody binding site remains exposed and capable of binding its antigen.
  • Substances ranging from porous polysaccharide based beads to plastic polymers to inorganic materials are substances to which the antibodies may be immobilized.
  • Preferred substances are those which provide a maximal surface area to volume ratio and do not adversely affect the antibodies or protein fragment.
  • Polysaccharide matrices formed into various sized beads are more highly preferred because they are porous, provide high surface area to volume ratios, are easy to handle and are well known and understood in the biochemical purification art.
  • Immobilization can be accomplished by covalently coupling the antibody directly to the desired substance or by bridging the antibody to the substance.
  • CNBr and carbodiimide coupling of antibodies to polysaccharide based beads such as ⁇ epharose' are illustrative of direct coupling schemes that are consistent with the invention.
  • Direct couplings generally do not orient the antibodies in any particular fashion; however, some types of direct couplings are able to reproducibly orient the antibody on the immobilizing substance.
  • Preferred coupling schemes orient the antibody such that its antigen binding regions remain exposed.
  • One such scheme utilizes the natural carbohydrate found on the heavy chains of the antibody. By first oxidizing the carbohydrate moieties to the corresponding aldehydes then reacting the aldehyde with a primary amino group on the surface, it is possible to link the antibody in an advantageous orientation.
  • bridges are possible and include small organic linkers which covalently bind the antibody to the immobilizing substance. Such spacer arms are acceptable and preferably should not interact with proteins once the bridge has been formed.
  • Protein-A is an example of a specific immunoadsorbant that is capable of orienting the antibody.
  • the above discussion is in no way meant to limit the scope of the invention. Numerous other well known schemes for linking antibodies to immobilizing substances are consistent with the invention.
  • the stream containing the protein fragment to be purified must be contacted with the surface. Batch methods are adequate, though column chromatography is preferable. Batch mode separation can be accomplished by filtering, centrifuging or decanting. When using column chromatography, a simple washing step serves to separate the mixture from the immobilized antibodies.
  • Removing the ob gene protein fragment from the immunoaffinity surface can be accomplished by subjecting the surface-bound complex to a solution capable of disrupting the interactions between the antibody and peptide of interest. The solution preferably will not adversely affect the immunoaffinity surface or the peptide. More preferred solutions are buffered, isotonic, salt solutions at near neutral pH which contain millimolar concentrations of 2-ME.
  • Antibodies labeled with a reporting group can be used to identify the presence of antigens in a variety of milieus.
  • Antibodies labeled with radioisotopes have been used for decades in radioimmune assays to identify, with great precision and sensitivity, the presence of antigens in a variety of biological fluids. More recently, enzyme labeled antibodies have been used as a substitute for radio- labeled antibodies in the popular ELISA.
  • Antibodies of the present invention can be bound to an immobilizing substance such as a polystyrene well or particle and used in immunoassays to determine whether a particular ob gene protein fragment is present in a test sample.
  • a sample is contacted with the immunoaffinity surface and allowed to incubate. After a washing step, the ob gene protein fragment that has bound to the immunoaffinity surface is detected by contacting the surface with another antibody of the invention labeled with a reporting grou .
  • a test sample suspected of containing a protein fragment of interest is dried onto a surface, forming an immobilized test sample.
  • a labeled antibody of the invention is then contacted with the immobilized test sample and allowed to incubate. If the sample contains a protein fragment of interest, the labeled antibody will bind to the immobilized fragment.
  • This method can also be done using an unlabeled antibody of the invention followed by a labeled secondary antibody that binds to an antibody of the invention which has already bound to a protein fragment of interest. After washing, the immobilized test sample is measured to detect the presence of any reporting groups.
  • Reporting groups are typically enzymes such as alkaline phosphatase, horseradish peroxidase or beta-D- galactosidase. Suitable substrates produce a color change when reacted with the enzyme. In so doing, measurements of the color intensity can be quantitated using a spectrophotometer. If the reporting group is a radioisotope, an appropriate gamma or beta ray detecting instrument can be used to quantitate the reporting group. The intensity of the reporting group directly correlates, with the amount of the protein fragment of interest in the test sample.
  • SEQ ID NO:l was prepared and used to make a lysine core multiple antigenic peptide (MAP) according to U.S. Patent Number 5,229,490, herein incorporated by reference, and Applied Biosystems product procedure Number 34, May 1992 (PE-Applied Biosystems, Foster City, CA) .
  • MAP lysine core multiple antigenic peptide
  • a 0.25 mg/ml adjuvant emulsion was prepared by adding 375 ⁇ ls of a 1.0 mg/ml solution of (22-41 MAPS) to 375 ⁇ ls of phospate buffered saline (PBS) and 750 ⁇ ls of Complete Freund's Adjuvant (CFA) . The solution was mixed until fully emulsivied.
  • Six BALB/c mice were then given a 50 ⁇ g primary immunization by subcutaneously injecting 0.2 cc of the above immunogen into each mouse. At three week intervals, the mice are boosted with 50 ⁇ gs of (22-41 MAPS) in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum.
  • SEQ ID NO:2 was prepared by solid phase synthesis on a Model 430A peptide synthesizer (PE-Applied Biosystems, Foster City, CA) using the Boc protecting strategy.
  • the side chain protecting groups were: Asp (Chxl), Glu (OBzl), Ser (Bzl), Thr (Bzl), Lys (Cl-Z), His (BOM), Trp (CHO) , Tyr (Br-Z) , and Arg (Tos) . All except for Asp (Chxl) (Peptides International) were obtained from PE-Applied Biosystems. Each residue was double coupled using either DCC initiated symmetric anhydride or HOBT activation.
  • the modified peptityl resin prepared was treated with 20 ml of 20% piperidine in DMF (dimethyl formamide) at 4°C for 1 hour to remove the Trp(CHO) protection.
  • the modified peptidyl resin was washed several times with CH 2 C1 2 - transfered to a teflon reaction vessel and dried in vacuo.
  • Two ml of m-cresol and a magnetic stir bar were added to the the vessel which was attached to an HF apparatus (Pennisula Laboraties, Inc.), cooled to -78°C, evacuated, and 20-25 ml HF was condensed into the vessel.
  • the reaction mixture was stirred for 60 min in an ice bath and the HF was then removed by vacuum.
  • the peptide residue was suspended in ethyl ether and stirred briefly. The solid material was then filtered using a 60 ml glass fritted filter funnel. After washing the solids twice with ethyl ether, the peptide was solubilized by washing the solids with 40 ml each of 50% aqueous acetic acid and 10% aqueous acetic acid. 100 ul of the combined aqueous filtrate was removed and prepared for standard HPLC analysis using the following conditions:
  • the peptide was then coupled to ovalbumin by first dissolving 3.0 mg of peptide into 1 ml water and solubilizing by adding 2 ⁇ l of 5 N NaOH. A 7.0 mg/ml solution of ovalbumin in 50% PBS/50% water was then prepared. In the dark, 8, 25 ⁇ l aliquotes of a 1:400 dilution of glutaraldehyde in PBS was added to the solublized peptide solution over a 2 hr period at room temperature. The reaction was then allowed to stand overnight at room temperature after which the reaction was quenched by adding 200 ⁇ ls of 1M TRIS® pH 8.5.
  • the final yield of the ovalbumin-coupled peptide solution was 2.1 ml at 1.4 mg/ml.
  • An adjuvant emulsion was then prepared as in Example 1 by adding 230 ⁇ ls of the ovalbumin-coupled peptide to 420 uls PBS and emulsifying in 650 ⁇ ls CFA for a final concentration of approximately 0.25 mg/ml.
  • Five, 18-21 gm, BALB/c mice were then given a primary immunization by subcutaneously injecting 0.2 cc of the above immunogen into each mouse. At three week intervals, the mice are boosted with 50 ⁇ gs of immunogen in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum.
  • IFA Incomplete Freund's Adjuvant
  • Intrachain disulfide bonded SEQ ID NO:3 was prepared and purified in substantial accordance with Example 2 and the general teachings herein disclosed.
  • the peptide was coupled to ovalbumin in substantial accordance with Example 2.
  • the final yield of the ovalbumin-coupled peptide solution was 2.45 ml at 2.0 mg/ml.
  • Six , 18-21 gm, BALB/c mice were then given a primary immunization by subcutaneously injecting 0.2 cc of immunogen, prpared in accordance with the previous examples, into each mouse. At three week intervals, the mice are boosted with 50 ⁇ gs of immunogen in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum.
  • IFA Incomplete Freund's Adjuvant
  • Both forms of SEQ ID NO:4 were prepared in an E. coli B121 strain using standard recombinant DNA techniques herein described. Transformed cells were grown and induced such that the heterologous protein was overexpressed. The cells were then lysed and protein granules were extracted. 50 ⁇ ls of wet granules were diluted in 450 ⁇ ls of water then solublized by adding 5 ⁇ ls of 5N NaOH. The solution was neutralized with 495 ⁇ ls of 1M TRIS®, pH 7.5, and 0.2 ⁇ ls of 2-mercaptoethanol was added to prevent cysteine disulfide bridging.
  • mice were each immunized with 50 ⁇ gs of immunogen as described above. At three week intervals, the mice are boosted with 50 ⁇ gs of immunogen in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum.
  • IFA Incomplete Freund's Adjuvant

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Abstract

Novel monoclonal and polyclonal antibodies that selectively bind to various biologically active fragments and fragment analogs of the mammalian ob gene product are claimed. The invention is useful for purifying the ob gene product, its fragments, and analogs. The claimed antibodies are also useful in diagnostic tests to monitor therapy.

Description

OB GENE PRODUCT ANTIBODIES
The invention belongs to the general field of immunology as applied to biopharmaceutical research and development. The invention includes both monoclonal and polyclonal antibodies that specifically bind fragments of the human ob gene product as well as certain analogs of the protein fragments.
Obesity, especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the U.S. population and 27% of the Canadian population are over weight. Kuczmarski, Amer. J. of Clin. Nut. 55: 495S - 502S (1992); Reeder ec al . , Can. Med. Ass. J.. 21- 226-233 (1992). Upper body obesity carries the highest risk factor known for Type II Diabetes and is a significant risk factor for cardiovascular disease and cancer as well. Recent cost estimates for medical complications associated with obesity are $150 billion world wide. The problem has now become so serious that the Surgeon General has begun a national initiative to combat obesity in America.
Hypertension, dyslipidemia, and insulin resistance are the primary pathologies associated with obesity. Many studies have demonstrated that weight reduction through diet and exercise dramatically improves these serious medical conditions. Unfortunately, obese individuals generally fail to significantly reduce their body mass through diet and exercise and have a near 95% failure rate. This failure may be due to genetically inherited factors that contribute to increased appetite, preference for high calorie foods, reduced physical activity, reduced lipolytic metabolism, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition. Therefore, new pharmacological agents that can reverse obesity in spite of genetic predisposition are needed. that can reverse obesity in spite of genetic predisposition are needed.
The ob lob mouse model of obesity and diabetes is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Zhang and co-workers published the positional cloning of the mouse gene linked to this condition. Zhang et al . Nature 372: 425-32 (1994). This report discloses the mouse cDNA sequence encoding a 167 amino acid protein that is expressed exclusively in adipose tissue and compares the mouse ob gene product to its human homolog. The report also discloses a point mutation resulting in the conversion of an Arg codon to a stop codon at position 105. This mutant gene is postulated to expresses a truncated protein that lacks the biological function of the complete intact protein.
Physiologist have long postulated that excess fat cells laid down through overeating signals the brain that the body is obese which, in turn, causes the body to eat less and burn more fuel. G. R. Hervey, Nature 227: 629-631 (1969). Parabiotic experiments support a "feedback" model and suggest that a circulating peptide hormone may regulate the size of the body's fat depot. The newly disclosed ob gene product mentioned above is now believed to be such a hormone.
The present specification discloses novel ob gene product fragments and analogs of such fragments that have the same biological activity as the full protein with varying potencies. Those skilled in the art will readily appreciate the value and utility of antibodies that selectively and specifically bind to these novel peptides. For example, the antibodies of the present invention can be used to purify the ob gene product fragments herein disclosed. The claimed antibodies may also be used as a means for diagnosing whether obese patients are expressing the ob gene product. Thus, the present invention solves various problems associated with purifying potentially valuable pharmaceutical peptides and with identifying individuals who require therapy using such peptides. The present invention encompasses monoclonal or polyclonal antibodies that specifically bind to proteins and peptides set forth in SEQ ID NOS: 1-31. The invention further includes a method for isolating the aforementioned proteins and peptides comprising immobilizing the antibody onto a surface and contacting the immobilized antibody with a mixture containing one of the proteins or peptides then separating the immobilized protein from the mixture and recovering the protein by removing it from the immobilized antibody complex. There is also claimed a method for quantitating such proteins and peptides in biological samples comprising radio-labeling a purified sample of the protein to be quantitated then mixing the antibody with the biological sample followed by adding a known amount of the labeled protein to the mixture then separating bound antibody from free antibody and quantitating the amount of radio-label in the bound fraction.
The specification describes how to prepare and isolate monoclonal and polyclonal antibodies that specifically bind to the mammalian ob gene product, its analogs and fragments thereof. For purposes of this specification these protein and peptides are defined by the following amino acid sequences:
SEQ ID NO:l 1 5 10 15 Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys
20 Thr lie Val Thr Arg
Figure imgf000005_0001
1 5 10 15
Val Leu Gin lie Ala Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu
20 His Leu Leu Ala Phe Ser Lys Ser
SEQ IP NQ.3, 1 5 10 15
Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys Ser Leu 96/23815
-4 -
20 25 30
Pro Gin Thr Ser Gly Leu Gin Lys Pro Glu Ser Leu Asp Gly Val
35 40 45 Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
50 55 60
Leu Gin Gly Ser Leu Gin Asp lie Leu Gin Gin Leu Asp Val Ser 63
Pro Glu Cys
SEQ IP NQ;4 1 5 10 15 Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys
20 25 30
Thr lie Val Thr Arg lie Asn Asp lie Ser His Thr Xaa Ser Val 35 40 45
Ser Ala Lys Gin Arg Val Thr Gly Leu Asp Phe lie Pro Gly Leu
50 55 60
His Pro lie Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala Val
65 70 75
Tyr Gin Gin Val Leu Thr Ser Leu Pro Ser Gin Asn Val Leu Gin
80 85 90 lie Ala Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu
95 100 105
Ala Phe Ser Lys Ser Cys Ser Leu Pro Gin Thr Ser Gly Leu Gin 110 115 120
Lys Pro Glu Ser Leu Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser
125 130 135
Thr Glu Val Val Ala Leu Ser Arg Leu Gin Gly Ser Leu Gin Asp
140 145 lie Leu Gin Gin Leu Asp Val Ser Pro Glu Cys wherein Xaa is Gin or is absent.
SEQ IP NQ.5
1 5 10 15 lie Asn Asp lie Ser His Thr Gin Ser Val Ser Ser Lys Gin Lys
18 Val Thr Cys SEQ ID NO:6 1 5 10 15
Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu
20 25 30
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu
35 40 45
Val Val Ala Leu Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu
50 Trp Gin Leu Asp Leu Ser Pro Gly Cys
SEQ IP NQ;7
1 5 10 15
Val Pro lie Xaa Lys Val Xaa Asp Asp Thr Lys Thr Leu lie Lys
20 25 30 Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val
35 40 45
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu 50 55 60
His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
65 70 75 Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa
80 85 90
He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu 95 100 105
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu
110 115 120
Thr Leu Xaa Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser
125 130 135
Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp
140 145 Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 4 is Gin or Glu; Xaa at position 7 is Gin or Glu; Xaa at position 22 is Gin, Asn, or Asp; Xaa at position 28 is Gin or Glu; Xaa at position 34 is Gin or Glu; Xaa at position 54 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly; Xaa at position 56 is Gin or Glu; Xaa at position 62 is Gin or Glu; Xaa at position 63 is Gin or Glu; Xaa at position 68 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly; Xaa at position 72 is Gin, Asn, or Asp; Xaa at position 75 is Gin or Glu; Xaa at position 78 is Gin, Asn, or Asp; Xaa at position 82 is Gin, Asn, or Asp; Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or
Leu;
Xaa at position 108 is Asp or Glu; Xaa at position 130 is Gin or Glu; Xaa at position 134 is Gin or Glu; Xaa at position 136 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly; Xaa at position 138 is Gin, Trp, Tyr, Phe, He, Val, or
Leu;
Xaa at position 139 is Gin or Glu;
SEQ ID NO:8 1 5 10 15
He Xaa Lys Val Xaa Asp Asp Thr Lys Thr Leu He Lys Thr He
20 25 30
Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val Ser Ser
35 40 45
Lys Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro
50 55 60
He Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa
65 70 75
Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa He Ser 80 85 90
Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe
95 100 105 Ser Lys Ser Cys Trp Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu
110 115 120
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu 125 130 135
Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Trp Xaa Leu
140 Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 2 is Gin or Glu;
Xaa at position 5 is Gin or Glu;
Xaa at position 20 is Asn, Asp or Gin;
Xaa at position 26 is Gin or Glu; Xaa at position 32 is Gin or Glu;
Xaa at position 52 is He, Leu, Met or methionine sulfoxide;
Xaa at position 54 is Gin or Glu;
Xaa at position 60 is Gin or Glu;
Xaa at position 61 is Gin or Glu; Xaa at position 66 is He, Leu, Met or methionine sulfoxide;
Xaa at position 70 is Asn, Asp or Gin;
Xaa at position 73 is Gin or Glu;
Xaa at position 76 is Asn, Asp or Gin;
Xaa at position 80 is Asn, Asp or Gin; Xaa at position 98 is Trp or Gin;
Xaa at position 128 is Gin or Glu;
Xaa at position 132 is Gin or Glu;
Xaa at position 134 is He, Leu, Met or methionine sulfoxide;
Xaa at position 136 is Trp or Gin; and Xaa at position 137 is Gin or Glu.
SEQ IP NQ ; 9 1 5 10 15
Val Xaa Asp Asp Thr Lys Thr Leu He Lys Thr He Val Thr Arg
20 25 30
He Xaa Asp He Ser His Thr Xaa Ser Val Ser Ser Lys Xaa Lys 35 40 45
Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He Leu Thr
50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa He Leu
65 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa He Ser Xaa Asp Leu
80 85 90
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
95 100 105
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
125 130 135 Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140 Asp Leu Ser Pro Gly Cys wherein: Xaa at position 2 is Gin or Glu;
Xaa at position 17 is Asn, Asp or Gin;
Xaa at position 23 is Gin or Glu;
Xaa at position 29 is Gin or Glu;
Xaa at position 49 is He, Leu, Met or methionine sulfoxide; Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is He, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin; Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu; Xaa at position 129 is Gin or Glu;
Xaa at position 131 is He, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu. SEQ IP NQ;1Q 1 5 10 15
Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val
20 25 30
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu
35 40 45
His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
50 55 60
Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa
65 70 75 He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
80 85 90
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu 95 100 105
Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser
110 115 120
Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp
125 130
Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 7 is Asn, Asp or Gin; Xaa at position 13 is Gin or Glu;
Xaa at position 19 is Gin or Glu;
Xaa at position 39 is He, Leu, Met or methionine sulfoxide;
Xaa at position 41 is Gin or Glu;
Xaa at position 47 is Gin or Glu; Xaa at position 48 is Gin or Glu;
Xaa at position 53 is He, Leu, Met or methionine sulfoxide;
Xaa at position 57 is Asn, Asp or Gin;
Xaa at position 60 is Gin or Glu;
Xaa at position 63 is Asn, Asp or Gin; ; Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 85 is Trp or Gin;
Xaa at position 115 is Gin or Glu;
Xaa at position 119 is Gin or Glu;
Xaa at position 121 is He, Leu, Met or methionine sulfoxide; Xaa at position 123 is Trp or Gin; and Xaa at position 124 is Gin or Glu.
SEQ ID NO : 11 1 5 10 15
He Xaa Asp He Ser His Thr Xaa Ser Val Ser Ser Lys Xaa Lys
20 25 30
Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He Leu Thr
35 40 45
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa He Leu
50 55 60
Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa He Ser Xaa Asp Leu
65 70 75
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
80 85 90
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
95 100 105
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
110 115 120
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
125 Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 2 is Asn, Asp or Gin;
Xaa at position 8 is Gin or Glu;
Xaa at position 14 is Gin or Glu;
Xaa at position 34 is He, Leu, Met or methionine sulfoxide;
Xaa at position 36 is Gin or Glu;
Xaa at position 42 is Gin or Glu;
Xaa at position 43 is Gin or Glu;
Xaa at position 48 is He, Leu, Met or methionine sulfoxide;
Xaa at position 52 is Asn, Asp or Gin;
Xaa at position 55 is Gin or Glu;
Xaa at position 58 is Asn, Asp or Gin;
Xaa at position 62 is Asn, Asp or Gin;
Xaa at position 80 is Trp or Gin;
Xaa at position 110 is Gin or Glu; Xaa at position 114 is Gin or Glu;
Xaa at position 116 is He, Leu, Met or methionine sulfoxide;
Xaa at position 118 is Trp or Gin; and
Xaa at position 119 is Gin or Glu.
SEQ ID NO: 12 1 5 10 15
Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 20 25 30
Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa
35 40 45
He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa He Ser Xaa
50 55 60
Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser
65 70 75 Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp
80 85 90
Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val 95 100 105
Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa
110 Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 1 is Gin or Glu;
Xaa at position 21 is He, Leu, Met or methionine sulfoxide;
Xaa at position 23 is Gin or Glu;
Xaa at position 29 is Gin or Glu; Xaa at position 30 is Gin or Glu;
Xaa at position 35 is He, Leu, Met or methionine sulfoxide;
Xaa at position 39 is Asn, Asp or Gin;
Xaa at position 42 is Gin or Glu;
Xaa at position 45 is Asn, Asp or Gin; Xaa at position 49 is Asn, Asp or Gin;
Xaa at position 67 is Trp or Gin;
Xaa at position 97 is Gin or Glu;
Xaa at position 101 is Gin or Glu;
Xaa at position 103 is He, Leu, Met or methionine sulfoxide; Xaa at position 105 is Trp or Gin; Xaa at position 106 is Gin or Glu.
SEQ IP NQ.13 1 5 10 15
Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He Leu Thr
20 25 30
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa He Leu
35 40 45
Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa He Ser Xaa Asp Leu
50 55 60
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
65 70 75
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
80 85 90
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
95 100 105
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
110 Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 19 is He, Leu, Met or methionine sulfoxide;
Xaa at position 21 is Gin or Glu;
Xaa at position 27 is Gin or Glu;
Xaa at position 28 is Gin or Glu;
Xaa at position 33 is He, Leu, Met or methionine sulfoxide; ;
Xaa at position 37 is Asn, Asp or Gin;
Xaa at position 40 is Gin or Glu;
Xaa at position 43 is Asn, Asp or Gin;
Xaa at position 47 is Asn, Asp or Gin;
Xaa at position 65 is Trp or Gin;
Xaa at position 95 is Gin or Glu;
Xaa at position 99 is Gin or Glu;
Xaa at position 101 is He, Leu, Met or methionine sulfoxide;
Xaa at position 103 is Trp or Gin; and
Xaa at position 104 is Gin or Glu. SEQ ID NO: 14 1 5 10 15
He Pro Gly Leu His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa
20 25 30
Thr Leu Ala Val Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg
35 40 45
Xaa Val He Xaa He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
50 55 60
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala
65 70 75
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
80 85 90
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
95 100 105
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 13 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 15 is Gin or Glu;
Xaa at position 21 is Gin or Glu;
Xaa at position 22 is Gin or Glu;
Xaa at position 27 is He, Leu, Met or methionine sulfoxide;
Xaa at position 31 is Asn, Asp or Gin;
Xaa at position 34 is Gin or Glu;
Xaa at position 37 is Asn, Asp or Gin;
Xaa at position 41 is Asn, Asp or Gin;
Xaa at position 59 is Trp or Gin;
Xaa at position 89 is Gin or Glu;
Xaa at position 93 is Gin or Glu;
Xaa at position 95 is He, Leu, Met or methionine sulfoxide;
Xaa at position 97 is Trp or Gin; and
Xaa at position 98 is Gin or Glu.
SEQ ID NO : 15
1 5 10 15
Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa He Leu Thr Ser Xaa
20 25 30 Pro Ser Arg Xaa Val He Xaa He Ser Xaa Asp Leu Glu Xaa Leu 35 40 45
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
50 55 60 Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val
65 70 75
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 80 85 90
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
Pro Gly Cys
wherein:
Xaa at position 1 is He, Leu, Met, methionine sulfoxide or absent;
Xaa at position 3 is Gin or Glu; Xaa at position 9 is Gin or Glu;
Xaa at position 10 is Gin or Glu;
Xaa at position 15 is He, Leu, Met or methionine sulfoxide;
Xaa at position 19 is Asn, Asp, or Gin;
Xaa at position 22 is Gin or Glu; Xaa at position 25 is Asn, Asp, or Gin;
Xaa at position 29 is Asn, Asp, or Gin;
Xaa at position 47 is Trp or Gin;
Xaa at position 77 is Gin or Glu;
Xaa at position 81 is Gin or Glu; Xaa at position 83 is- He, Leu, Met or methionine sulfoxide;
Xaa at position 85 is Trp or Gin; and
Xaa at position 86 is Gin or Glu.
SEQ ID NO:16 1 5 10 15
Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa He
20 25 30
Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala
35 40 45
Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr
50 55 60
Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr -15 -
65 70 75
Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa 80 85
Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 1 is Gin or Glu; Xaa at position 2 is Gin or Glu;
Xaa at position 7 is He, Leu, Met or methionine sulfoxide;
Xaa at position 11 is Asn, Asp, or Gin;
Xaa at position 14 is Gin or Glu;
Xaa at position 17 is Asn, Asp, or Gin; Xaa at position 21 is Asn, Asp, or Gin;
Xaa at position 39 is Trp or Gin;
Xaa at position 69 is Gin or Glu;
Xaa at position 73 is Gin or Glu;
Xaa at position 75 is He, Leu, Met or methionine sulfoxide; Xaa at position 77 is Trp or Gin; and
Xaa at position 78 is Gin or Glu.
SEQ IP NQ . 3.7 1 5 10 15 Xaa Val He Xaa He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
20 25 30
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala 35 40 45
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
50 55 60
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
65 70 75
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 1 is Asn, Asp or Gin; Xaa at position 4 is Gin or Glu;
Xaa at position 7 is Asn, Asp or Gin;
Xaa at position 11 is Asn, Asp or Gin; Xaa at position 29 is Trp or Gin;
Xaa at position 59 is Gin or Glu;
Xaa at position 63 is Gin or Glu;
Xaa at position 65 is He, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Trp or Gin; and
Xaa at position 68 is Gin or Glu.
SEQ IP NQ.18 1 5 10 15 Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro
20 25 30
Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu ' 35 40 45
Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu
50 55 60
Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro
Gly Cys wherein:
Xaa at position 16 is Trp or Gin; Xaa at position 46 is Gin or Glu;
Xaa at position 50 is Gin or Glu;
Xaa at position 52 is He, Leu, Met or methionine sulfoxide;
Xaa at position 54 is Trp or Gin; and
Xaa at position 55 is Gin or Glu.
SEQ IP NQ;19 1 5 10 15
Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu 20 25 30
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu
35 40 45
Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu
50 Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 8 is Trp or Gin; Xaa at position 38 is Gin or Glu; Xaa at position 42 is Gin or Glu;
Xaa at position 44 is He, Leu, Met or methionine sulfoxide;
Xaa at position 46 is Trp or Gin; and
Xaa at position 47 is Gin or Glu.
SEQ ID NO : 20 1 5 10 15
Thr Leu He Lys Thr He Val Thr Arg He Xaa Asp He Ser His 20 25 ,30
Thr Xaa Ser Val Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe
35 40 45
He Pro Gly Leu His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa
50 55 60
Thr Leu Ala Val Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg
65 70 75 Xaa Val He Xaa He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
80 85 90
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala 95 100 105
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
110 115 120
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
125 130 135
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 11 is Asn, Asp, or Gin; Xaa at position 17 is Gin or Glu;
Xaa at position 23 is Gin or Glu;
Xaa at position 43 is He, Leu, Met or methionine sulfoxide;
Xaa at position 45 is Gin or Glu;
Xaa at position 51 is Gin or Glu; Xaa at position 52 is Gin or Glu;
Xaa at position 57 is He, Leu, Met or methionine sulfoxide;
Xaa at position 61 is Asn, Asp, or Gin;
Xaa at position 64 is Gin or Glu;
Xaa at position 67 is Asn, Asp, or Gin; Xaa at position 71 is Asn, Asp, or Gin;
Xaa at position 89 is Trp or Gin;
Xaa at position 119 is Gin or Glu;
Xaa at position 123 is Gin or Glu;
Xaa at position 125 is He, Leu, Met or methionine sulfoxide;
Xaa at position 127 is Trp or Gin; and
Xaa at position 128 is Gin or Glu.
SEQ ID NO:21 1 5 10 15
Val He Xaa He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu
20 25 30
His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser
35 40 45
Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
50 55 60
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser
65 70
Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 3 is Gin or Glu;
Xaa at position 6 is Asn, Asp or Gin;
Xaa at position 10 is Asn, Asp or Gin;
Xaa at position 28 is Trp or Gin;
Xaa at position 58 is Gin or Glu;
Xaa at position 62 is Gin or Glu;
Xaa at position 64 is He, Leu, Met or methionine sulfoxide;
Xaa at position 66 is Trp or Gin; and
Xaa at position 67 is Gin or Glu.
SEQ IP NQ.22 1 5 10 15
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
20 25 30
Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val
35 40 45
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 50 55 60
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
Pro Gly Cys wherein:
Xaa at position 17 is Trp or Gin;
Xaa at position 47 is Gin or Glu;
Xaa at position 51 is Gin or Glu;
Xaa at position 53 is He, Leu, Met or methionine sulfoxide;
Xaa at position 55 is Trp or Gin; and
Xaa at position 56 is Gin or Glu.
SEQ ID NO:.23 1 5 10 15
Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser
20 25 30
Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
35 40 45
Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa
50 Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 6 is Trp or Gin;
Xaa at position 36 is Gin or Glu;
Xaa at position 40 is Gin or Glu; Xaa at position 42 is He, Leu, Met or methionine sulfoxide;
Xaa at position 44 is Trp or Gin; and
Xaa at position 45 is Gin or Glu.
SEQ IP Q;24 1 5 10 15
Val Pro He Xaa Lys Val Xaa Asp Asp Thr Lys Thr Leu He Lys
20 25 30
Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val
35 40 45
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu
50 55 60 His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val 65 70 75
Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa 80 85 90
He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
95 100
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa wherein:
Xaa at position 4 is Gin or Glu;
Xaa at position 7 Gin or Glu;
Xaa at position 22 is Gin, Asn, or Asp;
Xaa at position 28 is Gin or Glu; Xaa at position 34 is Gin or Glu;
Xaa at position 54 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly;
Xaa at position 56 Gin or Glu;
Xaa at position 62 Gin or Glu; Xaa at position 63 Gin or Glu;
Xaa at position 68 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly;
Xaa at position 72 is Gin, Asn, or Asp;
Xaa at position 75 is Gin or Glu; Xaa at position 78 is Gin, Asn, or Asp;
Xaa at position 82 is Gin, Asn, or Asp; and
Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or Leu.
SEQ ID NO:25
1 5 10 15
Ala Ser Gly Leu Glu Thr Leu Xaa Ser Leu Gly Gly Val Leu Glu
20 25 30 Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa
35 40 45
Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly
Cys wherein:
Xaa at position 8 is Asp or Glu; Xaa at position 30 is Gin or Glu;
Xaa at position 34 is Gin or Glu;
Xaa at position 36 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly; Xaa at position 38 is Gin, Trp, Tyr, Phe, He, Val, or
Leu; and
Xaa at position 39 is Gin or Glu.
SEQ ID NO:26 1 5 10 15
Val Pro He Xaa Lys Val Xaa Asp Asp Thr Lys Thr Leu He Lys
20 25 30
Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val
35 40 45
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu
50 55 60 His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
65 70 75
Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa 80 85 90
He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
95 100 105
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu
110 115
Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr
wherein: Xaa at position 4 is Gin or Glu;
Xaa at position 7 is Gin or Glu;
Xaa at position 22 is Asn, Asp or Gin;
Xaa at position 28 is Gin or Glu;
Xaa at position 34 is Gin or Glu; Xaa at position 54 is He, Leu, Met or methionine sulfoxide;
Xaa at position 56 is Gin or Glu;
Xaa at position 62 is Gin or Glu;
Xaa at position 63 is Gin or Glu; Xaa at position 68 is He, Leu, Met or methionine sulfoxide;
Xaa at position 72 is Asn, Asp or Gin;
Xaa at position 75 is Gin or Glu; Xaa at position 78 is Asn, Asp or Gin;
Xaa at position 82 is Asn, Asp or Gin; and
Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or Leu.
SEQ ID NO:27
1 5 10 15
Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa
20 25 Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 11 is Gin or Glu;
Xaa at position 15 is Gin or Glu;
Xaa at position 17 is Met or methionine sulfoxide, Leu, He, Val, Ala, or Gly;
Xaa at position 19 is Trp or Gin; and
Xaa at position 20 is Gin or Glu.
SEQ IP NQ;2§ 1 5 10 15
Val Pro He Xaa Lys Val Xaa Asp Asp Thr Lys Thr Leu He Lys
20 25 30
Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val
35 40 45
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu
50 55 60
His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
65 70 75
Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa 80 85 90
He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
95 100 105
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu 110 115 120
Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser
125
Thr Glu Val Val Ala Leu Ser Arg
wherein:
Xaa at position 4 is Gin or Glu;
Xaa at position 7 is Gin or Glu;
Xaa at position 22 is Asn, Asp or Gin;
Xaa at position 28 is Gin or Glu;
Xaa at position 34 is Gin or Glu;
Xaa at position 54 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly;
Xaa at position 56 is Gin or Glu;
Xaa at position 62 is Gin or Glu;
Xaa at position 63 is Gin or Glu;
Xaa at position 68 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly;
Xaa at position 72 is Asn, Asp or Gin;
Xaa at position 75 is Gin or Glu;
Xaa at position 78 is Asn, Asp or Gin;
Xaa at position 82 is Asn, Asp or Gin; and
Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or
Leu.
SEQ ID NO:29
1 5 10 15
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
Pro Gly Cys
Xaa at position 2 is Gin or Glu;
Xaa at position 6 is Gin or Glu;
Xaa at position 8 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly;
Xaa at position 10 is Trp or Gin; and
Xaa at position 11 is Gin or Glu. 1
Figure imgf000026_0001
Val Pro He Xaa Lys Val Xaa Asp Asp Thr Lys Thr Leu He Lys
20 25 30
Thr He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val
35 40 45
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu
50 55 60
His Pro He Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
65 70 75
Tyr Xaa Xaa He Leu Thr Ser Xaa Pro Ser Arg Xaa Val He Xaa
80 85 90
He Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
95 100 105
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu
110 115 120
Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser
125 130 135 Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp
Xaa Leu Xaa wherein:
Xaa at position 4 is Gin or Glu;
Xaa at position 7 is Gin or Glu;
Xaa at position 22 is Asn, Asp or Gin; Xaa at position 28 is Gin or Glu;
Xaa at position 34 is Gin or Glu;
Xaa at position 54 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly;
Xaa at position 56 is Gin or Glu; Xaa at position 62 is Gin or Glu;
Xaa at position 63 is Gin or Glu;
Xaa at position 68 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly;
Xaa at position 72 is Asn, Asp or Gin; Xaa at position 75 is Gin or Glu;
Xaa at position 78 is Asn, Asp or Gin; Xaa at position 82 is Asn, Asp or Gin;
Xaa at position 100 is Gin, Trp, Tyr, Phe, He, Val, or Leu;
Xaa at position 130 is Gin or Glu; Xaa at position 134 is Gin or Glu;
Xaa at position 136 is Met, methionine sulfoxide, Leu,
He, Val, Ala, or Gly; and
Xaa at position 138 is Trp or Gin.
SEQ ID NO: 31
1 5
Xaa Leu Asp Leu Ser Pro Gly Cys wherein: Xaa at position 1 is Gin or Glu.
For purposes of this application, the term antibody includes antibody fragments which retain their specific binding capacity. Antibodies that specifically bind to SEQ ID NOS: 1-6 are preferred.
The specification will further show how to use antibodies to purify certain ob gene product fragments from complex biological mixtures and how to identify and quantitate the presence of these fragments in various environments. The preparation of antibodies against proteins of interest is well known in the art. See Galfre and Milstein, Methods in Enzvmoloσv. Vol. 73, Academic Press, New York (1981);
James W. Goding, Monoclonal Antibodies: Princ les and.
Practice, Academic Press, Orlando, Florida (1986); Current Protocols in Molecular Bioloσv. F. M. Ausubel, e_ζ. al. ed. , Wiley Interscience, New York, (1987) .
To prepare antibodies reactive with a protein or peptide of interest, the protein or peptide must be first be either purified, synthesized or recombinantly expressed. Relatively crude antigenic preparations of the protein or peptide may be used for immunization purposes. However, highly purified protein is required to determine accurately if hybridomas are producing the sought after monoclonal antibodies (mabs) or to assay the antibody titers of immune serum.
Given the sequence information herein disclosed and the state of the art in solid phase protein synthesis, the peptide fragments herein disclosed can be prepared via chemical synthesis. Also, recombinant DNA techniques may be used to express these proteins.
The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Dugas, H. and Penney, C, Bioorσanic Chemistry (1981) Springer-Verlag, New York, pgs. 54-92, Merrifield, J.M., Chem . Soc , &5.:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco, 1969) . For example, any given peptide set forth in the
SEQUENCE LISTING may be synthesized by solid-phase methodology utilizing a 430A peptide synthesizer (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) and synthesis cycles supplied by PE-Applied Biosystems. Boc amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical supply houses. Sequential Boc chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides. For the production of C-terminal acids, the corresponding PAM resin is used. Asn, Gin, and Arg are coupled using preformed hydroxy benzotriazole esters. The following side chain protecting groups may be used: Arg, Tosyl Asp, cyclohexyl
Glu, cyclohexyl Ser, Benzyl
Thr, Benzyl Tyr, 4-bromo carbobenzoxy
Boc deprotection may be accomplished with trifluoroacetic acid in methylene chloride. Following completion of the synthesis the peptides may be deprotected and cleaved from the resin with anhydrous hydrogen fluoride (HF) containing 10% meta-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at -5°C to 5°C, preferably on ice for 60 minutes. After removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and lyophilized.
Likewise, the state of the art in molecular biology provides the ordinarily skilled artisan another means by which the protein fragments necessary to practice the present invention can be obtained. Although such peptides may be produced by solid phase peptide synthesis or recombinant methods, recombinant methods may be preferable because higher yields are possible. The basic steps in recombinant production are:
a) constructing a synthetic or semi-synthetic DNA coding sequence for the protein fragment, b) placing the coding sequence into an expression vector in a manner suitable for expressing proteins either alone or as a fusion proteins, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, culturing the transformed host cell under conditions that will permit expression, and e) recovering and purifying the recombinantly produced protein.
Synthetic genes, the in vi tro or in vivo transcription and translation of which results in the production of any of the peptides described in the SEQUENCE
LISTING, may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the ordinarily skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed, all of which encode any one given peptide set forth in the SEQUENCE LISTING. The methodology of synthetic gene construction is well known in the art. See Brown, et al . (1979) Methods in Enzymology, Academic Press, N.Y. , Vol. 68, pgs. 109-151. A O 9623815
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vast array of DNA sequences can be designed based on the amino acid sequences herein disclosed. Once designed, the sequence itself may be generated using conventional DNA synthesizing apparatus such as the Model 380A or 380B DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404).
To effect expression of a polypeptide of interest, one inserts the engineered synthetic DNA sequence into any one of many appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases. See generally Maniatis et al . (1989) Molecular Cloning; A Laboratory Manual , Cold Springs Harbor Laboratory Press, N.Y. , Vol. 1-3. Restriction endonuclease cleavage sites are engineered into either end of the coding region to facilitate isolation from, and integration into, known amplification and expression vectors. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The choice of restriction sites are chosen so as to properly orient and position the coding sequence with control sequences to achieve expression of the protein of interest.
To achieve efficient transcription of the synthetic gene, it must be operably associated with a promoter-operator region. Therefore, the promoter-operator region of the synthetic gene is placed in the same sequential orientation with respect to the ATG start codon of the synthetic gene.
A variety of expression vectors useful for transforming prokaryotic and eukaryotic cells are well known in the art. See The Promega Biological Research Products Catalogue (1992) (Promega Corp., 2800 Woods Hollow Road, Madison, WI, 53711-5399); and The Stratagene Cloning Systems Catalogue (1992) (Stratagene Corp., 11011 North Torrey Pines Road, La Jolla, CA, 92037). Also, U.S. Patent No. 4,710,473 describes circular DNA plasmid transformation vectors useful for expression of exogenous genes in E. coli at high levels. These plasmids are useful as transformation vectors in recombinant DNA procedures and
(a) confer on the plasmid the capacity for autonomous replication in a host cell;
(b) control autonomous plasmid replication in relation to the temperature at which host cell cultures are maintained;
(c) stabilize maintenance of the plasmid in host cell populations;
(d) direct synthesis of a protein prod, indicative of plasmid maintenance in a host cell population;
(e) provide in series restriction endonuclease recognition sites unique to the plasmid; and (f) terminate mRNA transcription.
These circular DNA plasmids are useful as vectors in recombinant DNA procedures for .securing high levels of expression of exogenous genes. Having constructed an expression vector for any given polypeptide set forth in the SEQUENCE LISTING, the next step is to place the vector into a suitable cell and thereby construct a recombinant host cell useful for expressing the polypeptide. Techniques for transforming cells with recombinant DNA vectors are well known in the art and may be found in such general references as Maniatis, et al . supra . Host cells made be constructed from either eukaryotic or prokaryotic cells.
Prokaryotic host cells generally produce the protein at higher rates and are easier to culture. Proteins which are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Such protein aggregates typically must be solubilized, denatured and refolded using techniques well known in the art. See Kreuger, et al . (1990) in Protein Folding, Gierasch and King, eds. , pgs 136-142, American 9623815
-30 -
Association for the Advancement of Science Publication No. 89-18S, Washington, D.C.; and U.S. Patent No. 4,923,967.
Antiserum Preparation:
After obtaining milligram quantities of the peptide of interest, antibodies specific for the peptide may be raised by conventional methods that are well known in the art. Repeated injections into a host of choice over a period of weeks or months generally elicits an immune response and results in significant serum titers. Preferred hosts are mammalian species and more highly preferred species are rabbits, goats, sheep and mice. The most preferred species is rabbit. Blood drawn from such immunized animals may be processed by established methods to obtain antiserum (polyclonal antibodies) reactive with the chosen immunogen. The antiserum may then be affinity purified by adsorption to the immunogen according to techniques known in the art. Affinity purified antiserum may be further purified by isolating the immunoglobulin fraction within the antiserum using procedures known in the art. The resulting material will be a heterogeneous population of immunoglobulins that specifically bind to the immunogen.
Since some of the protein fragments are short, antibodies of interest may also be generated by preparing a semi-synthetic immunogen consisting of a peptide fragment bound to an immunogenic carrier. Many suitable immunogenic carriers such as bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin are also well known in the art as are techniques for coupling the two proteins. Once the semi-synthetic immunogen has been constructed, the procedure for making antibodies is identical to those used for making antibodies to unconjugated proteins.
Preparation of monoclonal antibodies:
Mabε of the present invention are readily prepared using isolated peptide fragments (immunogens) set forth in the SEQUENCE LISTING. Methods for producing mabs have been practiced for over 15 years and are well known to those of ordinary skill in the art. Repeated intraperitoneal or subcutaneous injections of immunogen in adjuvant will elicit an immune response in most animals. Hyperimmunized B-lymphocytes are removed from the animal and fused with a suitable fusion partner cell line capable of being cultured indefinitely.
Preferred animals whose B-lymphocytes may be hyperimmunized and used in the production of mabs are mammals. More preferred animals are rats and mice and most preferred is the BALB/c mouse strain. Numerous mammalian cell lines are suitable fusion partners for the production of hybridomas. Many such lines are commercially available from the ATCC and commercial suppliers. Preferred fusion partner cell lines are derived from mouse myelomas and the HL-1' Friendly myeloma-653 cell line (Ventrex, Portland, ME) is most preferred.
Once fused, the resulting hybridomas are cultured in a selective growth medium for one to two weeks. Two well known selection systems are available for eliminating unfused myeloma cells, or fusions between myeloma cells, from the mixed hybridoma culture. The choice of selection system depends on the strain of mouse immunized and myeloma fusion partner used. The AAT selection system, described by Taggart and Samloff, Science 219. 1228 (1982), may be used; however, the HAT
(hypoxanthine, aminopterin, thymidine) selection system, described by Littlefield, Science 145. 709 (1964), is preferred because of its compatibility with the preferred mouse strain and fusion partner mentioned above.
Spent growth medium is then screened for immunospecific mab secretion. Enzyme linked immunosorbant assay (ELISA) procedures are best suited for this purpose; though, radioimmune assays adapted for large volume screening are also acceptable. Multiple screens designed to consecutively pare down the considerable number of irrelevant or less desired cultures should be performed. Cultures that secrete mabs reactive with the immunogen should be screened for cross- reactivity with other proteins such as the immunogenic carrier. O 96/23815
-32 -
Mabs that preferentially bind to the immunogen of interest may be isotyped using commercially available assays. Binding constants may also be determined by well known methods such a Scatchard analysis. Those skilled in the art will then be able to choose a particular antibody in light of these characteristics based on the intended use for the mab.
Hybridoma cultures which secrete the sought-after mabs should be sub-cloned several times to establish monoclonality and stability. Well known methods for sub-cloning eukaryotic, non-adherent cell cultures include limiting dilution, soft agarose and fluorescence activated cell sorting techniques the latter of which is exemplified in U.S. Patent No. 4,264,341. After each sub-cloning, the resultant cultures must be re- assayed for antibody secretion and isotype to ensure that a stable antibody-secreting culture has been established. Immunoaffinitv purification^
The claimed antibodies are essential reagents for preparing an immunoa finity surface necessary to practice the isolation method of the invention. The immunoaffinity surface is formed by immobilizing a claimed antibody to a substance so that at least some of the antibody binding site remains exposed and capable of binding its antigen. Substances ranging from porous polysaccharide based beads to plastic polymers to inorganic materials are substances to which the antibodies may be immobilized. Preferred substances are those which provide a maximal surface area to volume ratio and do not adversely affect the antibodies or protein fragment. Polysaccharide matrices formed into various sized beads are more highly preferred because they are porous, provide high surface area to volume ratios, are easy to handle and are well known and understood in the biochemical purification art. See Affinity Chromatoσraphv Principles & Methods. Pharmacia Fine Chemicals, (1983). A wide assortment of schemes for immobilizing antibodies has developed over the past few decades. Immobilization can be accomplished by covalently coupling the antibody directly to the desired substance or by bridging the antibody to the substance.
CNBr and carbodiimide coupling of antibodies to polysaccharide based beads such as Ξepharose' (Pharmacia, Pistcataway, NJ) are illustrative of direct coupling schemes that are consistent with the invention. Direct couplings generally do not orient the antibodies in any particular fashion; however, some types of direct couplings are able to reproducibly orient the antibody on the immobilizing substance. Preferred coupling schemes orient the antibody such that its antigen binding regions remain exposed. One such scheme utilizes the natural carbohydrate found on the heavy chains of the antibody. By first oxidizing the carbohydrate moieties to the corresponding aldehydes then reacting the aldehyde with a primary amino group on the surface, it is possible to link the antibody in an advantageous orientation.
Many types of bridges are possible and include small organic linkers which covalently bind the antibody to the immobilizing substance. Such spacer arms are acceptable and preferably should not interact with proteins once the bridge has been formed.
Larger multivalent molecules bound to the immobilizing substance and which are capable of binding several antibodies describe another type of bridge. Specific immunoadsorbants bound to the immobilizing surface and which are capable of non- covalently binding the antibody represents yet another type of bridge. Protein-A is an example of a specific immunoadsorbant that is capable of orienting the antibody. By chemically bonding Protein-A to an immobilizing surface then allowing the antibodies to bind to it, the Fab antibody portions will be oriented away from the immunoaffinity surface resulting in a favorable configuration.
The above discussion is in no way meant to limit the scope of the invention. Numerous other well known schemes for linking antibodies to immobilizing substances are consistent with the invention. Once the immunoaffinity surface has been prepared, the stream containing the protein fragment to be purified must be contacted with the surface. Batch methods are adequate, though column chromatography is preferable. Batch mode separation can be accomplished by filtering, centrifuging or decanting. When using column chromatography, a simple washing step serves to separate the mixture from the immobilized antibodies. Removing the ob gene protein fragment from the immunoaffinity surface can be accomplished by subjecting the surface-bound complex to a solution capable of disrupting the interactions between the antibody and peptide of interest. The solution preferably will not adversely affect the immunoaffinity surface or the peptide. More preferred solutions are buffered, isotonic, salt solutions at near neutral pH which contain millimolar concentrations of 2-ME.
Identification of ob σene protein fragments:
It is well known that antibodies labeled with a reporting group can be used to identify the presence of antigens in a variety of milieus. Antibodies labeled with radioisotopes have been used for decades in radioimmune assays to identify, with great precision and sensitivity, the presence of antigens in a variety of biological fluids. More recently, enzyme labeled antibodies have been used as a substitute for radio- labeled antibodies in the popular ELISA. Antibodies of the present invention can be bound to an immobilizing substance such as a polystyrene well or particle and used in immunoassays to determine whether a particular ob gene protein fragment is present in a test sample. In this embodiment of the invention, a sample is contacted with the immunoaffinity surface and allowed to incubate. After a washing step, the ob gene protein fragment that has bound to the immunoaffinity surface is detected by contacting the surface with another antibody of the invention labeled with a reporting grou . In another mode of the assay embodiment, a test sample suspected of containing a protein fragment of interest is dried onto a surface, forming an immobilized test sample. A labeled antibody of the invention is then contacted with the immobilized test sample and allowed to incubate. If the sample contains a protein fragment of interest, the labeled antibody will bind to the immobilized fragment. This method can also be done using an unlabeled antibody of the invention followed by a labeled secondary antibody that binds to an antibody of the invention which has already bound to a protein fragment of interest. After washing, the immobilized test sample is measured to detect the presence of any reporting groups.
Reporting groups are typically enzymes such as alkaline phosphatase, horseradish peroxidase or beta-D- galactosidase. Suitable substrates produce a color change when reacted with the enzyme. In so doing, measurements of the color intensity can be quantitated using a spectrophotometer. If the reporting group is a radioisotope, an appropriate gamma or beta ray detecting instrument can be used to quantitate the reporting group. The intensity of the reporting group directly correlates, with the amount of the protein fragment of interest in the test sample.
By way of illustration, the following examples are provided to help describe how to make and practice the various embodiments of the invention. These example are in no way meant to limit the scope of the invention.
EXAMPLE i
SEQ ID NO:l was prepared and used to make a lysine core multiple antigenic peptide (MAP) according to U.S. Patent Number 5,229,490, herein incorporated by reference, and Applied Biosystems product procedure Number 34, May 1992 (PE-Applied Biosystems, Foster City, CA) .
A 0.25 mg/ml adjuvant emulsion was prepared by adding 375 μls of a 1.0 mg/ml solution of (22-41 MAPS) to 375 μls of phospate buffered saline (PBS) and 750 μls of Complete Freund's Adjuvant (CFA) . The solution was mixed until fully emulsivied. Six BALB/c mice were then given a 50 μg primary immunization by subcutaneously injecting 0.2 cc of the above immunogen into each mouse. At three week intervals, the mice are boosted with 50 μgs of (22-41 MAPS) in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum.
EXAMP E 2.
SEQ ID NO:2 was prepared by solid phase synthesis on a Model 430A peptide synthesizer (PE-Applied Biosystems, Foster City, CA) using the Boc protecting strategy. The side chain protecting groups were: Asp (Chxl), Glu (OBzl), Ser (Bzl), Thr (Bzl), Lys (Cl-Z), His (BOM), Trp (CHO) , Tyr (Br-Z) , and Arg (Tos) . All except for Asp (Chxl) (Peptides International) were obtained from PE-Applied Biosystems. Each residue was double coupled using either DCC initiated symmetric anhydride or HOBT activation.
The modified peptityl resin prepared was treated with 20 ml of 20% piperidine in DMF (dimethyl formamide) at 4°C for 1 hour to remove the Trp(CHO) protection. The modified peptidyl resin was washed several times with CH2C12- transfered to a teflon reaction vessel and dried in vacuo. Two ml of m-cresol and a magnetic stir bar were added to the the vessel which was attached to an HF apparatus (Pennisula Laboraties, Inc.), cooled to -78°C, evacuated, and 20-25 ml HF was condensed into the vessel. The reaction mixture was stirred for 60 min in an ice bath and the HF was then removed by vacuum. The peptide residue was suspended in ethyl ether and stirred briefly. The solid material was then filtered using a 60 ml glass fritted filter funnel. After washing the solids twice with ethyl ether, the peptide was solubilized by washing the solids with 40 ml each of 50% aqueous acetic acid and 10% aqueous acetic acid. 100 ul of the combined aqueous filtrate was removed and prepared for standard HPLC analysis using the following conditions:
Buffers: A) 0.1% TFA
B) 0.1% TFA / 50% CH3CN Column: Vydac C18 (0.46 x 15 cm) Temperature* 45°C Flow: 1.0 ml/min.
Detector: 214 nm Gradient: 0% B for 5 min., then 0 to 100% B over 60 min.
The remaining aqueous filtrate was loaded onto a
2.2 X 25 cm Vydac C18 column and preparatively chromatographed
(Pharmacia FPLC) at room temperature while monitoring at 214 nm. Fractions were collected every 5 min at a flow rate of 4 ml/min in a gradient beginning with 20% B (A and B were the same as above) and ending with 100% B over 790 min at room temperature. UV absorbing fractions were analysed by HPLC and selected fractions were combined and lyophilized to yield the title compound.
The peptide was then coupled to ovalbumin by first dissolving 3.0 mg of peptide into 1 ml water and solubilizing by adding 2 μl of 5 N NaOH. A 7.0 mg/ml solution of ovalbumin in 50% PBS/50% water was then prepared. In the dark, 8, 25 μl aliquotes of a 1:400 dilution of glutaraldehyde in PBS was added to the solublized peptide solution over a 2 hr period at room temperature. The reaction was then allowed to stand overnight at room temperature after which the reaction was quenched by adding 200 μls of 1M TRIS® pH 8.5. The final yield of the ovalbumin-coupled peptide solution was 2.1 ml at 1.4 mg/ml. An adjuvant emulsion was then prepared as in Example 1 by adding 230 μls of the ovalbumin-coupled peptide to 420 uls PBS and emulsifying in 650 μls CFA for a final concentration of approximately 0.25 mg/ml. Five, 18-21 gm, BALB/c mice were then given a primary immunization by subcutaneously injecting 0.2 cc of the above immunogen into each mouse. At three week intervals, the mice are boosted with 50 μgs of immunogen in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum. O 96/2 1
-38 -
Intrachain disulfide bonded SEQ ID NO:3 was prepared and purified in substantial accordance with Example 2 and the general teachings herein disclosed. The peptide was coupled to ovalbumin in substantial accordance with Example 2. The final yield of the ovalbumin-coupled peptide solution was 2.45 ml at 2.0 mg/ml. Six , 18-21 gm, BALB/c mice were then given a primary immunization by subcutaneously injecting 0.2 cc of immunogen, prpared in accordance with the previous examples, into each mouse. At three week intervals, the mice are boosted with 50 μgs of immunogen in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum.
EXAMPLE 4
Both forms of SEQ ID NO:4 were prepared in an E. coli B121 strain using standard recombinant DNA techniques herein described. Transformed cells were grown and induced such that the heterologous protein was overexpressed. The cells were then lysed and protein granules were extracted. 50 μls of wet granules were diluted in 450 μls of water then solublized by adding 5 μls of 5N NaOH. The solution was neutralized with 495 μls of 1M TRIS®, pH 7.5, and 0.2 μls of 2-mercaptoethanol was added to prevent cysteine disulfide bridging. The protein concentrations were determined to be approximately 11 mg/ml for the des-Gln form and 7.6 for the Gin form. An adjuvant emulsion was prepared for each protein form in susbstantial accordance with the previous examples. Eight BALB/c mice were each immunized with 50 μgs of immunogen as described above. At three week intervals, the mice are boosted with 50 μgs of immunogen in Incomplete Freund's Adjuvant (IFA) until sufficient titers (approximately 1:1000) of antibody are detected in the serum.

Claims

1. A monoclonal or polyclonal antibody that specifically binds to a protein selected from the group consisting of SEQ ID N0:1 through SEQ ID NO:31.
2. A monoclonal or polyclonal antibody that specifically binds to a protein selected from the group consisting of SEQ ID NO:l through SEQ ID NO:6.
An antibody of Claim 1 which is a monoclonal antibody.
4. An antibody of Claim 2 which is a monoclonal antibody.
5. An antibody of Claim 1 which is a polyclonal antibody.
An antibody of Claim 2 which is a polyclonal antibody.
7. An antibody of Claim 1 which is a monoclonal antibody of the IgG class.
8. An antibody of Claim 2 which is a monoclonal antibody of the IgG class.
9. A method of using an antibody of Claim 1 for isolating a protein of Claim 1: a) immobilizing said antibody onto a surface; b) contacting a said immobilized antibody with a mixture containing a protein of Claim 1; c) separating said immobilized antibody bound to a protein of Claim 1 from said mixture, and; d) recovering a protein of Claim 1 by removing the antibody-bound protein of step c) from said immoblized antibody.
10. A method of using an antibody of Claim 1 for identifying the presence of a protein of Claim 1 in a biological fluid suspected of containing said protein comprising: a) immobilizing said antibody onto an assay surface; b) contacting said assay surface with the biological fluid and washing said assay surface with a suitable solution; c) contacting said assay surface with an antibody of a Claim 1 labeled with a reporting group and washing said assay surface with a suitable solution, and; d) detecting the presence of said reporting group.
11. A method of using an antibody of Claim 1 for identifying the presence of a protein of Claim 1 comprising: a) immobilizing a sample suspected of containing a protein of Claim 1 onto an assay surface; b) contacting said assay surface with an antibody of Claim 1 directly or indirectly labeled with a reporting group and washing said assay surface with a suitable solution, and; c) detecting the presence of said reporting group.
12. A method of using an antibody of Claim 1 to quantitate the amount of a protein of Claim 1 in a biological fluid suspected of containing said protein comprising: a) radio-labelling the protein; b) mixing the antibody with the biological fluid; -41-
c) adding a know amount of the labelled protein to the mixture of step b) ; d) seperating bound antibody from free antibody, and; e) quantitating the amount of radio-label in the bound fraction.
PCT/US1996/000957 1995-01-31 1996-01-29 Ob gene product antibodies WO1996023815A1 (en)

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WO1996034885A2 (en) * 1995-05-05 1996-11-07 Smithkline Beecham Plc Biologically active peptide fragments of ob protein
WO1997016550A1 (en) * 1995-11-02 1997-05-09 Bristol-Myers Squibb Company Polypeptide fragments derived from the obese gene product
WO1997046585A2 (en) * 1996-06-06 1997-12-11 Smithkline Beecham P.L.C. Fragments of leptin (ob protein)
WO1998036762A1 (en) * 1997-02-25 1998-08-27 Eli Lilly And Company Method of modulating hypothalamic somatostatin release
WO1998036763A1 (en) * 1997-02-25 1998-08-27 Eli Lilly And Company Treatment of infertility with leptin receptor ligands
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
WO2000021574A2 (en) 1998-10-14 2000-04-20 Amgen Inc. Site-directed dual pegylation of proteins
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives
US7786265B2 (en) * 1998-08-21 2010-08-31 Albany Medical College Isolated antibodies against biologically active leptin-related peptides
JP4841037B2 (en) * 1999-02-12 2011-12-21 アムジエン・インコーポレーテツド Glycosylated leptin compositions and related methods
CN110183530A (en) * 2019-06-21 2019-08-30 深圳市亚辉龙生物科技股份有限公司 Leptin immunogene, hybridoma, monoclonal antibody, polyclonal antibody and application
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Cited By (23)

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Publication number Priority date Publication date Assignee Title
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives
US7544492B1 (en) 1994-08-17 2009-06-09 The Rockefeller University OB polypeptides, modified forms and derivatives
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
US6001968A (en) * 1994-08-17 1999-12-14 The Rockefeller University OB polypeptides, modified forms and compositions
US7521258B2 (en) 1994-08-17 2009-04-21 The Rockefeller University Methods of detecting, measuring, and evaluating modulators of body weight in biological samples, and diagnostic, monitoring, and therapeutic uses thereof
WO1996034885A3 (en) * 1995-05-05 1997-05-09 Smithkline Beecham Plc Biologically active peptide fragments of ob protein
WO1996034885A2 (en) * 1995-05-05 1996-11-07 Smithkline Beecham Plc Biologically active peptide fragments of ob protein
US6187751B1 (en) 1995-05-05 2001-02-13 Smithkline Beecham P.L.C. Biologically active peptide of ob protein
WO1997016550A1 (en) * 1995-11-02 1997-05-09 Bristol-Myers Squibb Company Polypeptide fragments derived from the obese gene product
WO1997046585A2 (en) * 1996-06-06 1997-12-11 Smithkline Beecham P.L.C. Fragments of leptin (ob protein)
WO1997046585A3 (en) * 1996-06-06 1998-04-23 Smithkline Beecham Plc Fragments of leptin (ob protein)
WO1998036762A1 (en) * 1997-02-25 1998-08-27 Eli Lilly And Company Method of modulating hypothalamic somatostatin release
WO1998036763A1 (en) * 1997-02-25 1998-08-27 Eli Lilly And Company Treatment of infertility with leptin receptor ligands
US7786265B2 (en) * 1998-08-21 2010-08-31 Albany Medical College Isolated antibodies against biologically active leptin-related peptides
US8022189B2 (en) 1998-08-21 2011-09-20 Albany Medical College Isolated antibodies against biologically active leptin-related peptides
US8067545B2 (en) 1998-08-21 2011-11-29 Albany Medical College Isolated antibodies against biologically active leptin-related peptides
US6420339B1 (en) 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
WO2000021574A2 (en) 1998-10-14 2000-04-20 Amgen Inc. Site-directed dual pegylation of proteins
JP4841037B2 (en) * 1999-02-12 2011-12-21 アムジエン・インコーポレーテツド Glycosylated leptin compositions and related methods
US11535659B2 (en) 2010-09-28 2022-12-27 Amryt Pharmaceuticals Inc. Engineered polypeptides having enhanced duration of action
CN110183530A (en) * 2019-06-21 2019-08-30 深圳市亚辉龙生物科技股份有限公司 Leptin immunogene, hybridoma, monoclonal antibody, polyclonal antibody and application
WO2020253187A1 (en) * 2019-06-21 2020-12-24 深圳市亚辉龙生物科技股份有限公司 Leptin immunogen, hybridoma cell, monoclonal antibody, polyclonal antibody and use thereof
EP3988564A4 (en) * 2019-06-21 2023-06-14 Shenzhen Yhlo Biotech Co., Ltd. Leptin immunogen, hybridoma cell, monoclonal antibody, polyclonal antibody and use thereof

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