EP0923604A1 - Peptides contenant de la cysteine ou de la methionine et possedant des effets immunomodulateurs - Google Patents

Peptides contenant de la cysteine ou de la methionine et possedant des effets immunomodulateurs

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Publication number
EP0923604A1
EP0923604A1 EP97919833A EP97919833A EP0923604A1 EP 0923604 A1 EP0923604 A1 EP 0923604A1 EP 97919833 A EP97919833 A EP 97919833A EP 97919833 A EP97919833 A EP 97919833A EP 0923604 A1 EP0923604 A1 EP 0923604A1
Authority
EP
European Patent Office
Prior art keywords
leu
pro
cys
seq
gly
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP97919833A
Other languages
German (de)
English (en)
Inventor
Hakan Bergstrand
Tomas Eriksson
Magnus Lindvall
Bengt Särnstrand
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AstraZeneca AB
Original Assignee
Astra AB
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
Priority claimed from SE9601422A external-priority patent/SE9601422D0/xx
Priority claimed from SE9603469A external-priority patent/SE9603469D0/xx
Application filed by Astra AB filed Critical Astra AB
Publication of EP0923604A1 publication Critical patent/EP0923604A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the field of the invention is non-antigen-specific immunomodulation, including both immunosuppression and immunostimulation.
  • the immune system when working properly, protects the individual from infection and from the growth of cancers. In order to carry out these functions, it must be able to recognize and mount an attack against foreign antigens, including cancer-specific antigens, but not against antigens that are normally present on cells throughout the body. It is possible to stimulate the immune system in order to improve the level of protection it affords. Immune stimulation is potentially beneficial where the individual is under attack from a chronic or an acute infection, or a malignant disease. Vaccines, including single-protein antigens such as diphtheria toxoid, are widely used to generate immunity against a specific antigen and thus against a specific disease.
  • the iiTunune response may be generally suppressed by treatment with corticosteroids, azathioprine, cyclosporine, tacrolimus (FK506) , rapamycin, or mycophenolate mofetil.
  • certain immunoglobulins including the monoclonal antibody OKT3 , have been used for this purpose. It may also be possible to suppress the immune response to a specific antigen. This procedure, which has been called “tolerance induction, " can be achieved by intravenous or repeated topical administration of the antigen in dilute form, treatment with a very high dose of the antigen, or oral administration of the antigen.
  • DNA molecules encoding certain immunoactive peptides can be used to treat animals in need of immunomodulation. When introduced into cells of the animal, the DNA molecules are transcribed, and a therapeutic amount of the peptide is produced at an appropriate site. Some of the peptides are immunostimulatory, and so are useful for treating conditions such as cancer. Other peptides are immunosuppressive, and so would be used to treat, e.g., autoimmune diseases or transplant rejection. The immunomodulatory effect appears to be generalized rather than antigen-specific, and is believed to be related to the function of T lymphocytes.
  • the DNA molecules of the invention encode Cys-containing or Met-containing peptides that fall within one of five motifs described by the formulas below.
  • the peptide is optionally linked to a signal peptide that is cleaved off by cellular proteases.
  • the DNA molecule of the invention encodes a peptide consisting of 4-30 amino acid residues (preferably 4-10, and more preferably 4-8, e.g., 4-7 or 4-6 residues) that conforms to the motif represented by Formula I:
  • X is selected from the group consisting of Ala, Val, Leu, lie, Gly, Asp, Glu, Asn, Gin, His, and Pro;
  • Y is selected from the group consisting Ala, Val, Leu, lie, Gly, Ser, Thr, Asp, Glu, Asp, Gin, Tyr, Phe, and Pro; n and m are whole integers chosen with the proviso that the sum of n and m is zero to twenty-six, inclusive; and the nucleic acid molecule optionally further encodes a mammalian signal peptide linked to the immunoactive peptide at A n .
  • X is Gly, Pro, lie, Val, Asp, Leu, Glu, Gin, or Ala
  • Y is Gly, Pro, lie, Val, Asp, Leu, Glu, Ser, Phe, Tyr, or Thr
  • the nucleic acid molecule optionally further encodes a mammalian signal peptide linked to the immunoactive peptide at A n .
  • the DNA molecule of the invention encodes a peptide conforming to the motif of Formula I, optionally linked to a signal peptide, wherein X is Gly and Y is Gly,
  • X is Pro and Y is Pro
  • X is lie and Y is Leu, X is Pro and Y is Glu,
  • X is Glu and Y is Tyr
  • X is Glu and Y is Phe
  • X is Ala and Y is Val, X is Val and Y is lie,
  • X is Gin and Y is Ser
  • X is Leu and Y is Asp, or
  • X is Asp and Y is lie; A and B vary according to the parameters above; and the sum of n and m is zero to eleven, inclusive.
  • the peptide represented by Formula I consists of 6-8 amino acid residues.
  • the nucleic acid molecule of the invention could encode a peptide where, for example, A is Gly, Lys, Arg, Cys, Ser, Val, Ala, Thr, Glu, Pro, Trp, Leu, Asp, Phe, or lie; B is Leu, Arg, lie, Val, Pro, Ala, Tyr, Gly, Trp, Thr, Lys, Met, Asp, Glu, or Phe; and the sum of n and m is two to four, inclusive.
  • the nucleic acid molecule could also encode a peptide where, for example, A is Pro, Gly, Glu, Ala, Val, Lys, Thr, Leu, or Ser; B is Tyr, Pro, Gly, Thr, Arg, Val, Ala, Leu, Lys, or lie; n is one; and m is one.
  • the DNA molecule of the invention may encode a peptide consisting of 5-30 amino acid residues (preferably 5-10, more preferably 5-9, e.g., 5, 6, 7, or 8 residues) that conforms to the motif represented by Formula II:
  • X is selected from the group consisting of Ala, Val, Leu, lie, Gly, Asp, Glu, Asn, Gin, Lys, Phe, His, and Pro;
  • Z is selected from the group consisting of Ala, Val, Leu, lie, Gly, Ser, Thr, Lys, His, Phe, Tyr, Arg, and Pro
  • Y is selected from the group consisting of Ala, Val, Leu, lie, Gly, Asp, Glu, Lys, Arg, Gin, Tyr, Phe, Ser, Thr, and Pro
  • n and m are whole integers chosen with the proviso that the sum of n and m is zero to twenty-five, inclusive; and the nucleic acid molecule optionally further encodes a mammalian signal peptide linked to the immunoactive peptide at A n .
  • X is Gly, Pro, lie, Val, Asp, Leu, Glu, Gin, or Ala
  • Y is selected from the group consisting of Gly, Glu, Val, Gin, Arg, Leu, Tyr, Phe, lie, Ser, Thr, Asp, and Pro
  • Z is selected from the group consisting of lie, Gly, Thr, Ala, Arg, and Lys
  • the sum of n and m is zero to ten, inclusive
  • the nucleic acid molecule optionally further encodes a mammalian signal peptide linked to the immunoactive peptide at A n .
  • the DNA molecule of the invention encodes a peptide conforming to the motif represented by Formula II, optionally linked to a signal peptide, wherein X is Gly and Y is Gly,
  • X is Pro and Y is Pro
  • X is Pro and Y is Glu, X is Glu and Y is Tyr,
  • X is Glu and Y is Phe
  • X is Ala and Y is Val
  • X is Val and Y is lie, X is Gin and Y is Ser, X is lie and Y is Thr, X is Leu and Y is Asp, or
  • X is Asp and Y is lie; Z is lie, Gly, Thr, Ala, or Lys; A and B vary according to the parameters above; the sum of n and m is zero to ten, inclusive; and the nucleic acid molecule optionally further encodes a mammalian signal peptide linked to the immunoactive peptide at A n .
  • Examples of such peptides of Formula II include the following: Val-Cys-Ile-Cys-Gln (SEQ ID N0..18) ,
  • Val-Cys-Gly-Cys-Arg (SEQ ID N0..19), Lys-Cys-Arg-Cys-Lys (SEQ ID NO..20), Asp-Cys-Ile-Cys-Gln (SEQ ID NO.:21) , Ile-Cys-Thr-Cys-Glu (SEQ ID NO. :22) , Ile-Cys-Thr-Cys-Arg (SEQ ID N0..23) ,
  • the nucleic acid molecule of the invention could encode a peptide where, for example, X is Val, Ala, Leu, lie, Lys, Asp, Phe or Pro; Y is Glu, Val, Gin, Arg, Lys, or Pro; Z is Gly, Ala, lie, Arg, Thr, or Lys; and the sum of n and m is one to three, inclusive.
  • the DNA molecule of the invention may encode a peptide consisting of 4-30 amino acid residues that conforms to the motif represented by Formula III :
  • X is selected from the group consisting of Ala, Val, Leu, lie, Gly, Ser, Thr, Asp, Glu, Lys, Arg, His, Trp, Tyr, and Phe;
  • Y is selected from the group consisting of Ala, Val, Leu, lie, Gly and Pro;
  • Z is selected from the group consisting of Ala, Val, Leu, He, Gly, Lys, Arg, His, Phe, and Pro; n and m are whole integers chosen with the proviso that the sum of n and m is zero to twenty-six, inclusive; and the nucleic acid molecule optionally further encodes a mammalian signal peptide linked to the immunoactive peptide at A n .
  • X is Gly, Ala, He, Asp, Thr, Ser, Arg, or Trp; Y is He, Gly, or Pro; Z is Lys, He, Phe, Pro, Ala, Tyr or Gly; and the sum of n and m is zero to eleven, inclusive .
  • the DNA molecule of the invention encodes a peptide conforming to the motif represented by Formula III, optionally linked to a signal peptide, where
  • X is Gly, Y is Pro, and Z is He,
  • X is Gly
  • Y is Pro
  • Z is Gly
  • X is Ala, Y is Pro, and Z is Ala, X is He, Y is Pro, and Z is Tyr,
  • X is Ala, Y is Pro, and Z is He,
  • X is Arg, Y is Pro, and Z is He
  • X is He
  • Y is Pro
  • Z is He
  • X is Asp, Y is Pro, and Z is He, X is Trp, Y is Pro, and Z is He, X is Trp, Y is Pro, and Z is Gly,
  • X is Gly, Y is He, and Z is He,
  • X is Thr, Y is Pro, and Z is Tyr,
  • X is Ala
  • Y is Pro
  • Z is Phe
  • X is Ser, Y is Pro, and Z is Phe,
  • X is Gly, Y is Pro, and Z is Pro, or
  • X is Gly, Y is Pro, and Z is Tyr; A and B vary according to the parameters above; and the sum of n and m is zero to eleven, inclusive.
  • Trp-Pro-Cys-Gly SEQ ID N0..31
  • the nucleic acid molecule of the invention may encode, for example, a peptide where X is Gly, Ala, He, Arg, Asp, Trp, Thr, or Ser; Y is Pro, Gly, or He; Z is Gly, Ala, He, Tyr, Phe, or Pro; and the sum of n and m is one to three, inclusive.
  • the DNA molecule of the invention encodes an immunoactive peptide consisting of 3-30 amino acid residues that conforms to the motif represented by Formula IV:
  • X is selected from the group consisting of Ser, Glu, Gly, Ala, Leu, Pro, Thr, Val, Asn, and Lys;
  • Y is selected from the group consisting of Leu, Arg, Pro, Tyr, He, Val, Ser, Ala, and Phe;
  • Z is selected from the group consisting of Met, Trp, Tyr, Phe, Gly, Pro, Arg, Asn, Gin, Ala, and Lys; n, , p, and q are whole integers chosen with the following provisos: p and q are independently zero or 1 but are not both simultaneously zero; when q is zero, m is zero; and the sum of n, m, p, and q is 1 to 28, inclusive.
  • the nucleic acid molecule encoding a peptide conforming to Formula IV optionally further encodes a mammalian signal peptide linked to the amino terminus of the immunoactive peptide.
  • both p and q are 1, and the peptide consists of 4-20 amino acid residues. More preferably, the peptide consists of 4-15 amino acid residues (e.g., 4-9 or
  • the peptide consists of 4-7 amino acid residues, optionally linked to a signal peptide.
  • the DNA molecule of the invention encodes a peptide conforming to the motif represented by Formula IV, optionally linked to a signal peptide, where
  • X is Glu
  • Y is Pro
  • Z is Met
  • X is Gly, Y is Pro, and Z is Met
  • X is Ala, Y is Pro, and Z is Trp, X is Ala, Y is Pro, and Z is Met,
  • X is Glu
  • Y is Pro
  • Z is Trp
  • X is Ser, Y is Pro, and Z is Trp,
  • X is Leu
  • Y is Leu
  • Z is Gly
  • X is Pro, Y is Arg, and Z is Arg, X is Gly, Y is Tyr, and Z is Pro, X is Val, Y is Val, and Z is Asn,
  • X is Leu
  • Y is Ser
  • Z is Gin
  • X is Ser, Y is Pro, and Z is Tyr,
  • X is Ala, Y is Leu, and Z is Arg, X is Ala, Y is Pro, and Z is Tyr,
  • X is Gly, Y is Ala, and Z is Pro,
  • X is Lys
  • Y is Ser
  • Z is Lys
  • X is Glu
  • Y is Pro
  • Z is Phe
  • X is Glu, Y is Pro, and Z is Tyr, X is Ser, Y is Pro, and Z is Met,
  • X is Ala, Y is Pro, and Z is Tyr,
  • X is absent, Y is Leu, and Z is Phe, or
  • X is Gly, Y is Pro, and Z is Trp; A and B are selected independently from the 20 common, naturally occurring amino acids; and n, m, p, and q are whole integers chosen with the provisos specified above.
  • peptides conforming to Formula IV are chosen with the proviso that: when Y is Pro or He, and q is 1, and Z is Tyr, Phe, Gly, Pro, or Ala, then
  • peptides of Formula IV include the following:
  • Ala-Ala-Trp-Ser-Pro-Cys-Met (SEQ ID NO. :96), Val-Ala-Tyr-Gly-Pro-Cys-Trp (SEQ ID NO.:97) Leu-Arg-Pro-Arg-Cys-Arg-Pro-He (SEQ ID NO. :98), Ala-Gly-Tyr-Cys-Pro-Thr-Met-Thr (SEQ ID NO.:99), Pro-Gln-Val-Val-Cys-Asn-Tyr-Arg (SEQ ID NO..100), or
  • Ala-Asn-Phe-Cys-Ala-Gly-Ala-Cys-Pro-Tyr-Leu-Trp (SEQ ID NO. :101) ;
  • a and B are selected independently from the 20 common, naturally occurring amino acids; and
  • n, m, p, and q are whole integers chosen with the provisos specified above.
  • peptides of Formula IV that contain a mammalian signal peptide sequence include the following:
  • the DNA molecule of the invention encodes an immunoactive peptide consisting of 3-30 amino acid residues that conforms to the motif represented by Formula V:
  • W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, Trp, Ser, Met, Cys, and Glu;
  • X is selected from the group consisting of Cys, Pro, He, Met, Tyr, Thr, and Arg; Y is selected from the group consisting of Cys and Met;
  • Z is selected from the group consisting of Gly, Phe, Val, He, Pro, Tyr, Trp, Glu, Leu, and Met;
  • W, X, and Y are chosen with the proviso that at least one of W, X, or Y is Met, and not more than one of W, X, or Y is Cys; n, m, and p are whole integers chosen with the provisos that p is zero or 1; when p is zero, m is zero; and the sum of n, m, and p is zero to 27, inclusive.
  • the nucleic acid molecule encoding a peptide conforming to Formula V optionally further encodes a mammalian signal peptide linked to the amino terminus of the immunoactive peptide.
  • p is 1, and the peptide consists of 4-20 amino acid residues. More preferably, the peptide consists of 4-15 amino acid residues (e.g., 4-9 or 4-10 amino acid residues) . Most preferably, the peptide consists of 4-7 amino acid residues.
  • the DNA molecule of the invention encodes a peptide conforming to the motif of Formula V, optionally linked to a signal peptide, wherein
  • W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, Trp, and Ser;
  • X is selected from the group consisting of Cys, Pro, He, and Met
  • Y is selected from the group consisting of Cys and Met
  • Z is selected from the group consisting of Gly, Phe, Val, He, Pro, and Leu.
  • the DNA molecule of the invention encodes a peptide conforming to the motif of Formula V, optionally linked to a signal peptide, wherein
  • W is selected from the group consisting of Gly, Asp, Arg, Ala, Trp, and Ser;
  • X is selected from the group consisting of Pro and He;
  • Y is Met ;
  • Z is selected from the group consisting of Phe, He, and Pro.
  • the DNA molecule of the invention encodes a peptide conforming to the motif of Formula V, optionally linked to a signal peptide, wherein
  • W is selected from the group consisting of Gly and Ser;
  • X is Pro
  • Y is Met; and Z is selected from the group consisting of Phe, He, and
  • the nucleic acid molecule of the invention may encode a peptide having Met and Cys or Met and Met aligned contiguously.
  • the encoded peptide may conform in sequence to A-W-Met-Met-Z-B,
  • A-W-Met-Cys-Z-B or A-W-Cys-Met-Z-B.
  • the nucleic acid molecule of the invention may encode a peptide in which Met and Cys are separated by no more than one amino acid.
  • the encoded peptide may conform in sequence to A-Met-X-Cys-Z-B or A-Cys-X-Met-Z-B.
  • Gly-Pro-Met-He (SEQ ID N0..114) , Lys-Met-Arg-Met-Lys (SEQ ID NO. : 115) Phe-Met-He-Met-Lys (SEQ ID N0..116), Ile-Cys-Thr-Met-Glu (SEQ ID N0..117), Leu-Met-Ala-Met-Val (SEQ ID N0. :118), He-Met-Tyr-Met-Glu (SEQ ID NO. :119) ,
  • each of the peptides conforming to the motifs represented by Forumula III, IV, or V are selected with the proviso that the following peptides are excluded:
  • the following sequences are excluded: Leu-Glu-Cys-Gly-Pro-Cys-Phe-Leu (SEQ ID NO.:157), Leu-Cys-Ala-Gly-Pro-Cys-Phe-Leu (SEQ ID NO.:158) , Tyr-Ile-Pro-Cys-Phe-Pro-Ser-Ser-Leu-Lys-Arg-Leu-Leu-Ile
  • Thr-Pro-Pro-Thr-Pro-Cys-Pro-Ser (SEQ ID NO.:162) , Asp-Pro-Cys-He-Ile (SEQ ID NO. :163), Cys-Gly-Gly-Ile-Cys-He-Ala-Arg (SEQ ID N0..164) , Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQ ID NO. :165) ,
  • Tyr-Arg-Arg-Gly-Arg-Cys-Gly-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO. :170)
  • Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Gly-Leu-Cys- Leu-Ala-Arg (SEQ ID NO. :171) ,
  • Tyr-Arg-Arg-Gly-Arg-Cys-Gly-Gly-Gly-Gly-Gly-Leu-Cys-Leu-Ala- Arg (SEQ ID NO. :172) , Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Gly-Gly-Leu-
  • Ser-Pro-Tyr-Met-Glu-Ala (SEQ ID NO. :175) .
  • the nucleic acid molecule of the invention can be RNA (e.g., in a retrovirus) or DNA. It preferably encodes an immunoactive peptide that is not a naturally occurring human polypeptide nor a fragment of a naturally occurring human polypeptide. Even where the sequence of the immunoactive peptide happens to be that of a fragment of a naturally occurring polypeptide, the nucleic acid molecule of the invention differs from any naturally occurring nucleic acid molecule in that the coding sequence encodes just that peptide, optionally linked to a signal peptide. Of course, multiple coding sequences can be linked in tandem, separated by stop codons and potentially other noncoding sequence.
  • the nucleic acid molecule may include a sequence encoding a mammalian signal peptide. That signal peptide would, when linked to the amino terminus of the immunoactive peptide within a mammalian cell, direct the secretion of the immunoactive peptide out of the cell.
  • the signal peptide is typically enzymatically cleaved from the immunoactive peptide during the process of secretion. Selection of a particular signal peptide depends upon the species of the animal to be treated, and the amino-terminal amino acid sequence of the immunoactive peptide to be expressed. Numerous examples of secretory signal sequences linked to immunoactive gene sequences are shown in Figure 2.
  • the signal peptide When a nucleic acid molecule is to be administered to a human patient, as described below, the signal peptide will usually be a human secretory signal peptide.
  • the nucleic acid molecule of the invention will generally also include a eukaryotic expression control sequence, e.g. a mammalian expression control sequence, operatively linked to the coding sequence.
  • the expression control sequence may be an inducible or constitutively active promoter that directs the expression of one or more immunoactive peptides encoded on the nucleic acid molecule in a tissue- or cell-specific manner. Selecting appropriate secretory signal sequences and expression control sequences is well within the abilities of skilled artisans, and further guidance regarding this selection is given below.
  • a related aspect of the invention is a mammalian expression vector, such as a viral, e.g. a retroviral, adenoviral, or adeno-associated vector, that has been modified by standard recombinant techniques to encode an immunoactive peptide.
  • viral vectors may be a part of a viral particle that is capable of infecting mammalian cells.
  • the expression vector of the invention can be used to produce an immunoactive peptide by, for example, introducing the expression vector into a cultured mammalian cell, culturing the cell in vi tro under conditions that permit expression of the immunoactive peptide, and harvesting the immunoactive peptide from the cell.
  • the immunoactive peptide may instead be harvested from the medium surrounding the cells.
  • an immunoactive peptide may be produced in a mammal (e.g., a human, simian, mouse, rat, guinea pig, hamster, rabbit, dog, cat, cow, pig, goat, sheep or horse) by introducing into the mammal either (1) the nucleic acid molecule of the invention, (2) an expression vector containing the nucleic acid molecule of the invention, or (3) a cell that contains and expresses the nucleic acid. In the latter case, the cell or its descendent would be transduced with the nucleic acid ex vi vo .
  • Such cells particularly mammalian cells such as human cells are considered to be within the invention.
  • non-integrating viral vectors include herpes simplex virus-based vectors, which have a broad cell specificity and can accept up to 36 kb of nonviral sequence, and the SV40 vector, which also targets a wide range of tissues.
  • viruses known to be useful for gene transfer include adenoviruses, adeno associated virus, mumps virus, poliovirus, retroviruses, Sindbis virus, and vaccinia virus such as canary pox virus.
  • Well-known methods of transducing cells that do not require a viral vector include calcium phosphate precipitation, lipofection, electroporation, or biolistic methods.
  • the invention features a method for modulating the immune response in a patient by administering to the patient a nucleic acid molecule encoding at least one peptide that functions either as an immunosuppressant or as an immunostimulant.
  • the method may be carried out by administering to the patient either (1) the isolated nucleic acid molecule consisting essentially of the coding sequence linked to expression control elements, (2) the nucleic acid molecule within an expression vector, or (3) a cell that secretes the immunoactive peptide.
  • cells of the patient could be transduced ex vivo by standard techniques, such as those described herein.
  • the nucleic acid molecule, the vector containing it, or a cell secreting the immunoactive peptide could be administered to the patient by any route commonly known to skilled pharmacologists. These include introduction into the patient's bloodstream or cerebrospinal fluid, into the synovial fluid, into a tumor, or into the vicinity of a tumor. It will be apparent to skilled artisans that the nucleic acid molecule of the invention can be contained within a therapeutic composition that is formulated with a pharmaceutically acceptable carrier.
  • Nucleic acid molecules encoding peptides that function as immunosuppressants may be administered to a patient who has received a biological transplant e.g., of an organ such as a kidney, heart, liver, eye, or lung; of a tissue such as skin or bone marrow; or of cells such as fibroblasts, neural cells, islet cells, hepatocytes, or chondrocytes .
  • a biological transplant e.g., of an organ such as a kidney, heart, liver, eye, or lung; of a tissue such as skin or bone marrow; or of cells such as fibroblasts, neural cells, islet cells, hepatocytes, or chondrocytes .
  • immunosuppressant-expressing nucleic acids can also be used to treat a person suffering from an autoimmune disease, including but not limited to the following: (1) a rheumatic disease such as rheumatoid arthritis, systemic lupus erythematosis, Sjogren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome, or Behcet ' s disease, (2) type I diabetes; (3) an autoimmune disease of the thyroid, such as Hashimoto's thyroiditis or Graves' Disease; (4) an autoimmune disease of the central nervous system, such as multiple sclerosis, myasthenia gravis, or encephalomyelitis; and (5) phemphigus such as phemphigus vulgaris, phemphigus vegetans, phemphigus foliaceus, Senear- Usher syndrome, or Brazilian phemphigus.
  • Nucleic acids encoding peptides that function as immunostimulants may be administered to a patient who is thought to be suffering from a chronic infection, an acute infection, or a cancer such as cancer of the breast, lung, colon stomach, skin, brain, cervix, uterus, liver, bone, pancreas, or hemotopoietic system.
  • nucleic acid molecule of the invention in the preparation of a medicament useful in treating any of the above conditions.
  • peptide is meant any chain of more than two amino acid residues, regardless of post-translational modification such as glycosylation or phosphorylation.
  • naturally occurring amino acids are L-glycine (Gly; G) , L-alanine (Ala; A), L-valine (Val; V) , L-leucine (Leu; L) , L-isoleucine (He; I) , L-serine (Ser; S) , L-threonine (Thr; T) , L-aspartic acid (Asp; D) , L-glutamic acid (Glu; E) , L-lysine (Lys; K) , L-arginine (Arg; R) , L-histidine (His; H) , L-methionine (Met; M) , L-cysteine (Cys; C) , L- asparagine (Asn; N) , L-glutamine (Gly; G) ,
  • Fig. 1 is a list of examples of nucleic acid molecules of the invention and the peptides they encode.
  • An asterisk demarks the boundary between a rat secretory signal sequence and the sequence of each immunoactive peptide.
  • Fig. 2 is a schematic diagram of the Moloney murine sarcoma virus retroviral vector pLXSN.
  • nucleic acid molecules that encode peptides which can be used to modulate the immune response. These nucleic acid molecules were cloned into expression vectors, transduced into mammalian cells, and shown to inhibit the formation of tumors in vivo, presumably by upregulating the activity of T lymphocytes in the treated animal .
  • Each vector described below includes a sequence encoding (a) an immunoactive peptide that conforms to the motif represented in Formula I, II, III, IV, or V, and (b) a signal sequence that targets the peptide for export from the transduced cell, and a eukaryotic expression control sequence. Standard recombinant techniques were used to link these sequences and clone them into the vector of choice.
  • a first single-stranded oligodeoxynucleotide was synthesized, using standard techniques for DNA synthesis.
  • This oligodeoxynucleotide consisted of, from the 5' end: 3-4 adenosine residues, a restriction enzyme site, a sequence encoding a signal peptide, a sequence encoding an immunoactive peptide, two stop codons, a second restriction enzyme site, and another 3-4 adenosine residues.
  • the restriction enzyme sites were chosen to facilitate ligation between the oligodeoxynucleotide and the vector of choice. In the examples below, the restriction enzymes were chosen from the following: EcoRI, BamHl, Xhol, and Hpal.
  • the signal sequence was chosen on the basis of the first N-terminal amino acid of the immunoactive peptide.
  • immunoactive peptides having alanine as their N-terminal amino acid were linked to signal sequences that are naturally associated with peptides that have alanine as the N-terminal amino acid.
  • signal sequences were chosen from those which occur naturally in rat cells.
  • appropriate signal sequences would be selected in an analogous way. Further guidance in selecting these sequences for use in humans is given below.
  • the DNA sequence encoding each signal peptide used in the experiments described below was the naturally occurring rat DNA sequence, except where it was necessary to modify it to avoid including a restriction enzyme site that would complicate the cloning strategy.
  • the DNA sequence encoding each immunoactive peptide was chosen in part to avoid introducing problematic restriction sites.
  • the single-stranded oligodeoxynucleotide prepared as described above was made double-stranded as follows.
  • An antisense oligodeoxynucleotide complementary to approximately 15 nucleotides at the 3 ' end of the first oligodeoxynucleotide was synthesized by standard synthetic means.
  • T7 DNA polymerase buffer United States Biochemicals
  • they were placed in a solution of T7 DNA polymerase buffer (United States Biochemicals) , gradually heated to 60°C, and held at that temperature for 30 minutes. Once annealed, a complete double-stranded molecule was enzymatically generated with T7 DNA polymerase, according to the manufacturer's instructions (United States Biochemicals) .
  • the double-stranded DNA was purified by passing it over a Sephadex G50 (Pharmacia, Uppsala, Sweden) column in TE buffer (10 mM Tris, 1 mM EDTA at pH 7.5) and digested with restriction enzymes corresponding to the restriction sites that had been placed at each end of the oligodeoxynucleotide.
  • the DNA was extracted from the digest with phenol-chloroform, precipitated with absolute ethanol at - 70°C, and collected by centrifugation, according to standard methods.
  • the DNA pellet was washed with 70% ethanol, dried under vacuum, and redissolved in TE buffer.
  • DNA prepared as described above can be inserted into any vector that has compatible restriction sites.
  • the DNA was inserted into the Moloney murine sarcoma virus retroviral vector pLXSN (Fig. 2) which had been digested with restriction enzymes to create cohesive ends complementary to those created by digestion of the insert, i.e., with one of the following pairs of restriction enzymes: (1) EcoRI-BamHI, (2) EcoRI-Xhol, (3) EcoRI-Hpal, (4) Hpal-BamHl, (5) Hpal-Xhol, or (6) XhoI-BamHI.
  • a ligation reaction containing approximately 20 ng of vector DNA and 4 ng of insert DNA was carried out at 16°C with T4 DNA ligase (Boehringer Mannheim) .
  • the ligation reaction was then used to transform electrocompetent E. coli cells (DH5 ⁇ strain) . Individual colonies that developed from transformed cells were picked at random and checked by the polymerase chain reaction (PCR) for the presence of vectors that contained insert. Colonies consisting of a clone of cells that contained the desired construct (vector with insert) were amplified, and the DNA construct was isolated and sequenced by standard methods .
  • Cultured cells were obtained from two types of mammary carcinomas: SPMW1 cells were obtained from a tumor that developed spontaneously in a female Wistar rat, and Ad9-101 cells were obtained from a tumor that developed after newborn female Wistar rats were inoculated with adenovirus type 9
  • Cell lines were established from the tumors as follows. The tumor was excised from the animal, minced with a pair of scissors, and treated with Dispase grade II for 30 minutes (2.4 mg/ml; Boehringer Mannheim) in RPMI 1640 medium to disaggregate the cells. The SPMW1 cell line was established from the nineteenth in vivo passage of a tumor, and the Ad9-101 cell line was established at the fifth in vivo passage.
  • the disaggregated cells were cultured in RPMI 1640 medium supplemented with 4 mM l-glutamine, 1 mM pyruvate, 10 mM HEPES buffer, 10 mM NaHC0 3 , and 5% fetal calf serum (FCS) in vessels obtained from NUNC (Roskilde, Denmark) .
  • DNA constructs were prepared as described above and used to transfect a retroviral packaging cell line GP+E or Psi2 with TRANSFECTAMTM (Promega, USA) , according to the manufacturer's instructions. Transfectants were selected with G418 (300 ⁇ g/ml) in RPMI 1640 with 10% FCS. Virus-laden supernatant from the transfectants was then used to infect either SPMWl cells or Ad9-101 cells. Successfully transduced cells were selected in the presence of G418, and clonal cell lines were developed.
  • Transduced tumor cells were harvested from the culture vessels by the addition of trypsin, collected by centrifugation, and resuspended in phosphate buffered saline (PBS) supplemented with 5% normal syngeneic rat serum.
  • PBS phosphate buffered saline
  • Each rat in the experimental group received a subcutaneous injection in the right hindlimb of approximately 200 ml of resuspended cells.
  • comparable rats received subcutaneous injections of the same type of tumor cells, but which had not been transduced, and thus did not express or secrete an immunoactive peptide.
  • SPMWl mammary carcinoma cells in cell culture were infected with a retroviral vector containing an insert encoding the immunoactive peptide D22175AX.
  • Transduced cells were selected in G418, as described above, but not cloned.
  • Approximately 25,000 transduced tumor cells were subcutaneously injected into the hindlimbs of each of 8 rats.
  • an equivalent group of rats was similarly injected with approximately 25,000 wild type (i.e., non-transduced) SPMWl cells.
  • the size of the tumor that developed in vivo was estimated according to the above formula in both groups for up to fifteen days following injection.
  • D22175AX-expressing SPMWl cells were also injected subcutaneously into the right hindlimb of "nude" rats. These animals do not have a thymus and thus do not produce T lymphocytes. Five animals were injected subcutaneously with approximately 25,000 uncloned D22175AX-expressing SPMWl cells and five were injected with a comparable number of wild type SPMWl cells. The tumors that developed in these two groups of animals following inoculation grew at a comparable rate (Table 2), suggesting that the inhibition of tumor growth seen when immunocompetent rats are inoculated with D22175AX- expressing SPMWl cells involves a T cell-mediated immune response. Table 2: Estimated Volume (mm ) of Palpated Tumor
  • Example 3 Immunostimulation by D22175AX-expressing Ad9-101 Cells
  • Ad9-101 mammary carcinoma cells in cell culture were infected with a retroviral vector containing an insert encoding the immunoactive peptide D22175AX. As described in Example 1, the cells were selected in G418, but they were not cloned. Approximately 10,000 cells were subcutaneously injected into the hindlimbs of each of 5 rats. As a control, an equivalent group of rats was injected with 10,000 wild type Ad9-101 cells. On any given day after injection, the average size of the tumor that had developed from D22175AX-expressing Ad9-101 cells was less than one-tenth the size of the tumor that developed from wild type Ad9-101 cells (Table 3) . Therefore, expression of D22175AX significantly impedes tumor growth in at least two model systems.
  • Table 3 Estimated Volume (mm 3 ) of Palpated Tumor
  • Example 4 Immunostimulation by two D22175AX- expressing Ad9-101 clones; clone 8 and clone 9 Cells from four different D22175AX-expressing Ad9-101 clonal cell lines were injected into rats in order to determine whether different transduced clones expressing the same peptide were equally effective in impeding tumor growth. Wild type Ad9- 101 cells served as the control for this experiment. Five animals in each group received subcutaneous injections containing approximately 50,000 cells of uncloned, clone 6, clone 7, clone 8, clone 9, or wild type Ad9-101 lines. As shown in Table 4, all transduced clones exhibited significantly slower tumor growth than did wild type Ad9-101 cells, at least after day 13. Table 4: Estimated Volume (mm 3 ) of Palpated Tumor
  • Example 5 Immunostimulation by D22175AX-expressing Ad9-101 Cells is Mediated by T Lymphocytes
  • D22175AX-expressing Ad9-101 cells were injected subcutaneously with either wild type Ad9-101 cells or D22175AX-expressing Ad9- 101 cells (clone 8 or clone 9) .
  • Each animal was killed when its tumor reached 50-100 mm 3 in size, and approximately 2.5 x IO 5 cells were harvested from the lymph nodes associated with the tumor, i.e., the inguinal and para-aortal lymph nodes ipsilateral to the tumor.
  • lymphatic cells were also harvested from the lymph nodes of normal rats (which were free of tumors) .
  • the homogeneous and heterogeneous cultures were established in parallel in a total of 10 wells of a 96 well plate (NUNC, Roskilde, Denmark) , and grown for 5 days in RPMI 1640 medium supplemented with 4 mM L-glutamine, 1 mM pyruvate, 10 mM Hepes buffer, 15 mM NaHC0 3 , 50 ⁇ M ⁇ -mercaptoethanol, and 10% FCS.
  • the cells were exposed to [ 3 H] -thymidine (0.5 ⁇ Ci) for 6 hours.
  • T lymphocytes can respond with mitotic activity on day 5.
  • the radioactivity incorporated into acid-insoluble material which reflects the mitotic activity of the cells in culture, was measured with a scintillation counter.
  • the amount of radioactivity incorporated into homogeneously cultured lymph cells was subtracted from the amount of radioactivity incorporated into heterogeneous cultures containing lymphatic and irradiated tumor cells. (The incorporated radioactivity is attributable solely to proliferation of the lymphatic cells, because the tumor cells were lethally irradiated prior to co- culture and so could not proliferate.)
  • the data obtained from two trials, and expressed as counts per minute (cpm) are presented in Table 5.
  • rats were inoculated with either Ad9-101 wild type cells that had been irradiated with
  • Example 7 Immunomodulation by D22139AA-expressing, D22069AX-expressing, or D7208-expressing Ad9-101 cells
  • D22139AA (without signal sequence) has previously been shown to be immunostimulatory when administered directly in a delayed-type hypersensitivity (DTH) assay, while D22069AX and D7208 (each without signal sequence) have shown activity consistent with immunosuppression.
  • DTH delayed-type hypersensitivity
  • D22069AX and D7208 each without signal sequence have shown activity consistent with immunosuppression.
  • the tumors that developed from uncloned D22139AX-expressing Ad9-101 cells were on average somewhat smaller than the tumors that developed from wild type Ad9-101 cells, consistent with the results of the experiment shown in Table 7, while the tumors that developed from clones 6 and 7 were substantially smaller.
  • clones 1, 2, 3, 5, and 9 showed essentially no tumor outgrowth through day 35 (data from clone 2 is shown in Table 8) .
  • DTH Delayed Type Hypersensitivity
  • DTH delayed type hypersensitivity
  • mice The detailed protocol for this assay can be found, for example, in Carlsten et al . (1986, Int. Arch. Allergy Appl. Immunol. 81:322) . Briefly, male or female mice, such as Balb/c mice, are sensitized by exposure to 4-ethoxymethylene-2-phenyloxazolin-5- one (OXA; Sigma Chemical Co. ) . On Day 0, 150 ⁇ l of an absolute ethanol-acetone (3:1) solution containing 3% OXA is applied to the animal's shaved abdomen.
  • OXA 4-ethoxymethylene-2-phenyloxazolin-5- one
  • Treatment with the immunoactive peptide itself e.g., by topical or IV administration
  • gene therapy using a nucleic acid encoding the peptide is then begun (methods of administration are discussed below) .
  • the thickness of the animal's ears is measured with an Oditest spring caliper before both ears are challenged by topical application of 20 ⁇ l of 1% OXA dissolved in an oil, such as peanut oil. Ear thickness is measured again 24 and 48 hours after the challenge. To minimize discomfort, challenges and measurements are performed under light anesthesia.
  • the intensity of the DTH reaction is measured as described by van Loveren et al. (1984, J. Immunol. Methods 67:311), and expressed according to the formula: Tt24/48 - TtO (in mm units) where tO, t24, and t48 represent ear thickness before, 24 hours after, and 48 hours after the challenge, respectively.
  • the ability of the peptide or gene therapy to modulate ear thickness is an indication of its ability to modulate the immune response: a relative increase in thickness indicates a heightened response, while a relative decrease in thickness indicates immune suppression. Inhibition of Tumor Growth
  • Nucleic acids encoding peptides that stimulate the immune response can be identified using any model system analogous to the mammary carcinoma models described above. These additional model systems could be developed with immortalized cells from an established cell line or an induced or spontaneous tumor of an animal. Other cell lines that would be amenable to an assay for tumor growth are readily available from the American Type Culture Collection (A.T.C.C.) , which maintains cell lines established from a wide variety of tumors that developed in many different species. Transgenic animals that develop tumors due to, for example, overexpression of an oncogene or inhibition of a tumor suppressor gene provide a second source of tumor cells suitable for tumor growth assays.
  • A.T.C.C. American Type Culture Collection
  • a spontaneous or induced tumor from an animal e.g., a spontaneous tumor from a human
  • Cells from any of these sources could be placed in culture, transduced with a nucleic acid encoding a candidate immunoactive peptide, and transplanted into a test animal.
  • Genes encoding peptides that stimulate the immune response can also be identified in various model systems by the "immunization" procedure described in Example 6.
  • Immortalized cells obtained from the A.T.C.C. or established from primary tumor tissue as described above, would be transduced with the gene of interest, lethally irradiated, and injected into animals.
  • animals could be injected with irradiated wild type tumor cells.
  • both groups of animals would be challenged with wild type tumor cells and examined for tumor formation. If the peptide is an immunostimulant with therapeutic potential, the growth of the tumor following the challenge with wild type cells would be impeded in animals that had been immunized with peptide- expressing cells.
  • transgenic animals e.g., mice
  • Transduced, irradiated tumor cells from such an animal can be used to immunize other animals of the same line, before they begin to develop any tumors .
  • Subsequent spontaneous or induced tumor formation and growth are then assessed in the immunized animals.
  • Another way to assay a peptide-encoding gene is to administer it to an animal after a tumor has formed.
  • the animal can be one that is a transgenic model for tumor formation, or one which has developed a tumor following injection of wild type tumor cells.
  • the animal is injected with the peptide-encoding vector or with peptide-expressing cells, which could either be viable or lethally irradiated.
  • a reduction in tumor size or number compared to control, or an extended time to death, would provide very strong evidence for the utility of genes encoding immunostimulatory peptides in the treatment of cancer.
  • a peptide In order to determine whether a peptide is capable of functioning as an immunosuppressant, it can be administered, directly or by genetic therapy, in the context of well- established transplantation paradigms.
  • a putative immunosuppressing peptide, or a nucleic acid molecule encoding it could be systemically or locally administered by standard means to any conventional laboratory animal, such as a rat, mouse, rabbit, guinea pig, or dog, before an allogeneic or xenogeneic skin graft, organ transplant, or cell implantation is performed on the animal.
  • the graft itself could be transduced with the nucleic acid of the invention.
  • mice such as C57B1-10, B10.BR, and B10.AKM (Jackson Laboratory, Bar Harbor, ME) , which have the same genetic background but are mismatched for the H-2 locus, are well suited for assessing various organ grafts .
  • the aorta of a donor heart is anastomosed to the abdominal aorta of the host, and the pulmonary artery of the donor heart is anastomosed to the adjacent vena cava using standard microvascular techniques.
  • the heart is grafted in place, and warmed to 37°C with Ringer's lactate solution, normal sinus rhythm will resume.
  • Function of the transplanted heart can be assessed frequently by palpation of ventricular contractions through the abdominal wall. Rejection is defined as the cessation of myocardial contractions.
  • a given peptide would be considered a successful immunosuppressant if it prolonged the time the grafted organ was tolerated by the host .
  • the effectiveness of an immunoactive peptide can also be assessed following a skin graft.
  • a donor animal is anesthetized and the full thickness skin is removed from a part of the tail.
  • the recipient animal is also anesthetized, and a graft bed is prepared by removing a portion of skin from the shaved flank.
  • the patch is approximately 0.5 x 0.5 cm.
  • the skin from the donor is shaped to fit the graft bed, positioned, covered with gauze, and bandaged.
  • the grafts can be inspected daily beginning on the sixth post-operative day, and are considered rejected when more than half of the transplanted epithelium appears non-viable.
  • Another technique for assaying immunosuppression is with cells that have been tranduced with a nucleic acid of the invention, then implanted into an allogeneic or xenogeneic animal . If the transduced implanted cells survive longer than control implanted cells which have not been transduced, then the nucleic acid molecule presumably encodes an immunosuppressive peptide.
  • Models of autoimmune disease provide another means to assess peptides in vivo . These models are well known to skilled artisans and can be used to determine whether a given peptide is an immunosuppressant that would be therapeutically useful in treating a specific autoimmune disease when delivered via genetic therapy.
  • rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosis (SLE) , type I diabetes, and autoimmune diseases of the thyroid and central nervous system.
  • SLE systemic lupus erythematosis
  • animal models of SLE include MRL mice, BXSB mice, and NZB mice and their FI hybrids. These animals can be crossed in order to study particular aspects of the rheumatic disease process; the NZB strain develops severe lupus glomerulonephritis when crossed with NZW mice (Bielschowsky et al., 1959, Proc. Univ. Otago Med. Sch.
  • MRL- lpr/lpr One of the MRL strains of mice that develops SLE, MRL- lpr/lpr, also develops a form of arthritis that resembles rheumatoid arthritis in humans (Theofilopoulos et al . , 1985, Adv. Immunol. 37:269) .
  • an experimental arthritis can be induced in rodents by injecting rat type II collagen (2 mg/ml) mixed 1:1 in Freund's complete adjuvant (100 ⁇ l total) into the base of the tail. Arthritis develops 2-3 weeks after immunization.
  • T lymphocytes The ability of genes encoding immunoactive peptides to combat the arthritic condition can be assessed by targeting the genes to T lymphocytes and/or to synovial cells of the joint.
  • One way to target T lymphocytes is the following: spleen cell suspensions are prepared 2-3 days after the onset of arthritis and incubated with collagen (100 ⁇ g/ml) for 48 hours to induce proliferation of collagen-activated T lymphocytes. During this time, the cells are transduced with a vector encoding the peptide of interest. As a control, parallel cultures are untransduced or transduced with the "empty" vector. The cells are then injected intraperiotoneally (5 x IO 7 cells/animal) .
  • the effectiveness of the treatment is assessed by following the disease symptoms during the subsequent 2 weeks, as described by Chernajovsky et al . (1995, Gene Therapy 2:731-735) .
  • a decrease in symptoms compared to control indicates that the peptide of interest, and the gene encoding it, function as an immunosuppressant potentially useful in treating autoimmune disease.
  • adenoviral vector e.g., an adenoviral vector
  • An effective control in this instance would entail injecting a joint on the opposite side of the same animal or the joint of a second animal, with an adenovirus that carries the vector sequence only (Evans et al . , 1995, Trends in Mol. Med. 27:543-546) .
  • the ability of genes encoding immunoactive peptides to suppress the immune response in the case of Type I diabetes can be tested in the BB rat strain, which was developed from a commercial colony of Wistar rats at the Bio-Breeding Laboratories in Ottawa.
  • peptide-encoding genes to the treatment of cancer, infection, autoimmune disease, or graft rejection in humans can utilize either in vivo or ex vivo based therapeutic approaches.
  • This approach would entail harvesting cells (e.g., tumor cells, synovial cells, or T lymphocytes) from a patient, establishing them in culture, and transducing them with a nucleic acid of the invention.
  • the transduction step could be accomplished by any standard means used for ex vivo gene therapy, including calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer.
  • Cells that have been successfully transduced are then selected, for example via a drug resistance gene. The cells may then be lethally irradiated if desired (e.g., for tumor cells) and injected or implanted into the patient.
  • T lymphocytes obtained from the synovial fluid of an affected joint are stimulated ex vivo with IL-2 or anti-human CD3 monoclonal antibody and simultaneously infected with a relevant gene construct.
  • the cells are reintroduced into the patient, e.g., by intravenous administration, where they should home to the diseased joints and produce the peptide at the site of the inflammation.
  • a retroviral or adeno-associated viral vector would be appropriate for this ex vivo infection procedure.
  • the in vivo approach requires delivery of the construct of the invention directly into the patient, targeting it to the cells or tissue of interest.
  • residual cells may be targeted by treating the vicinity of the tumor with a composition containing a retroviral vector encoding an immunostimulatory peptide.
  • the primary tumor could be treated by in si tu injection of the vector directly into the tumor.
  • Malignant cells distal to the primary tumor site may be reached by delivering the vector intravenously.
  • Targeting of tumor cells can be accomplished by the use of a retrovirus, which targets proliferating cells.
  • a retrovirus which targets proliferating cells.
  • Non-viral vector system is a molecular conjugate composed of a plasmid attached to poly-L-lysine by electrostatic forces.
  • Poly-L-lysine covalently binds to a ligand that can bind to a receptor on tumor cells (Cristiano et al., 1995, J. Mol. Med 73:479-486) .
  • a promoter inducing relatively tumor-specific expression can be used to achieve a further level of targeting: for example, ⁇ -fetoprotein promoter for hepatocellular carcinoma (Huber et al . , 1991, Proc. Natl. Acad. Sci.
  • telomeres for melanoma
  • a constitutively active promoter could be used, e.g., the SV40 promoter or CMV promoter.
  • Adeno-associated viral vectors may be used if long-term expression is desired, or an adenoviral vector for shorter-term expression.
  • MOLECULE TYPE peptide

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Abstract

On décrit des molécules d'acide nucléique qui codent des peptides contenant de la cystéine ou de la méthionine et ont pour fonction de stimuler ou de supprimer la réponse immune chez un mammifère. De manière classique, ces peptides ne contiennent pas plus de 30 restes d'acides aminés et ils peuvent, le cas échéant, être liés à un peptide signal sécrétoire mammifère au niveau de leur extrémité terminale amino.
EP97919833A 1996-04-12 1997-04-04 Peptides contenant de la cysteine ou de la methionine et possedant des effets immunomodulateurs Withdrawn EP0923604A1 (fr)

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CA2250707A1 (fr) 1997-10-23
JP2000508898A (ja) 2000-07-18
WO1997039023A1 (fr) 1997-10-23
ID16421A (id) 1997-09-25
AR006641A1 (es) 1999-09-08
AU2417597A (en) 1997-11-07

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